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Sample records for adult drosophila midgut

  1. Intestinal stem cells in the adult Drosophila midgut

    SciTech Connect

    Jiang, Huaqi; Edgar, Bruce A.

    2011-11-15

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: Black-Right-Pointing-Pointer The homeostasis and regeneration of adult fly midguts are mediated by ISCs. Black-Right-Pointing-Pointer Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). Black-Right-Pointing-Pointer EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. Black-Right-Pointing-Pointer Notch signaling regulates ISC self-renewal and differentiation.

  2. Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

    SciTech Connect

    Lee, Shin-Hae; Park, Joung-Sun; Kim, Young-Shin; Chung, Hae-Young; Yoo, Mi-Ae

    2012-03-10

    Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: Black-Right-Pointing-Pointer Mmp1 is expressed in the adult midgut. Black-Right-Pointing-Pointer Mmp1 is involved in the regulation of ISC proliferation activity. Black-Right-Pointing-Pointer Mmp1-related ISC proliferation is associated with EGFR signaling. Black-Right-Pointing-Pointer Mmp1 in the gut is required for the intestinal homeostasis and longevity.

  3. Debra-mediated Ci degradation controls tissue homeostasis in Drosophila adult midgut.

    PubMed

    Li, Zhouhua; Guo, Yueqin; Han, Lili; Zhang, Yan; Shi, Lai; Huang, Xudong; Lin, Xinhua

    2014-02-11

    Adult tissue homeostasis is maintained by resident stem cells and their progeny. However, the underlying mechanisms that control tissue homeostasis are not fully understood. Here, we demonstrate that Debra-mediated Ci degradation is important for intestinal stem cell (ISC) proliferation in Drosophila adult midgut. Debra inhibition leads to increased ISC activity and tissue homeostasis loss, phenocopying defects observed in aging flies. These defects can be suppressed by depleting Ci, suggesting that increased Hedgehog (Hh) signaling contributes to ISC proliferation and tissue homeostasis loss. Consistently, Hh signaling activation causes the same defects, whereas depletion of Hh signaling suppresses these defects. Furthermore, the Hh ligand from multiple sources is involved in ISC proliferation and tissue homeostasis. Finally, we show that the JNK pathway acts downstream of Hh signaling to regulate ISC proliferation. Together, our results provide insights into the mechanisms of stem cell proliferation and tissue homeostasis control.

  4. Hindsight/RREB-1 functions in both the specification and differentiation of stem cells in the adult midgut of Drosophila

    PubMed Central

    Baechler, Brittany L.; McKnight, Cameron; Pruchnicki, Porsha C.; Biro, Nicole A.; Reed, Bruce H.

    2016-01-01

    ABSTRACT The adult Drosophila midgut is established during the larval/pupal transition from undifferentiated cells known as adult midgut precursors (AMPs). Four fundamental cell types are found in the adult midgut epithelium: undifferentiated intestinal stem cells (ISCs) and their committed daughter cells, enteroblasts (EBs), plus enterocytes (ECs) and enteroendocrine cells (EEs). Using the Drosophila posterior midgut as a model, we have studied the function of the transcription factor Hindsight (Hnt)/RREB-1 and its relationship to the Notch and Egfr signaling pathways. We show that hnt is required for EC differentiation in the context of ISC-to-EC differentiation, but not in the context of AMP-to-EC differentiation. In addition, we show that hnt is required for the establishment of viable or functional ISCs. Overall, our studies introduce hnt as a key factor in the regulation of both the developing and the mature adult midgut. We suggest that the nature of these contextual differences can be explained through the interaction of hnt with multiple signaling pathways. PMID:26658272

  5. Ubx dynamically regulates Dpp signaling by repressing Dad expression during copper cell regeneration in the adult Drosophila midgut.

    PubMed

    Li, Hongjie; Qi, Yanyan; Jasper, Heinrich

    2016-11-15

    The gastrointestinal (GI) tract of metazoans is lined by a series of regionally distinct epithelia. To maintain structure and function of the GI tract, regionally diversified differentiation of somatic stem cell (SC) lineages is critical. The adult Drosophila midgut provides an accessible model to study SC regulation and specification in a regionally defined manner. SCs of the posterior midgut (PM) have been studied extensively, but the control of SCs in the middle midgut (MM) is less well understood. The MM contains a stomach-like copper cell region (CCR) that is regenerated by gastric stem cells (GSSCs) and contains acid-secreting copper cells (CCs). Bmp-like Decapentaplegic (Dpp) signaling determines the identity of GSSCs, and is required for CC regeneration, yet the precise control of Dpp signaling activity in this lineage remains to be fully established. Here, we show that Dad, a negative feedback regulator of Dpp signaling, is dynamically regulated in the GSSC lineage to allow CC differentiation. Dad is highly expressed in GSSCs and their first daughter cells, the gastroblasts (GBs), but has to be repressed in differentiating CCs to allow Dpp-mediated differentiation into CCs. We find that the Hox gene ultrabithorax (Ubx) is required for this regulation. Loss of Ubx prevents Dad repression in the CCR, resulting in defective CC regeneration. Our study highlights the need for dynamic control of Dpp signaling activity in the differentiation of the GSSC lineage and identifies Ubx as a critical regulator of this process.

  6. Isolating intestinal stem cells from adult Drosophila midguts by FACS to study stem cell behavior during aging.

    PubMed

    Tauc, Helen M; Tasdogan, Alpaslan; Pandur, Petra

    2014-12-16

    Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells' (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

  7. Hs3st-A and Hs3st-B regulate intestinal homeostasis in Drosophila adult midgut.

    PubMed

    Guo, Yueqin; Li, Zhouhua; Lin, Xinhua

    2014-11-01

    Intrinsic and extrinsic signals as well as the extracellular matrix (ECM) tightly regulate stem cells for tissue homeostasis and regenerative capacity. Little is known about the regulation of tissue homeostasis by the ECM. Heparan sulfate proteoglycans (HSPGs), important components of the ECM, are involved in a variety of biological events. Two heparin sulfate 3-O sulfotransferase (Hs3st) genes, Hs3st-A and Hs3st-B, encode the modification enzymes in heparan sulfate (HS) biosynthesis. Here we demonstrate that Hs3st-A and Hs3st-B are required for adult midgut homeostasis. Depletion of Hs3st-A in enterocytes (ECs) results in increased intestinal stem cell (ISC) proliferation and tissue homeostasis loss. Moreover, increased ISC proliferation is also observed in Hs3st-B null mutant alone, or in combination with Hs3st-A RNAi. Hs3st-A depletion-induced ISC proliferation is effectively suppressed by simultaneous inhibition of the EGFR signaling pathway, suggesting that tissue homeostasis loss in Hs3st-A-deficient intestines is due to increased EGFR signaling. Furthermore, we find that Hs3st-A-depleted ECs are unhealthy and prone to death, while ectopic expression of the antiapoptotic p35 is able to greatly suppress tissue homeostasis loss in these intestines. Together, our data suggest that Drosophila Hs3st-A and Hs3st-B are involved in the regulation of ISC proliferation and midgut homeostasis maintenance.

  8. A subset of neurons controls the permeability of the peritrophic matrix and midgut structure in Drosophila adults.

    PubMed

    Kenmoku, Hiroyuki; Ishikawa, Hiroki; Ote, Manabu; Kuraishi, Takayuki; Kurata, Shoichiro

    2016-08-01

    The metazoan gut performs multiple physiological functions, including digestion and absorption of nutrients, and also serves as a physical and chemical barrier against ingested pathogens and abrasive particles. Maintenance of these functions and structures is partly controlled by the nervous system, yet the precise roles and mechanisms of the neural control of gut integrity remain to be clarified in Drosophila Here, we screened for GAL4 enhancer-trap strains and labeled a specific subsets of neurons, using Kir2.1 to inhibit their activity. We identified an NP3253 line that is susceptible to oral infection by Gram-negative bacteria. The subset of neurons driven by the NP3253 line includes some of the enteric neurons innervating the anterior midgut, and these flies have a disorganized proventricular structure with high permeability of the peritrophic matrix and epithelial barrier. The findings of the present study indicate that neural control is crucial for maintaining the barrier function of the gut, and provide a route for genetic dissection of the complex brain-gut axis in adults of the model organism Drosophila.

  9. The fine structural morphology of the midgut of adult Drosophila: A morphometric analysis.

    PubMed

    Gartner, L P

    1985-01-01

    The midgut of one day old Drosophilia was examined morphometrically at the electron microscopic level. Results suggest that parenchymal cells, with the exception of basal cells, possess identical functions. Drosophilia midgut cells are smaller than those of other insects studied, and the surface densities of the rER was less, indicating that its protein synthetic activity is also less than that of other insects.

  10. Genomic Regions Required for Morphogenesis of the Drosophila Embryonic Midgut

    PubMed Central

    Bilder, D.; Scott, M. P.

    1995-01-01

    The Drosophila midgut is an excellent system for studying the cell migration, cell-cell communication, and morphogenetic events that occur in organ formation. Genes representative of regulatory gene families common to all animals, including homeotic, TGFβ, and Wnt genes, play roles in midgut development. To find additional regulators of midgut morphogenesis, we screened a set of genomic deficiencies for midgut phenotypes. Fifteen genomic intervals necessary for proper midgut morphogenesis were identified; three contain genes already known to act in the midgut. Three other genomic regions are required for formation of the endoderm or visceral mesoderm components of the midgut. Nine regions are required for proper formation of the midgut constrictions. The E75 ecdysone-induced gene, which encodes a nuclear receptor superfamily member, is the relevant gene in one region and is essential for proper formation of midgut constrictions. E75 acts downstream of the previously known constriction regulators or in parallel. Temporal hormonal control may therefore work in conjunction with spatial regulation by the homeotic genes in midgut development. Another genomic region is required to activate transcription of the homeotic genes Antp and Scr specifically in visceral mesoderm. The genomic regions identified by this screen provide a map to novel midgut development regulators. PMID:8582615

  11. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    SciTech Connect

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2014-07-25

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

  12. Transcriptional Signatures in Response to Wheat Germ Agglutinin and Starvation in Drosophila melanogaster Larval Midgut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One function of plant lectins such as wheat germ agglutinin (WGA) is to serve as defenses against herbivorous insects. The midgut is one critical site affected by dietary lectins. We observed marked cellular, structural, and gene expression changes in the midguts of Drosophila melanogaster third-i...

  13. Midgut malrotation causing intermittent intestinal obstruction in a young adult.

    PubMed

    Bektasoglu, Huseyin Kazim; Idiz, Ufuk Oguz; Hasbahceci, Mustafa; Yardimci, Erkan; Firat, Yurdakul Deniz; Karatepe, Oguzhan; Muslumanoglu, Mahmut

    2014-01-01

    Midgut malrotation is a congenital anomaly of intestinal rotation and fixation that is generally seen in neonatal population. Adult cases are rarely reported. Early diagnosis is crucial to avoid life threatening complications. Here, we present an adulthood case of midgut volvulus as a rare cause of acute abdomen.

  14. Injury-stimulated and self-restrained BMP signaling dynamically regulates stem cell pool size during Drosophila midgut regeneration.

    PubMed

    Tian, Aiguo; Wang, Bing; Jiang, Jin

    2017-03-13

    Many adult organs rely on resident stem cells to maintain homeostasis. Upon injury, stem cells increase proliferation, followed by lineage differentiation to replenish damaged cells. Whether stem cells also change division mode to transiently increase their population size as part of a regenerative program and, if so, what the underlying mechanism is have remained largely unexplored. Here we show that injury stimulates the production of two bone morphogenetic protein (BMP) ligands, Dpp and Gbb, which drive an expansion of intestinal stem cells (ISCs) by promoting their symmetric self-renewing division in Drosophila adult midgut. We find that BMP production in enterocytes is inhibited by BMP signaling itself, and that BMP autoinhibition is required for resetting ISC pool size to the homeostatic level after tissue repair. Our study suggests that dynamic BMP signaling controls ISC population size during midgut regeneration and reveals mechanisms that precisely control stem cell number in response to tissue needs.

  15. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus

    PubMed Central

    Li-Byarlay, Hongmei; Pittendrigh, Barry R.; Murdock, Larry L.

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant–insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology. PMID:27594789

  16. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus.

    PubMed

    Li-Byarlay, Hongmei; Pittendrigh, Barry R; Murdock, Larry L

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant-insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology.

  17. Adult midgut malrotation presented with acute bowel obstruction and ischemia

    PubMed Central

    Zengin, Akile; Uçar, Bercis İmge; Düzgün, Şükrü Aydın; Bayhan, Zülfü; Zeren, Sezgin; Yaylak, Faik; Şanal, Bekir; Bayhan, Nilüfer Araz

    2016-01-01

    Introduction Intestinal malrotation refers to the partial or complete failure of rotation of midgut around the superior mesenteric vessels in embryonic life. Arrested midgut rotation results due to narrow-based mesentery and increases the risk of twisting midgut and subsequent obstruction and necrosis. Presentation of case 40 years old female patient admitted to emergency service with acute abdomen and computerized tomography scan showed dilated large and small intestine segments with air-fluid levels and twisted mesentery around superior mesenteric artery and vein indicating “whirpool sign”. Discussion Malrotation in adults is a rare cause of midgut volvulus as though it should be considered in differential diagnosis in patients presented with acute abdomen and intestinal ischemia. Even though clinical symptoms are obscure, adult patients usually present with vomiting and recurrent abdominal pain due to chronic partial obstruction. Contrast enhanced radiograph has been shown to be the most accurate method. Typical radiological signs are corkscrew sign, which is caused by the dilatation of various duodenal segments at different levels and the relocation of duodenojejunal junction due to jejunum folding. As malrotation commonly causes intestinal obstruction, patients deserve an elective laparotomy. Conclusion Malrotation should be considered in differential diagnosis in patients presented with acute abdomen and intestinal ischemia. Surgical intervention should be prompt to limit morbidity and mortality. PMID:27015011

  18. Fine-structural changes in the midgut of old Drosophila melanogaster

    NASA Technical Reports Server (NTRS)

    Anton-Erxleben, F.; Miquel, J.; Philpott, D. E.

    1983-01-01

    Senescent fine-structural changes in the midgut of Drosophila melanogaster are investigated. A large number of midgut mitochondria in old flies exhibit nodular cristae and a tubular system located perpendicular to the normal cristae orientation. Anterior intestinal cells show a senescent accumulation of age pigment, either with a surrounding two-unit membrane or without any membrane. The predominant localization of enlarged mitochondria and pigment in the luminal gut region may be related to the polarized metabolism of the intestinal cells. Findings concur with previous observations of dense-body accumulations and support the theory that mitochondria are involved in the aging of fixed post-mitotic cells. Demonstrated by statistical analyses is that mitochondrial size increase is related to mitochondrial variation increase.

  19. Midgut-enriched receptor protein tyrosine phosphatase PTP52F is required for Drosophila development during larva-pupa transition.

    PubMed

    Santhanam, Abirami; Liang, Suh-Yuen; Chen, Dong-Yuan; Chen, Guang-Chao; Meng, Tzu-Ching

    2013-01-01

    To date our understanding of Drosophila receptor protein tyrosine phosphatases (R-PTPs) in the regulation of signal transduction is limited. Of the seven R-PTPs identified in flies, six are involved in the axon guidance that occurs during embryogenesis. However, whether and how R-PTPs may control key steps of Drosophila development is not clear. In this study we investigated the potential role of Drosophila R-PTPs in developmental processes outside the neuronal system and beyond the embryogenesis stage. Through systematic data mining of available microarray databases, we found the mRNA level of PTP52F to be highly enriched in the midgut of flies at the larva-pupa transition. This finding was confirmed by gut tissue staining with a specific antibody. The unique spatiotemporal expression of PTP52F suggests that it is possibly involved in regulating metamorphosis during the transformation from larva to pupa. To test this hypothesis, we employed RNA interference to examine the defects of transgenic flies. We found that ablation of endogenous PTP52F led to high lethality characterized by the pharate adult phenotype, occurring due to post pupal eclosion failure. These results show that PTP52F plays an indispensable role during the larva-pupa transition. We also found that PTP52F could be reclassified as a member of the subtype R3 PTPs instead of as an unclassified R-PTP without a human ortholog, as suggested previously. Together, these findings suggest that Drosophila R-PTPs may control metamorphosis and other biological processes beyond our current knowledge.

  20. Acute exposure of mercury chloride stimulates the tissue regeneration program and reactive oxygen species production in the Drosophila midgut.

    PubMed

    Chen, Zhi; Wu, Xiaochun; Luo, Hongjie; Zhao, Lingling; Ji, Xin; Qiao, Xianfeng; Jin, Yaping; Liu, Wei

    2016-01-01

    We used Drosophila as an animal model to study the digestive tract in response to the exposure of inorganic mercury (HgCl2). We found that after oral administration, mercury was mainly sequestered within the midgut. This resulted in increased cell death, which in turn stimulated the tissue regeneration program, including accelerated proliferation and differentiation of the intestinal stem cells (ISCs). We further demonstrated that these injuries correlate closely with the excessive production of the reactive oxygen species (ROS), as vitamin E, an antioxidant reagent, efficiently suppressed the HgCl2-induced phenotypes of midgut and improved the viability. We propose that the Drosophila midgut could serve as a suitable model to study the treatment of acute hydrargyrism on the digestive systems.

  1. Ecdysone-Induced Receptor Tyrosine Phosphatase PTP52F Regulates Drosophila Midgut Histolysis by Enhancement of Autophagy and Apoptosis

    PubMed Central

    Santhanam, Abirami; Peng, Wen-Hsin; Yu, Ya-Ting; Sang, Tzu-Kang

    2014-01-01

    The rapid removal of larval midgut is a critical developmental process directed by molting hormone ecdysone during Drosophila metamorphosis. To date, it remains unclear how the stepwise events can link the onset of ecdysone signaling to the destruction of larval midgut. This study investigated whether ecdysone-induced expression of receptor protein tyrosine phosphatase PTP52F regulates this process. The mutation of the Ptp52F gene caused significant delay in larval midgut degradation. Transitional endoplasmic reticulum ATPase (TER94), a regulator of ubiquitin proteasome system, was identified as a substrate and downstream effector of PTP52F in the ecdysone signaling. The inducible expression of PTP52F at the puparium formation stage resulted in dephosphorylation of TER94 on its Y800 residue, ensuring the rapid degradation of ubiquitylated proteins. One of the proteins targeted by dephosphorylated TER94 was found to be Drosophila inhibitor of apoptosis 1 (DIAP1), which was rapidly proteolyzed in cells with significant expression of PTP52F. Importantly, the reduced level of DIAP1 in response to inducible PTP52F was essential not only for the onset of apoptosis but also for the initiation of autophagy. This study demonstrates a novel function of PTP52F in regulating ecdysone-directed metamorphosis via enhancement of autophagic and apoptotic cell death in doomed Drosophila midguts. PMID:24550005

  2. FGF control of E-cadherin targeting in the Drosophila midgut impacts on primordial germ cell motility.

    PubMed

    Parés, Guillem; Ricardo, Sara

    2016-01-15

    Embryo formation requires tight regulation and coordination of adhesion in multiple cell types. By undertaking imaging, three-dimensional (3D) reconstructions and genetic analysis during posterior midgut morphogenesis in Drosophila, we find a new requirement for the conserved fibroblast growth factor (FGF) signaling pathway in the maintenance of epithelial cell adhesion through FGF modulation of zygotic E-cadherin. During Drosophila gastrulation, primordial germ cells (PGCs) are transported with the posterior midgut while it undergoes dynamic cell shape changes. In embryos mutant for the FGF signaling pathway components Branchless and Breathless, zygotic E-cadherin is not targeted to adherens junctions, causing midgut pocket collapse, which impacts on PGC movement. We find that the ventral midline also requires FGF signaling to maintain cell-cell adhesion. We show that FGF signaling regulates the distribution of zygotic E-cadherin during early embryonic development to maintain cell-cell adhesion in the posterior midgut and the ventral midline, a role that is likely crucial in other tissues undergoing active cell shape changes with higher adhesive needs.

  3. Barber Pole Sign in CT Angiography, Adult Presentation of Midgut Malrotation: A Case Report

    PubMed Central

    Garcelan-Trigo, Juan Arsenio; Tello-Moreno, Manuel; Rabaza-Espigares, Manuel Jesus; Talavera-Martinez, Ildefonso

    2015-01-01

    Adult midgut volvulus is a challenging diagnosis because of its low incidence and nonspecific symptoms. Diagnostic delay and long-term complaints are frequent in this clinical scenario. We reported a patient referred to our diagnostic imaging unit with intermittent abdominal pain, bloating and episodic vomiting for several years. He underwent barium gastrointestinal transit and abdominal ultrasound, which revealed severe gastric dilatation, food retention and slow transit until a depressed duodenojejunal flexure, with malrotation of the midgut and jejunal loops being located in the right upper quadrant. Computed tomography angiography was performed, showing rotation of the small intestine around the mesentery root, suggestive of midgut malrotation. In addition, an abnormal twisted disposition of superior mesenteric artery with corkscrew appearance was seen, shaping the pole-barber sign which was evident in volume rendering three-dimensional reconstructions. The patient underwent scheduled surgical treatment without any complication and had good outcome after hospital discharge and follow-up. Computed tomography plays an important role in evaluation of adult midgut volvulus. In addition, angiographic reconstructions can help us to assess the anatomic disposition of mesenteric vascular supply. Both of these assessments are useful in preoperative management. PMID:26557278

  4. Ecdysteroid receptors in Drosophila melanogaster adult females

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melanogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent w...

  5. Forces driven by morphogenesis modulate Twist Expression to determine Anterior Mid-gut Differentiation in Drosophila embryos

    NASA Astrophysics Data System (ADS)

    Farge, Emmanuel

    2008-03-01

    By combining magnetic tweezers to in vivo laser ablation, we locally manipulate Drosophila embryonic tissues with physiologically relevant forces. We demonstrate that high level of Twist expression in the stomodeal primordium is mechanically induced in response to compression by the 60±20 nN force developed during germ-band extension (GBE). We find that this force triggers the junctional release and nuclear translocation of Armadillo involved in Twist mechanical induction in the stomodeum in a Src42A dependent way. Finally, stomodeal-specific RNAi-mediated silencing of Twist during compression impairs the differentiation of midgut cells, as revealed by strong defects in Dve expression and abnormal larval lethality. Thus, mechanical induction of Twist overexpression in stomodeal cells is necessary for subsequent midgut differentiation. In collaboration with Nicolas Desprat, Willy Supatto, and Philippe-Alexandre Pouille, MGDET, UMR168 CNRS, Institut Curie11 rue Pierre et Marie Curie, F-75005, Paris, France; and Emmanuel Beaurepaire, LOB, Ecole Polytechnique, CNRS and INSERM U 696, 91128 Palaiseau, France.

  6. Study on Fungal Flora in the Midgut of the Larva and Adult of the Different Populations of the Malaria Vector Anopheles stephensi

    PubMed Central

    Tajedin, L; Hashemi, J; Abaei, MR; Hosseinpour, L; Rafei, F; Basseri, HR

    2009-01-01

    Background Many microorganisms in midgut of mosquito challenge with their host and also other pathogens present in midgut. The aim of this study was presence of non-pathogens microorganisms like fungal flora which may be crucial on interaction between vectors and pathogens. Methods: Different populations of Anopheles stephensi were reared in insectary and objected to determine fungal flora in their midguts. The midgut paunch of mosquito adults and larvae as well as breading water and larval food samples transferred on Subaru-dextrose agar, in order to detect the environment fungus. Results: Although four fungi, Aspergillus, Rhizopus, Geotrichum and Sacharomyces were found in the food and water, but only Aspiragilus observed in the midgut of larvae. No fungus was found in the midgut of adults. This is the first report on fungal flora in the midgut of the adults and larvae of An. stephensi and possible stadial transmission of fungi from immature stages to adults. Conclusion: The midgut environment of adults is not compatible for survivorship of fungi but the larval midgut may contain few fungi as a host or even pathogen. PMID:22808370

  7. Endocrine remodelling of the adult intestine sustains reproduction in Drosophila

    PubMed Central

    Reiff, Tobias; Jacobson, Jake; Cognigni, Paola; Antonello, Zeus; Ballesta, Esther; Tan, Kah Junn; Yew, Joanne Y; Dominguez, Maria; Miguel-Aliaga, Irene

    2015-01-01

    The production of offspring is energetically costly and relies on incompletely understood mechanisms that generate a positive energy balance. In mothers of many species, changes in key energy-associated internal organs are common yet poorly characterised functionally and mechanistically. In this study, we show that, in adult Drosophila females, the midgut is dramatically remodelled to enhance reproductive output. In contrast to extant models, organ remodelling does not occur in response to increased nutrient intake and/or offspring demands, but rather precedes them. With spatially and temporally directed manipulations, we identify juvenile hormone (JH) as an anticipatory endocrine signal released after mating. Acting through intestinal bHLH-PAS domain proteins Methoprene-tolerant (Met) and Germ cell-expressed (Gce), JH signals directly to intestinal progenitors to yield a larger organ, and adjusts gene expression and sterol regulatory element-binding protein (SREBP) activity in enterocytes to support increased lipid metabolism. Our findings identify a metabolically significant paradigm of adult somatic organ remodelling linking hormonal signals, epithelial plasticity, and reproductive output. DOI: http://dx.doi.org/10.7554/eLife.06930.001 PMID:26216039

  8. Molecular mechanism and functional significance of acid generation in the Drosophila midgut

    PubMed Central

    Overend, Gayle; Luo, Yuan; Henderson, Louise; Douglas, Angela E.; Davies, Shireen A.; Dow, Julian A. T.

    2016-01-01

    The gut of Drosophila melanogaster includes a proximal acidic region (~pH 2), however the genome lacks the H+/K+ ATPase characteristic of the mammalian gastric parietal cell, and the molecular mechanisms of acid generation are poorly understood. Here, we show that maintenance of the low pH of the acidic region is dependent on H+ V-ATPase, together with carbonic anhydrase and five further transporters or channels that mediate K+, Cl− and HCO3− transport. Abrogation of the low pH did not influence larval survival under standard laboratory conditions, but was deleterious for insects subjected to high Na+ or K+ load. Insects with elevated pH in the acidic region displayed increased susceptibility to Pseudomonas pathogens and increased abundance of key members of the gut microbiota (Acetobacter and Lactobacillus), suggesting that the acidic region has bacteriostatic or bacteriocidal activity. Conversely, the pH of the acidic region was significantly reduced in germ-free Drosophila, indicative of a role of the gut bacteria in shaping the pH conditions of the gut. These results demonstrate that the acidic gut region protects the insect and gut microbiome from pathological disruption, and shed light on the mechanisms by which low pH can be maintained in the absence of H+, K+ ATPase. PMID:27250760

  9. Assessing the genotoxic effects of two lipid peroxidation products (4-oxo-2-nonenal and 4-hydroxy-hexenal) in haemocytes and midgut cells of Drosophila melanogaster larvae.

    PubMed

    Demir, Eşref; Marcos, Ricard

    2017-03-22

    Lipid peroxidation products can induce tissue damage and are implicated in diverse pathological conditions, including aging, atherosclerosis, brain disorders, cancer, lung and various liver disorders. Since in vivo studies produce relevant information, we have selected Drosophila melanogaster as a suitable in vivo model to characterise the potential risks associated to two lipid peroxidation products namely 4-oxo-2-nonenal (4-ONE) and 4-hydroxy-hexenal (4-HHE). Toxicity, intracellular reactive oxygen species production, and genotoxicity were the end-points evaluated. Haemocytes and midgut cells were the evaluated targets. Results showed that both compounds penetrate the intestine of the larvae, affecting midgut cells, and reaching haemocytes. Significant genotoxic effects, as determined by the comet assay, were observed in both selected cell targets in a concentration/time dependent manner. This study highlights the importance of D. melanogaster as a model organism in the study of the different biological effects caused by lipid peroxidation products entering via ingestion. This is the first study reporting genotoxicity data in haemocytes and midgut cells of D. melanogaster larvae for the two selected compounds.

  10. Mesenchymal to epithelial transition during tissue homeostasis and regeneration: Patching up the Drosophila midgut epithelium.

    PubMed

    Antonello, Zeus A; Reiff, Tobias; Dominguez, Maria

    2015-01-01

    Stem cells are responsible for preserving morphology and function of adult tissues. Stem cells divide to self-renew and to generate progenitor cells to sustain cell demand from the tissue throughout the organism's life. Unlike stem cells, the progenitor cells have limited proliferation potential but have the capacity to terminally differentiate and thereby to substitute older or damaged mature cells. Recent findings indicate that adult stem cells can adapt their division kinetics dynamically to match changes in tissue demand during homeostasis and regeneration. However, cell turnover not only requires stem cell division but also needs timed differentiation of the progenitor cells, which has been much less explored. In this Extra View article, we discuss the ability of progenitor cells to actively postpone terminal differentiation in the absence of a local demand and how tissue demand activates terminal differentiation via a conserved mesenchymal-epithelial transition program revealed in our recent EMBO J paper and other published and unpublished data. The extent of the significance of these results is discussed for models of tissue dynamics during both homeostasis and regeneration.

  11. Identification of bacterial microflora in the midgut of the larvae and adult of wild caught Anopheles stephensi: a step toward finding suitable paratransgenesis candidates.

    PubMed

    Chavshin, Ali Reza; Oshaghi, Mohammad Ali; Vatandoost, Hasan; Pourmand, Mohammad Reza; Raeisi, Ahmad; Enayati, Ahmad Ali; Mardani, Nadia; Ghoorchian, Sadigheh

    2012-02-01

    To describe the midgut microbial diversity and to find the candidate bacteria for the genetic manipulation for the generation of paratransgenic Anopheline mosquitoes refractory to transmission of malaria, the microbiota of wild larvae and adult Anopheles stephensi mosquito midgut from southern Iran was studied using a conventional cell-free culture technique and analysis of a 16S ribosomal RNA (rRNA) gene sequence library. Forty species in 12 genera including seven Gram-negative Myroides, Chryseobacterium, Aeromonas, Pseudomonas, Klebsiella, Enterobacter and Shewanella and five Gram-positive Exiguobacterium, Enterococcus, Kocuria, Microbacterium and Rhodococcus bacteria were identified in the microbiota of the larvae midgut. Analysis of the adult midgut microbiota revealed presence of 25 Gram-negative species in five genera including Pseudomonas, Alcaligenes, Bordetella, Myroides and Aeromonas. Pseudomonas and Exiguobacterium with a frequency of 51% and 14% at the larval stage and Pseudomonas and Aeromonas with a frequency of 54% and 20% at the adult stage were the most common midgut symbionts. Pseudomonas, Aeromonas and Myroides genera have been isolated from both larvae and adult stages indicating possible trans-stadial transmission from larva to adult stage. Fast growth in cheap media, Gram negative, and being dominantly found in both larvae and adult stages, and presence in other malaria vectors makes Pseudomonas as a proper candidate for paratransgenesis of An. stephensi and other malaria vectors.

  12. An unexpected cause of small bowel obstruction in an adult patient: midgut volvulus

    PubMed Central

    Söker, Gökhan; Yılmaz, Cengiz; Karateke, Faruk; Gülek, Bozkurt

    2014-01-01

    The most important complication of intestinal malrotation is midgut volvulus because it may lead to intestinal ischaemia and necrosis. A 29-year-old male patient was admitted to the emergency department with abdominal pain. Ultrasonography (US), colour Doppler ultrasonography (CDUS), CT and barium studies were carried out. On US and CDUS, twisting of intestinal segments around the superior mesenteric artery (SMA) and superior mesenteric vein (SMV) and alteration of the SMA–SMV relationship were detected. CT demonstrated that the small intestine was making a rotation around the SMA and SMV, which amounted to more than 360°. The upper gastrointestinal barium series revealed a corkscrew appearance of the duodenum and proximal jejunum, which is a pathognomonic finding of midgut volvulus. Prior knowledge of characteristic imaging findings of midgut volvulus is essential in order to reach proper diagnosis and establish proper treatment before the development of intestinal ischaemia and necrosis. PMID:24811563

  13. Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila.

    PubMed

    Ma, Meifang; Zhao, Hang; Zhao, Hanfei; Binari, Richard; Perrimon, Norbert; Li, Zhouhua

    2016-03-15

    Adult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. We find that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of caspase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis.

  14. Drosophila adult and larval pheromones modulate larval food choice

    PubMed Central

    Farine, Jean-Pierre; Cortot, Jérôme; Ferveur, Jean-François

    2014-01-01

    Insects use chemosensory cues to feed and mate. In Drosophila, the effect of pheromones has been extensively investigated in adults, but rarely in larvae. The colonization of natural food sources by Drosophila buzzatii and Drosophila simulans species may depend on species-specific chemical cues left in the food by larvae and adults. We identified such chemicals in both species and measured their influence on larval food preference and puparation behaviour. We also tested compounds that varied between these species: (i) two larval volatile compounds: hydroxy-3-butanone-2 and phenol (predominant in D. simulans and D. buzzatii, respectively), and (ii) adult cuticular hydrocarbons (CHs). Drosophila buzzatii larvae were rapidly attracted to non-CH adult conspecific cues, whereas D. simulans larvae were strongly repulsed by CHs of the two species and also by phenol. Larval cues from both species generally reduced larval attraction and pupariation on food, which was generally—but not always—low, and rarely reflected larval response. As these larval and adult pheromones specifically influence larval food search and the choice of a pupariation site, they may greatly affect the dispersion and survival of Drosophila species in nature. PMID:24741012

  15. MiRNA profiling provides insights on adverse effects of Cr(VI) in the midgut tissues of Drosophila melanogaster.

    PubMed

    Chandra, Swati; Pandey, Ashutosh; Chowdhuri, Debapratim Kar

    2015-01-01

    Cr(VI), a well-known environmental chemical, is reported to cause various adverse effects on exposed organisms including genomic instability and carcinogenesis. Despite available information on the underlying mechanism of Cr(VI) induced toxicity, studies regarding toxicity modulation by epigenetic mechanisms are limited. It was therefore, hypothesized that the global miRNA profiling in Cr(VI) exposed Drosophila, a genetically tractable model organism, will provide information about mis-regulated miRNAs along with their targeted genes and relevant processes. Third instar larvae of Drosophila melanogaster (Oregon R(+)) were exposed to 5.0-20.0 μg/ml of Cr(VI) for 24 and 48 h. Following miRNA profile analysis on an Agilent platform, 28 of the 36 differentially expressed miRNAs were found to be significantly mis-regulated targeting major biological processes viz., DNA damage repair, oxidation-reduction processes, development and differentiation. Down-regulation of mus309 and mus312 under DNA repair, acon to oxidation-reduction and pyd to stress activated MAPK cascade respectively belonging to these gene ontology classes concurrent with up-regulation of dme-miR-314-3p, dme-miR-79-3p and dme-miR-12-5p confirm their functional involvement against Cr(VI) exposure. These findings assume significance since majority of the target genes in Drosophila have functional homologues in humans. The study further recommends Drosophila as a model to explore the role of miRNAs in xenobiotic induced toxicity.

  16. Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway.

    PubMed

    Na, Hyun-Jin; Park, Joung-Sun; Pyo, Jung-Hoon; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2015-07-01

    We delineated the mechanism regulating the inhibition of centrosome amplification by metformin in Drosophila intestinal stem cells (ISCs). Age-related changes in tissue-resident stem cells may be closely associated with tissue aging and age-related diseases, such as cancer. Centrosome amplification is a hallmark of cancers. Our recent work showed that Drosophila ISCs are an excellent model for stem cell studies evaluating age-related increase in centrosome amplification. Here, we showed that metformin, a recognized anti-cancer drug, inhibits age- and oxidative stress-induced centrosome amplification in ISCs. Furthermore, we revealed that this effect is mediated via down-regulation of AKT/target of rapamycin (TOR) activity, suggesting that metformin prevents centrosome amplification by inhibiting the TOR signaling pathway. Additionally, AKT/TOR signaling hyperactivation and metformin treatment indicated a strong correlation between DNA damage accumulation and centrosome amplification in ISCs, suggesting that DNA damage might mediate centrosome amplification. Our study reveals the beneficial and protective effects of metformin on centrosome amplification via AKT/TOR signaling modulation. We identified a new target for the inhibition of age- and oxidative stress-induced centrosome amplification. We propose that the Drosophila ISCs may be an excellent model system for in vivo studies evaluating the effects of anti-cancer drugs on tissue-resident stem cell aging.

  17. From Embryo to Adult: Hematopoiesis along the Drosophila Life Cycle.

    PubMed

    Ramond, Elodie; Meister, Marie; Lemaitre, Bruno

    2015-05-26

    Studies on Drosophila hematopoiesis have thus far focused on the embryonic and larval origin of hemocytes, the fly blood cells. In this issue of Developmental Cell, Ghosh et al. (2015) identify adult hematopoietic hubs containing progenitors that can differentiate into different blood cell types.

  18. Dietary glucose regulates yeast consumption in adult Drosophila males

    PubMed Central

    Lebreton, Sébastien; Witzgall, Peter; Olsson, Marie; Becher, Paul G.

    2014-01-01

    The adjustment of feeding behavior in response to hunger and satiety contributes to homeostatic regulation in animals. The fruit fly Drosophila melanogaster feeds on yeasts growing on overripe fruit, providing nutrients required for adult survival, reproduction and larval growth. Here, we present data on how the nutritional value of food affects subsequent yeast consumption in Drosophila adult males. After a period of starvation, flies showed intensive yeast consumption. In comparison, flies stopped feeding after having access to a nutritive cornmeal diet. Interestingly, dietary glucose was equally efficient as the complex cornmeal diet. In contrast, flies fed with sucralose, a non-metabolizable sweetener, behaved as if they were starved. The adipokinetic hormone and insulin-like peptides regulate metabolic processes in insects. We did not find any effect of the adipokinetic hormone pathway on this modulation. Instead, the insulin pathway was involved in these changes. Flies lacking the insulin receptor (InR) did not respond to nutrient deprivation by increasing yeast consumption. Together these results show the importance of insulin in the regulation of yeast consumption in response to starvation in adult D. melanogaster males. PMID:25566097

  19. Genotypic structure of a Drosophila population for adult locomotor activity

    SciTech Connect

    Grechanyi, G.V.; Korzun, V.M.

    1995-01-01

    Analysis of the variation of adult locomotor activity in four samples taken at different times from a natural population of Drosophila melanogaster showed that the total variation of this trait is relatively stable in time and has a substantial genetic component. Genotypic structure of the population for locomotor activity is characterized by the presence of large groups of genotypes with high and low values of this trait. A possible explanation for the presence of such groups in a population is cyclic density-dependent selection.

  20. Locomotion Induced by Spatial Restriction in Adult Drosophila

    PubMed Central

    Xiao, Chengfeng; Robertson, R. Meldrum

    2015-01-01

    Drosophila adults display an unwillingness to enter confined spaces but the behaviors induced by spatial restriction in Drosophila are largely unknown. We developed a protocol for high-throughput analysis of locomotion and characterized features of locomotion in a restricted space. We observed intense and persistent locomotion of flies in small circular arenas (diameter 1.27 cm), whereas locomotion was greatly reduced in large circular arenas (diameter 3.81 cm). The increased locomotion induced by spatial restriction was seen in male flies but not female flies, indicating sexual dimorphism of the response to spatial restriction. In large arenas, male flies increased locomotion in arenas previously occupied by male but not female individuals. In small arenas, such pre-conditioning had no effect on male flies, which showed intense and persistent locomotion similar to that seen in fresh arenas. During locomotion with spatial restriction, wildtype Canton-S males traveled slower and with less variation in speed than the mutant w1118 carrying a null allele of white gene. In addition, wildtype flies showed a stronger preference for the boundary than the mutant in small arenas. Genetic analysis with a series of crosses revealed that the white gene was not associated with the phenotype of boundary preference in wildtype flies. PMID:26351842

  1. Impact of floral feeding on adult Drosophila suzukii survival and nutrient status

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Drosophila suzukii, spotted wing drosophila, is a serious pest of small fruits and cherries in many regions of the world. While host usage has been well studied at the ovipositional and larval feeding stages, little is known about the feeding ecology of adults. This study addressed the impact of fee...

  2. Isolation of Undifferentiated Female Germline Cells from Adult Drosophila Ovaries.

    PubMed

    Lim, Robyn Su May; Osato, Motomi; Kai, Toshie

    2015-08-03

    This unit describes a method for isolating undifferentiated, stem cell-like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand-dissected ovaries in considerably large quantities. Tumor ovaries with expanded populations of undifferentiated germline cells are first removed from fly abdomens and dissociated into a cell suspension with the aid of protease treatment. The target cells, which express Vasa-green fluorescent protein (GFP) fusion protein under the control of the germline-specific vasa promoter, are specifically selected from the suspension via fluorescence-activated cell sorting (FACS). These protocols can be adapted to isolate other cell types from fly ovaries, such as somatic follicle cells or escort cells, by driving GFP expression in the respective target cells.

  3. The Drosophila Prosecretory Transcription Factor dimmed Is Dynamically Regulated in Adult Enteroendocrine Cells and Protects Against Gram-Negative Infection.

    PubMed

    Beebe, Katherine; Park, Dongkook; Taghert, Paul H; Micchelli, Craig A

    2015-05-20

    The endocrine system employs peptide hormone signals to translate environmental changes into physiological responses. The diffuse endocrine system embedded in the gastrointestinal barrier epithelium is one of the largest and most diverse endocrine tissues. Furthermore, it is the only endocrine tissue in direct physical contact with the microbial environment of the gut lumen. However, it remains unclear how this sensory epithelium responds to specific pathogenic challenges in a dynamic and regulated manner. We demonstrate that the enteroendocrine cells of the adult Drosophila melanogaster midgut display a transient, sensitive, and systemic induction of the prosecretory factor dimmed (dimm) in response to the Gram-negative pathogen Pseudomonas entomophila (Pe). In enteroendocrine cells, dimm controls the levels of the targets Phm, dcat-4, and the peptide hormone, Allatostatin A. Finally, we identify dimm as a host factor that protects against Pe infection and controls the expression of antimicrobial peptides. We propose that dimm provides "gain" in enteroendocrine output during the adaptive response to episodic pathogen exposure.

  4. Visualization of adult stem cells within their niches using the Drosophila germline as a model system.

    PubMed

    König, Annekatrin; Shcherbata, Halyna R

    2013-01-01

    The germaria of the fruit fly Drosophila melanogaster present an excellent model to study germline stem cell-niche interactions. Two to three adult stem cells are surrounded by a number of somatic cells that form the niche. Here we describe how Drosophilae germaria can be dissected and specifically immuno-stained to allow for identification and analysis of both the adult stem cells and their somatic niche cells.

  5. Associations of yeasts with spotted-wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in cherries and raspberries.

    PubMed

    Hamby, Kelly A; Hernández, Alejandro; Boundy-Mills, Kyria; Zalom, Frank G

    2012-07-01

    A rich history of investigation documents various Drosophila-yeast mutualisms, suggesting that Drosophila suzukii similarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts of D. suzukii. Terminal restriction fragment length polymorphism (TRFLP) technology and decimal dilution plating were used to identify and determine the relative abundance of yeast species present in fruit juice samples that were either infested with D. suzukii or not infested. Yeasts were less abundant in uninfested than infested samples. A total of 126 independent yeast isolates were cultivated from frass, midguts, and fruit hosts of D. suzukii, representing 28 species of yeasts, with Hanseniaspora uvarum predominating. This suggests an association between D. suzukii and H. uvarum that could be utilized for pest management of the highly pestiferous D. suzukii.

  6. Associations of Yeasts with Spotted-Wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in Cherries and Raspberries

    PubMed Central

    Hernández, Alejandro; Zalom, Frank G.

    2012-01-01

    A rich history of investigation documents various Drosophila-yeast mutualisms, suggesting that Drosophila suzukii similarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts of D. suzukii. Terminal restriction fragment length polymorphism (TRFLP) technology and decimal dilution plating were used to identify and determine the relative abundance of yeast species present in fruit juice samples that were either infested with D. suzukii or not infested. Yeasts were less abundant in uninfested than infested samples. A total of 126 independent yeast isolates were cultivated from frass, midguts, and fruit hosts of D. suzukii, representing 28 species of yeasts, with Hanseniaspora uvarum predominating. This suggests an association between D. suzukii and H. uvarum that could be utilized for pest management of the highly pestiferous D. suzukii. PMID:22582060

  7. Morphology of the midgut of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) adult ticks in different feeding stages.

    PubMed

    Remedio, R N; Sampieri, B R; Vendramini, M C R; Souza, N M; Anholeto, L A; Denardo, T A G B; Camargo-Mathias, M I

    2013-01-01

    The intestinal epithelial cells of ticks are fundamental for their full feeding and reproductive success, besides being considered important sites for the development of pathogens. Rhipicephalus sanguineus ticks are known for their great medical and veterinary importance, and for this reason, the knowledge of their intestinal morphology may provide relevant subsidies for the control of these animals, either by direct acaricidal action over these cells or by the production of vaccines. Therefore, this study aimed to describe the midgut morphology of male and female R. sanguineus ticks in different feeding stages, by means of histological analysis. Significant differences were observed between the genders, and such alterations may refer mainly to the distinct demands for nutrients, much higher in females, which need to develop and carry out the egg-laying process. In general, the midgut is coated by a thin muscle layer and presents a pseudostratified epithelium, in which two basic types of cells can be observed, connected to a basal membrane-generative or stem and digestive cells. The latter was classified as follows: residual, deriving from the phase anterior to ecdysis; pinocytic, with vesicles containing liquid or pre-digested components of blood; phagocytic, with entire cells or remnants of nuclear material inside cytoplasmic vesicles; and mature, free in the lumen. Digestion is presumably intracellular and asynchronous and corresponds to a process which starts with the differentiation of generative cells into pinocytic digestive cells, which subsequently start to phagocytize intact blood cells and finally detach from the epithelium, being eliminated with feces.

  8. The Glycolytic Enzymes Activity in the Midgut of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) adult and their Seasonal Changes

    PubMed Central

    Guzik, Joanna; Nakonieczny, Mirosław; Tarnawska, Monika; Bereś, Paweł K.; Drzewiecki, Sławomir; Migula, Paweł

    2015-01-01

    The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is an important pest of maize. The diet of the D. virgifera imago is rich in starch and other polysaccharides present in cereals such as maize. Therefore, knowledge about enzymes involved in digestion of such specific food of this pest seems to be important. The paper shows, for the first time, the activities of main glycolytic enzymes in the midgut of D. virgifera imago: endoglycosidases (α-amylase, cellulase, chitinase, licheninase, laminarinase); exoglycosidases (α- and β-glucosidases, α- and β-galactosidases) and disaccharidases (maltase, isomaltase, sucrase, trehalase, lactase, and cellobiase). Activities of α-amylase, α-glucosidase, and maltase were the highest among assayed endoglycosidases, exoglycosidases, and disaccharidases, respectively. This indicates that in the midgut of D. virgifera imago α-amylase, α-glucosidase and maltase are important enzymes in starch hydrolysis and products of its digestion. These results lead to conclusion that inhibition of most active glycolytic enzymes of D. virgifera imago may be another promising method for chemical control of this pest of maize.

  9. The glia of the adult Drosophila nervous system

    PubMed Central

    Kremer, Malte C.; Jung, Christophe; Batelli, Sara; Rubin, Gerald M.

    2017-01-01

    Glia play crucial roles in the development and homeostasis of the nervous system. While the GLIA in the Drosophila embryo have been well characterized, their study in the adult nervous system has been limited. Here, we present a detailed description of the glia in the adult nervous system, based on the analysis of some 500 glial drivers we identified within a collection of synthetic GAL4 lines. We find that glia make up ∼10% of the cells in the nervous system and envelop all compartments of neurons (soma, dendrites, axons) as well as the nervous system as a whole. Our morphological analysis suggests a set of simple rules governing the morphogenesis of glia and their interactions with other cells. All glial subtypes minimize contact with their glial neighbors but maximize their contact with neurons and adapt their macromorphology and micromorphology to the neuronal entities they envelop. Finally, glial cells show no obvious spatial organization or registration with neuronal entities. Our detailed description of all glial subtypes and their regional specializations, together with the powerful genetic toolkit we provide, will facilitate the functional analysis of glia in the mature nervous system. GLIA 2017 GLIA 2017;65:606–638 PMID:28133822

  10. Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector

    PubMed Central

    2009-01-01

    Background Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms. Results Lab-reared and field-collected A. stephensi male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught A. stephensi mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval A. stephensi, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared A. stephensi midgut, respectively. These OTUs had a Shannon diversity index value of 1.74–2.14 for lab-reared and in the range of 2.75–3.49 for field-caught A. stephensi mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant. Conclusion More than fifty percent of

  11. Octopamine mediates starvation-induced hyperactivity in adult Drosophila.

    PubMed

    Yang, Zhe; Yu, Yue; Zhang, Vivian; Tian, Yinjun; Qi, Wei; Wang, Liming

    2015-04-21

    Starved animals often exhibit elevated locomotion, which has been speculated to partly resemble foraging behavior and facilitate food acquisition and energy intake. Despite its importance, the neural mechanism underlying this behavior remains unknown in any species. In this study we confirmed and extended previous findings that starvation induced locomotor activity in adult fruit flies Drosophila melanogaster. We also showed that starvation-induced hyperactivity was directed toward the localization and acquisition of food sources, because it could be suppressed upon the detection of food cues via both central nutrient-sensing and peripheral sweet-sensing mechanisms, via induction of food ingestion. We further found that octopamine, the insect counterpart of vertebrate norepinephrine, as well as the neurons expressing octopamine, were both necessary and sufficient for starvation-induced hyperactivity. Octopamine was not required for starvation-induced changes in feeding behaviors, suggesting independent regulations of energy intake behaviors upon starvation. Taken together, our results establish a quantitative behavioral paradigm to investigate the regulation of energy homeostasis by the CNS and identify a conserved neural substrate that links organismal metabolic state to a specific behavioral output.

  12. Lactobacillus plantarum favors the early emergence of fit and fertile adult Drosophila upon chronic undernutrition

    PubMed Central

    Téfit, Mélisandre A.

    2017-01-01

    ABSTRACT Animals are naturally surrounded by a variety of microorganisms with which they constantly interact. Among these microbes, some live in close association with a host and form its microbiota. These communities are being extensively studied, owing to their contributions to shaping various aspects of animal physiology. One of these commensal species, Lactobacillus plantarum, and in particular the L.p.WJL strain, has been shown to promote the growth of Drosophila larvae upon nutrient scarcity, allowing earlier metamorphosis and adult emergence compared with axenic individuals. As for many insects, conditions surrounding the post-embryonic development dictate key adult life history traits in Drosophila, and adjusting developmental timing according to the environment is essential for adult fitness. Thus, we wondered whether the growth acceleration induced by L.p.WJL in a context of poor nutrition could adversely impact the fitness of Drosophila adults. Here, we show that the L.p.WJL-mediated acceleration of growth is not deleterious; adults emerging after an accelerated development are as fit as their axenic siblings. Additionally, the presence of L.p.WJL even leads to a lifespan extension in nutritionally challenged males. These results demonstrate that L.p.WJL is a beneficial partner for Drosophila melanogaster through its entire life cycle. Thus, commensal bacteria allow the earlier emergence and longer survival of fit and fertile individuals and might represent one of the factors contributing to the ecological success of Drosophila. PMID:28062579

  13. Laparoscopic Ileocolic Resection for Crohn's Disease Associated With Midgut Malrotation

    PubMed Central

    Biancone, Livia; Tema, Giorgia; Porokhnavets, Kristina; Tesauro, Manfredi; Gaspari, Achille L.; Sica, Giuseppe S.

    2014-01-01

    Midgut malrotation is an anomaly of fetal intestinal rotation. Its incidence in adults is rare. A case of midgut malrotation in a 51-year-old man with complicated Crohn's disease of the terminal ileum is presented. Symptoms, diagnosis, and treatment are reviewed. Preoperative workup led to correct surgical planning that ultimately allowed a successful laparoscopic resection. PMID:25419109

  14. Antibody staining of the central nervous system in adult Drosophila.

    PubMed

    Sweeney, Sean T; Hidalgo, Alicia; de Belle, J Steven; Keshishian, Haig

    2012-02-01

    The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at specific times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology. Antibody-labeling techniques are particularly useful for examining the Drosophila brain. These methods generally use primary antibodies specific to a protein or a structure of interest and a fluorescently labeled or enzyme-coupled secondary antibody to detect the primary antibodies. Immunofluorescence methods allow for simultaneous probing for multiple antigens using different fluorophores, as well as high-resolution confocal examination of deep structures. This protocol describes general procedures for antibody labeling of neural tissue from Drosophila, as well as visualization techniques for fluorescent and enzyme-linked probes.

  15. Raf-mediated cardiac hypertrophy in adult Drosophila.

    PubMed

    Yu, Lin; Daniels, Joseph; Glaser, Alex E; Wolf, Matthew J

    2013-07-01

    In response to stress and extracellular signals, the heart undergoes a process called cardiac hypertrophy during which cardiomyocytes increase in size. If untreated, cardiac hypertrophy can progress to overt heart failure that causes significant morbidity and mortality. The identification of molecular signals that cause or modify cardiomyopathies is necessary to understand how the normal heart progresses to cardiac hypertrophy and heart failure. Receptor tyrosine kinase (RTK) signaling is essential for normal human cardiac function, and the inhibition of RTKs can cause dilated cardiomyopathies. However, neither investigations of activated RTK signaling pathways nor the characterization of hypertrophic cardiomyopathy in the adult fly heart has been previously described. Therefore, we developed strategies using Drosophila as a model to circumvent some of the complexities associated with mammalian models of cardiovascular disease. Transgenes encoding activated EGFR(A887T), Ras85D(V12) and Ras85D(V12S35), which preferentially signal to Raf, or constitutively active human or fly Raf caused hypertrophic cardiomyopathy as determined by decreased end diastolic lumen dimensions, abnormal cardiomyocyte fiber morphology and increased heart wall thicknesses. There were no changes in cardiomyocyte cell numbers. Additionally, activated Raf also induced an increase in cardiomyocyte ploidy compared with control hearts. However, preventing increases in cardiomyocyte ploidy using fizzy-related (Fzr) RNAi did not rescue Raf-mediated cardiac hypertrophy, suggesting that Raf-mediated polyploidization is not required for cardiac hypertrophy. Similar to mammals, the cardiac-specific expression of RNAi directed against MEK or ERK rescued Raf-mediated cardiac hypertrophy. However, the cardiac-specific expression of activated ERK(D334N), which promotes hyperplasia in non-cardiac tissues, did not cause myocyte hypertrophy. These results suggest that ERK is necessary, but not sufficient, for

  16. The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium.

    PubMed

    Franzetti, Eleonora; Romanelli, Davide; Caccia, Silvia; Cappellozza, Silvia; Congiu, Terenzio; Rajagopalan, Muthukumaran; Grimaldi, Annalisa; de Eguileor, Magda; Casartelli, Morena; Tettamanti, Gianluca

    2015-08-01

    The midgut represents the middle part of the alimentary canal and is responsible for nutrient digestion and absorption in insect larva. Despite the growing interest in this organ for different purposes, such as studies on morphogenesis and differentiation, stem cell biology, cell death processes and transport mechanisms, basic information on midgut development is still lacking for a large proportion of insect species. Undoubtedly, this lack of data could hinder the full exploitation of practical applications that involve midgut as their primary target. This may represent in particular a significant problem for Lepidoptera, an insect order that includes some of the most important species of high economic importance. With the aim of overcoming this fragmentation of knowledge, we performed a detailed morphofunctional analysis of the midgut of the silkworm, Bombyx mori, a representative model among Lepidoptera, during its development from the larval up to the adult stage, focusing attention on stem cells. Our data demonstrate stem cell proliferation and differentiation, not only in the larval midgut but also in the pupal and adult midgut epithelium. Moreover, we present evidence for a complex trophic relationship between the dying larval epithelium and the new adult one, which is established during metamorphosis. This study, besides representing the first morphological and functional characterization of the changes that occur in the midgut of a lepidopteron during the transition from the larva to the moth, provides a detailed analysis of the midgut of the adult insect, a stage that has been neglected up to now.

  17. An integrated hybrid microfluidic device for oviposition-based chemical screening of adult Drosophila melanogaster.

    PubMed

    Leung, Jacob C K; Hilliker, Arthur J; Rezai, Pouya

    2016-02-21

    Chemical screening using Drosophila melanogaster (the fruit fly) is vital in drug discovery, agricultural, and toxicological applications. Oviposition (egg laying) on chemically-doped agar plates is an important read-out metric used to quantitatively assess the biological fitness and behavioral responses of Drosophila. Current oviposition-based chemical screening studies are inaccurate, labor-intensive, time-consuming, and inflexible due to the manual chemical doping of agar. In this paper, we have developed a novel hybrid agar-polydimethylsiloxane (PDMS) microfluidic device for single- and multi-concentration chemical dosing and on-chip oviposition screening of free-flying adult stage Drosophila. To achieve this, we have devised a novel technique to integrate agar with PDMS channels using ice as a sacrificial layer. Subsequently, we have conducted single-chemical toxicity and multiple choice chemical preference assays on adult Drosophila melanogaster using zinc and acetic acid at various concentrations. Our device has enabled us to 1) demonstrate that Drosophila is capable of sensing the concentration of different chemicals on a PDMS-agar microfluidic device, which plays significant roles in determining oviposition site selection and 2) investigate whether oviposition preference differs between single- and multi-concentration chemical environments. This device may be used to study fundamental and applied biological questions in Drosophila and other egg laying insects. It can also be extended in design to develop sophisticated and dynamic chemical dosing and high-throughput screening platforms in the future that are not easily achievable with the existing oviposition screening techniques.

  18. Starvation-Induced Dietary Behaviour in Drosophila melanogaster Larvae and Adults

    PubMed Central

    Ahmad, Muhammad; Chaudhary, Safee Ullah; Afzal, Ahmed Jawaad; Tariq, Muhammad

    2015-01-01

    Drosophila melanogaster larvae are classified as herbivores and known to feed on non-carnivorous diet under normal conditions. However, when nutritionally challenged these larvae exhibit cannibalistic behaviour by consuming a diet composed of larger conspecifics. Herein, we report that cannibalism in Drosophila larvae is confined not only to scavenging on conspecifics that are larger in size, but also on their eggs. Moreover, such cannibalistic larvae develop as normally as those grown on standard cornmeal medium. When stressed, Drosophila melanogaster larvae can also consume a carnivorous diet derived from carcasses of organisms belonging to diverse taxonomic groups, including Musca domestica, Apis mellifera, and Lycosidae sp. While adults are ill-equipped to devour conspecific carcasses, they selectively oviposit on them and also consume damaged cadavers of conspecifics. Thus, our results suggest that nutritionally stressed Drosophila show distinct as well as unusual feeding behaviours that can be classified as detritivorous, cannibalistic and/or carnivorous. PMID:26399327

  19. Drosophila dyskerin is required for somatic stem cell homeostasis.

    PubMed

    Vicidomini, Rosario; Petrizzo, Arianna; di Giovanni, Annamaria; Cassese, Laura; Lombardi, Antonella Anna; Pragliola, Caterina; Furia, Maria

    2017-03-23

    Drosophila represents an excellent model to dissect the roles played by the evolutionary conserved family of eukaryotic dyskerins. These multifunctional proteins are involved in the formation of H/ACA snoRNP and telomerase complexes, both involved in essential cellular tasks. Since fly telomere integrity is guaranteed by a different mechanism, we used this organism to investigate the specific role played by dyskerin in somatic stem cell maintenance. To this aim, we focussed on Drosophila midgut, a hierarchically organized and well characterized model for stemness analysis. Surprisingly, the ubiquitous loss of the protein uniquely affects the formation of the larval stem cell niches, without altering other midgut cell types. The number of adult midgut precursor stem cells is dramatically reduced, and this effect is not caused by premature differentiation and is cell-autonomous. Moreover, a few dispersed precursors found in the depleted midguts can maintain stem identity and the ability to divide asymmetrically, nor show cell-growth defects or undergo apoptosis. Instead, their loss is mainly specifically dependent on defective amplification. These studies establish a strict link between dyskerin and somatic stem cell maintenance in a telomerase-lacking organism, indicating that loss of stemness can be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction.

  20. big bang gene modulates gut immune tolerance in Drosophila.

    PubMed

    Bonnay, François; Cohen-Berros, Eva; Hoffmann, Martine; Kim, Sabrina Y; Boulianne, Gabrielle L; Hoffmann, Jules A; Matt, Nicolas; Reichhart, Jean-Marc

    2013-02-19

    Chronic inflammation of the intestine is detrimental to mammals. Similarly, constant activation of the immune response in the gut by the endogenous flora is suspected to be harmful to Drosophila. Therefore, the innate immune response in the gut of Drosophila melanogaster is tightly balanced to simultaneously prevent infections by pathogenic microorganisms and tolerate the endogenous flora. Here we describe the role of the big bang (bbg) gene, encoding multiple membrane-associated PDZ (PSD-95, Discs-large, ZO-1) domain-containing protein isoforms, in the modulation of the gut immune response. We show that in the adult Drosophila midgut, BBG is present at the level of the septate junctions, on the apical side of the enterocytes. In the absence of BBG, these junctions become loose, enabling the intestinal flora to trigger a constitutive activation of the anterior midgut immune response. This chronic epithelial inflammation leads to a reduced lifespan of bbg mutant flies. Clearing the commensal flora by antibiotics prevents the abnormal activation of the gut immune response and restores a normal lifespan. We now provide genetic evidence that Drosophila septate junctions are part of the gut immune barrier, a function that is evolutionarily conserved in mammals. Collectively, our data suggest that septate junctions are required to maintain the subtle balance between immune tolerance and immune response in the Drosophila gut, which represents a powerful model to study inflammatory bowel diseases.

  1. Wolbachia-mediated antiviral protection in Drosophila larvae and adults following oral infection.

    PubMed

    Stevanovic, Aleksej L; Arnold, Pieter A; Johnson, Karyn N

    2015-12-01

    Understanding viral dynamics in arthropods is of great importance when designing models to describe how viral spread can influence arthropod populations. The endosymbiotic bacterium Wolbachia spp., which is present in up to 40% of all insect species, has the ability to alter viral dynamics in both Drosophila spp. and mosquitoes, a feature that in mosquitoes may be utilized to limit spread of important arboviruses. To understand the potential effect of Wolbachia on viral dynamics in nature, it is important to consider the impact of natural routes of virus infection on Wolbachia antiviral effects. Using adult Drosophila strains, we show here that Drosophila-Wolbachia associations that have previously been shown to confer antiviral protection following systemic viral infection also confer protection against virus-induced mortality following oral exposure to Drosophila C virus in adults. Interestingly, a different pattern was observed when the same fly lines were challenged with the virus when still larvae. Analysis of the four Drosophila-Wolbachia associations that were protective in adults indicated that only the w1118-wMelPop association conferred protection in larvae following oral delivery of the virus. Analysis of Wolbachia density using quantitative PCR (qPCR) showed that a high Wolbachia density was congruent with antiviral protection in both adults and larvae. This study indicates that Wolbachia-mediated protection may vary between larval and adult stages of a given Wolbachia-host combination and that the variations in susceptibility by life stage correspond with Wolbachia density. The differences in the outcome of virus infection are likely to influence viral dynamics in Wolbachia-infected insect populations in nature and could also have important implications for the transmission of arboviruses in mosquito populations.

  2. Wolbachia-Mediated Antiviral Protection in Drosophila Larvae and Adults following Oral Infection

    PubMed Central

    Stevanovic, Aleksej L.; Arnold, Pieter A.

    2015-01-01

    Understanding viral dynamics in arthropods is of great importance when designing models to describe how viral spread can influence arthropod populations. The endosymbiotic bacterium Wolbachia spp., which is present in up to 40% of all insect species, has the ability to alter viral dynamics in both Drosophila spp. and mosquitoes, a feature that in mosquitoes may be utilized to limit spread of important arboviruses. To understand the potential effect of Wolbachia on viral dynamics in nature, it is important to consider the impact of natural routes of virus infection on Wolbachia antiviral effects. Using adult Drosophila strains, we show here that Drosophila-Wolbachia associations that have previously been shown to confer antiviral protection following systemic viral infection also confer protection against virus-induced mortality following oral exposure to Drosophila C virus in adults. Interestingly, a different pattern was observed when the same fly lines were challenged with the virus when still larvae. Analysis of the four Drosophila-Wolbachia associations that were protective in adults indicated that only the w1118-wMelPop association conferred protection in larvae following oral delivery of the virus. Analysis of Wolbachia density using quantitative PCR (qPCR) showed that a high Wolbachia density was congruent with antiviral protection in both adults and larvae. This study indicates that Wolbachia-mediated protection may vary between larval and adult stages of a given Wolbachia-host combination and that the variations in susceptibility by life stage correspond with Wolbachia density. The differences in the outcome of virus infection are likely to influence viral dynamics in Wolbachia-infected insect populations in nature and could also have important implications for the transmission of arboviruses in mosquito populations. PMID:26407882

  3. Clonal development and organization of the adult Drosophila central brain

    PubMed Central

    Yu, Hung-Hsiang; Awasaki, Takeshi; Schroeder, Mark David; Long, Fuhui; Yang, Jacob S.; He, Yisheng; Ding, Peng; Kao, Jui-Chun; Wu, Gloria Yueh-Yi; Peng, Hanchuan; Myers, Gene; Lee, Tzumin

    2013-01-01

    Summary Background The insect brain can be divided into neuropils that are formed by neurites of both local and remote origin. The complexity of the interconnections obscures how these neuropils are established and interconnected through development. The Drosophila central brain develops from a fixed number of neuroblasts (NBs) that deposit neurons in regional clusters. Results By determining individual NB clones and pursuing their projections into specific neuropils we unravel the regional development of the brain neural network. Exhaustive clonal analysis revealed 95 stereotyped neuronal lineages with characteristic cell body locations and neurite trajectories. Most clones show complex projection patterns, but despite the complexity, neighboring clones often co-innervate the same local neuropil(s) and further target a restricted set of distant neuropils. Conclusions These observations argue for regional clonal development of both neuropils and neuropil connectivity throughout the Drosophila central brain. PMID:23541733

  4. Elements of olfactory reception in adult Drosophila melanogaster.

    PubMed

    Martin, Fernando; Boto, Tamara; Gomez-Diaz, Carolina; Alcorta, Esther

    2013-09-01

    The olfactory system of Drosophila has become an attractive and simple model to investigate olfaction because it follows the same organizational principles of vertebrates, and the results can be directly applied to other insects with economic and sanitary relevance. Here, we review the structural elements of the Drosophila olfactory reception organs at the level of the cells and molecules involved. This article is intended to reflect the structural basis underlying the functional variability of the detection of an olfactory universe composed of thousands of odors. At the genetic level, we further detail the genes and transcription factors (TF) that determine the structural variability. The fly's olfactory receptor organs are the third antennal segments and the maxillary palps, which are covered with sensory hairs called sensilla. These sensilla house the odorant receptor neurons (ORNs) that express one or few odorant receptors in a stereotyped pattern regulated by combinations of TF. Also, perireceptor events, such as odor molecules transport to their receptors, are carried out by odorant binding proteins. In addition, the rapid odorant inactivation to preclude saturation of the system occurs by biotransformation and detoxification enzymes. These additional events take place in the lymph that surrounds the ORNs. We include some data on ionotropic and metabotropic olfactory transduction, although this issue is still under debate in Drosophila.

  5. Design and implementation of in vivo imaging of neural injury responses in the adult Drosophila wing.

    PubMed

    Fang, Yanshan; Soares, Lorena; Bonini, Nancy M

    2013-04-01

    Live-imaging technology has markedly advanced in the field of neural injury and axon degeneration; however, studies are still predominantly performed in in vitro settings such as cultured neuronal cells or in model organisms such as Caenorhabditis elegans in which axons lack glial wrappings. We recently developed a new in vivo model for adult-stage neural injury in Drosophila melanogaster, using the highly accessible wing of the animal. Because the Drosophila wing is translucent and dispensable for survival, it allows clear and direct visualization of injury-induced progressive responses of axons and glia highlighted by fluorescent protein (FP) markers in live animals over time. Moreover, unlike previous Drosophila models of neural injury, this procedure does not require dissection of the CNS. Thus, the key preparation steps for in vivo imaging of the neural injury response described in this protocol can be completed within 30 min.

  6. The glial investment of the adult and developing antennal lobe of Drosophila

    PubMed Central

    Oland, Lynne A.; Biebelhausen, John P.; Tolbert, Leslie P.

    2009-01-01

    In recent years, the Drosophila olfactory system, with its unparalleled opportunities for genetic dissection of development and functional organization, has been used to study the development of central olfactory neurons and the molecular basis of olfactory coding. The results of these studies have been interpreted in the absence of a detailed understanding of the steps in maturation of glial cells in the antennal lobe. Here, we present a high-resolution study of the glia associated with olfactory glomeruli in adult and developing antennal lobes. The study provides a basis for comparison of findings in Drosophila with those in the moth Manduca sexta that indicate a critical role for glia in antennal lobe development. Using flies expressing GFP under a Nervana2 driver to visualize glia for confocal microscopy, and probing at higher resolution with the electron microscope, we find that glial development in Drosophila differs markedly from that in moths: glial cell bodies remain in a rind around the glomerular neuropil; glial processes ensheathe axon bundles in the nerve layer but likely contribute little to axonal sorting; their processes insinuate between glomeruli only very late and then form only a sparse, open network around each glomerulus; and glial processes invade the synaptic neuropil. Taking our results in the context of previous studies, we conclude that glial cells in the developing Drosophila antennal lobe are unlikely to play a strong role in either axonal sorting or glomerulus stabilization and that in the adult, glial processes do not electrically isolate glomeruli from their neighbors. PMID:18537134

  7. An enhancer trap screen for ecdysone-inducible genes required for Drosophila adult leg morphogenesis.

    PubMed Central

    Gates, J; Thummel, C S

    2000-01-01

    Although extensive studies of Drosophila imaginal disc development have focused on proliferation and patterning, relatively little is known about how the patterned imaginal discs are transformed into adult structures during metamorphosis. Studies focused primarily on leg development have shown that this remarkable transformation is coordinated by pulses of the steroid hormone ecdysone and requires the function of ecdysone-inducible transcription factors as well as proteases and components of the contractile cytoskeleton and adherens junctions. Here, we describe a genetic screen aimed at expanding our understanding of the hormonal regulation of Drosophila adult leg morphogenesis. We screened 1300 lethal P-element enhancer trap insertions on the second chromosome for a series of sequential parameters including pupal lethality, defects in leg morphogenesis, and ecdysone-induced lacZ reporter gene expression. From this screen we identified four mutations, one of which corresponds to bancal, which encodes the Drosophila homolog of hnRNP K. We also identified vulcan, which encodes a protein that shares sequence similarity with a family of rat SAPAP proteins. Both bancal and vulcan are inducible by ecdysone, thus linking the hormone signal with leg morphogenesis. This screen provides new directions for understanding the hormonal regulation of leg development during Drosophila metamorphosis. PMID:11102372

  8. Active Hematopoietic Hubs in Drosophila Adults Generate Hemocytes and Contribute to Immune Response

    PubMed Central

    Ghosh, Saikat; Singh, Arashdeep; Mandal, Sudip; Mandal, Lolitika

    2015-01-01

    Summary Blood cell development in Drosophila shares significant similarities with vertebrate. The conservation ranges from biphasic mode of hematopoiesis to signaling molecules crucial for progenitor cell formation, maintenance, and differentiation. Primitive hematopoiesis in Drosophila ensues in embryonic head mesoderm, whereas definitive hematopoiesis happens in larval hematopoietic organ, the lymph gland. This organ, with the onset of pupation, ruptures to release hemocytes into circulation. It is believed that the adult lacks a hematopoietic organ and survives on the contribution of both embryonic and larval hematopoiesis. However, our studies revealed a surge of blood cell development in the dorsal abdominal hemocyte clusters of adult fly. These active hematopoietic hubs are capable of blood cell specification and can respond to bacterial challenges. The presence of progenitors and differentiated hemocytes embedded in a functional network of Laminin A and Pericardin within this hematopoietic hub projects it as a simple version of the vertebrate bone marrow. PMID:25959225

  9. Optogenetic Manipulation of Selective Neural Activity in Free-Moving Drosophila Adults.

    PubMed

    Hsiao, Po-Yen; Wu, Ming-Chin; Lin, Yen-Yin; Fu, Chein-Chung; Chiang, Ann-Shyn

    2016-01-01

    Activating selected neurons elicits specific behaviors in Drosophila adults. By combining optogenetics and laser-tracking techniques, we have recently developed an automated laser-tracking and optogenetic manipulation system (ALTOMS) for studying how brain circuits orchestrate complex behaviors. The established ALTOMS can independently target three lasers (473-nm blue laser, 593.5-nm yellow laser, and 1064-nm infrared laser) on any specified body part of two freely moving flies. Triggering light-sensitive proteins in real time, the blue laser and yellow laser can respectively activate and inhibit target neurons in artificial transgenic flies. Since infrared light is invisible to flies, we use the 1064-nm laser as an aversive stimulus in operant learning without perturbing visual inputs. Herein, we provide a detailed protocol for the construction of ALTOMS and optogenetic manipulation of target neurons in Drosophila adults during social interactions.

  10. Affecting Rhomboid-3 Function Causes a Dilated Heart in Adult Drosophila

    PubMed Central

    Yu, Lin; Lee, Teresa; Lin, Na; Wolf, Matthew J.

    2010-01-01

    Drosophila is a well recognized model of several human diseases, and recent investigations have demonstrated that Drosophila can be used as a model of human heart failure. Previously, we described that optical coherence tomography (OCT) can be used to rapidly examine the cardiac function in adult, awake flies. This technique provides images that are similar to echocardiography in humans, and therefore we postulated that this approach could be combined with the vast resources that are available in the fly community to identify new mutants that have abnormal heart function, a hallmark of certain cardiovascular diseases. Using OCT to examine the cardiac function in adult Drosophila from a set of molecularly-defined genomic deficiencies from the DrosDel and Exelixis collections, we identified an abnormally enlarged cardiac chamber in a series of deficiency mutants spanning the rhomboid 3 locus. Rhomboid 3 is a member of a highly conserved family of intramembrane serine proteases and processes Spitz, an epidermal growth factor (EGF)–like ligand. Using multiple approaches based on the examination of deficiency stocks, a series of mutants in the rhomboid-Spitz–EGF receptor pathway, and cardiac-specific transgenic rescue or dominant-negative repression of EGFR, we demonstrate that rhomboid 3 mediated activation of the EGF receptor pathway is necessary for proper adult cardiac function. The importance of EGF receptor signaling in the adult Drosophila heart underscores the concept that evolutionarily conserved signaling mechanisms are required to maintain normal myocardial function. Interestingly, prior work showing the inhibition of ErbB2, a member of the EGF receptor family, in transgenic knock-out mice or individuals that received herceptin chemotherapy is associated with the development of dilated cardiomyopathy. Our results, in conjunction with the demonstration that altered ErbB2 signaling underlies certain forms of mammalian cardiomyopathy, suggest that an

  11. [Adult of Drosophila melanogaster parasitized in human nasal cavity: a case report].

    PubMed

    Zhan, Xiaodong; Tang, Xiaoniu; Wang, Shaosheng

    2015-05-01

    We reported a case of adult Drosophila melanogaster parasitized in nasal cavity of a 81-year-old woman who was living in Xuancheng City, Anhui Province now. She was admitted for treatment of cerebral infarction and water accumulation in the lungs in 2014 June. The patient was also suffering from secretory otitis media, a history of hypertension and heart stents were placed in 2007. A foreign body was found in the left nasal cavity during the preoperative examination process, and then the part of the inflammatory tissue was removed through the nasal endoscopy, and sent to our department for identification. There are three adults of Drosophila in paraffin-embedded biopsy specimens. The parasites length is approximately 3mm, with huge red compound eyes. The end of the body is tip, with 5 ring lines in back, has no dark spots. The abdomen of the parasites have seven sections. Tarsus of foot I have no sex comb on base, and they are male adult of Drosophila melanogaster after identification. After a thorough reviewing of medical history, we knew the patient began to sneeze violently and frequently six years ago. But there was no clear or purulent nasal discharge flowing, therefore did not attract attention. After removing the parasites the sneezing symptoms were relieved, and had no abnormal symptoms in the follow-up 6 months.

  12. Oral magnetite nanoparticles disturb the development of Drosophila melanogaster from oogenesis to adult emergence.

    PubMed

    Chen, Hanqing; Wang, Bing; Feng, Weiyue; Du, Wei; Ouyang, Hong; Chai, Zhifang; Bi, Xiaolin

    2015-05-01

    The potential impacts of nanomaterials (NMs) on fetal development have attracted great concerns because of the increased potential exposure to NMs during pregnancy. Drosophila melanogaster oogenesis and developmental transitions may provide an attractive system to study the biological and environmental effects of NMs on the embryonic development. In this study, the effects of three types of magnetite (Fe3O4) nanoparticles (MNPs): UN-MNPs (pristine), CA-MNPs (citric acid modified) and APTS-MNPs (3-aminopropyltriethoxylsilane coated) on the development of Drosophila at 300 and 600 μg/g dosage were studied. The uptake of MNPs by female and male flies caused obvious reduction in the female fecundity, and the developmental delay at the egg-pupae and pupae-adult transitions, especially in those treated by the positive APTS-MNPs. Further investigation demonstrates that the parental uptake of MNPs disturbs the oogenesis period, induces ovarian defect, reduces the length of eggs, decreases the number of nurse cells and delays egg chamber development, which may contribute to the decrease of fecundity of female Drosophila and the development delay of their offspring. Using the synchrotron radiation-based micro-X-ray fluorescence (SR-μXRF), the dyshomeostasis of trace elements such as Fe, Ca and Cu along the anterior-posterior axis of the fertilized eggs was found, which may be an important reason for the development delay of Drosophila.

  13. Comparison of larval and adult Drosophila astrocytes reveals stage-specific gene expression profiles.

    PubMed

    Huang, Yanmei; Ng, Fanny S; Jackson, F Rob

    2015-02-04

    The analysis of adult astrocyte glial cells has revealed a remarkable heterogeneity with regard to morphology, molecular signature, and physiology. A key question in glial biology is how such heterogeneity arises during brain development. One approach to this question is to identify genes with differential astrocyte expression during development; certain genes expressed later in neural development may contribute to astrocyte differentiation. We have utilized the Drosophila model and Translating Ribosome Affinity Purification (TRAP)-RNA-seq methods to derive the genome-wide expression profile of Drosophila larval astrocyte-like cells (hereafter referred to as astrocytes) for the first time. These studies identified hundreds of larval astrocyte-enriched genes that encode proteins important for metabolism, energy production, and protein synthesis, consistent with the known role of astrocytes in the metabolic support of neurons. Comparison of the larval profile with that observed for adults has identified genes with astrocyte-enriched expression specific to adulthood. These include genes important for metabolism and energy production, translation, chromatin modification, protein glycosylation, neuropeptide signaling, immune responses, vesicle-mediated trafficking or secretion, and the regulation of behavior. Among these functional classes, the expression of genes important for chromatin modification and vesicle-mediated trafficking or secretion is overrepresented in adult astrocytes based on Gene Ontology analysis. Certain genes with selective adult enrichment may mediate functions specific to this stage or may be important for the differentiation or maintenance of adult astrocytes, with the latter perhaps contributing to population heterogeneity.

  14. A Subset of Serotonergic Neurons Evokes Hunger in Adult Drosophila.

    PubMed

    Albin, Stephanie D; Kaun, Karla R; Knapp, Jon-Michael; Chung, Phuong; Heberlein, Ulrike; Simpson, Julie H

    2015-09-21

    Hunger is a complex motivational state that drives multiple behaviors. The sensation of hunger is caused by an imbalance between energy intake and expenditure. One immediate response to hunger is increased food consumption. Hunger also modulates behaviors related to food seeking such as increased locomotion and enhanced sensory sensitivity in both insects and vertebrates. In addition, hunger can promote the expression of food-associated memory. Although progress is being made, how hunger is represented in the brain and how it coordinates these behavioral responses is not fully understood in any system. Here, we use Drosophila melanogaster to identify neurons encoding hunger. We found a small group of neurons that, when activated, induced a fed fly to eat as though it were starved, suggesting that these neurons are downstream of the metabolic regulation of hunger. Artificially activating these neurons also promotes appetitive memory performance in sated flies, indicating that these neurons are not simply feeding command neurons but likely play a more general role in encoding hunger. We determined that the neurons relevant for the feeding effect are serotonergic and project broadly within the brain, suggesting a possible mechanism for how various responses to hunger are coordinated. These findings extend our understanding of the neural circuitry that drives feeding and enable future exploration of how state influences neural activity within this circuit.

  15. Avoidance of heat and attraction to optogenetically induced sugar sensation as operant behavior in adult Drosophila.

    PubMed

    Nuwal, Nidhi; Stock, Patrick; Hiemeyer, Jochen; Schmid, Benjamin; Fiala, André; Buchner, Erich

    2012-09-01

    Animals have to perform adequate behavioral actions dependent on internal states and environmental situations, and adjust their behavior according to positive or negative consequences. The fruit fly Drosophila melanogaster represents a key model organism for the investigation of neuronal mechanisms underlying adaptive behavior. The authors are using a behavioral paradigm in which fruit flies attached to a manipulator can walk on a Styrofoam ball whose movements are recorded such that intended left or right turns of the flies can be registered and used to operantly control heat stimuli or optogenetic activation of distinct subsets of neurons. As proof of principle, the authors find that flies in this situation avoid heat stimuli but prefer optogenetic self-stimulation of sugar receptors. Using this setup it now should be possible to study the neuronal network underlying positive and negative value assessment of adult Drosophila in an operant setting.

  16. Muscle niche-driven Insulin-Notch-Myc cascade reactivates dormant Adult Muscle Precursors in Drosophila.

    PubMed

    Aradhya, Rajaguru; Zmojdzian, Monika; Da Ponte, Jean Philippe; Jagla, Krzysztof

    2015-12-09

    How stem cells specified during development keep their non-differentiated quiescent state, and how they are reactivated, remain poorly understood. Here, we applied a Drosophila model to follow in vivo behavior of adult muscle precursors (AMPs), the transient fruit fly muscle stem cells. We report that emerging AMPs send out thin filopodia that make contact with neighboring muscles. AMPs keep their filopodia-based association with muscles throughout their dormant state but also when they start to proliferate, suggesting that muscles could play a role in AMP reactivation. Indeed, our genetic analyses indicate that muscles send inductive dIlp6 signals that switch the Insulin pathway ON in closely associated AMPs. This leads to the activation of Notch, which regulates AMP proliferation via dMyc. Altogether, we report that Drosophila AMPs display homing behavior to muscle niche and that the niche-driven Insulin-Notch-dMyc cascade plays a key role in setting the activated state of AMPs.

  17. The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling.

    PubMed

    Takashima, Shigeo; Mkrtchyan, Marianna; Younossi-Hartenstein, Amelia; Merriam, John R; Hartenstein, Volker

    2008-07-31

    The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.

  18. 20-Hydroxyecdysone stimulates the accumulation of translatable yolk polypeptide gene transcript in adult male Drosophila melanogaster.

    PubMed

    Shirk, P D; Minoo, P; Postlethwait, J H

    1983-01-01

    Yolk polypeptide (YP) synthesis is hormonally stimulated during maturation of adult female Drosophila melanogaster. Synthesis of the three YPs is sex specific and occurs in fat body cells and follicle cells of adult females. However, males have been shown to produce YPs when treated with the steroid hormone 20-hydroxyecdysone (20-HE). By using a cell-free translation system as an assay for YP mRNA, we found that 20-HE also causes the accumulation of translatable YP message in males. In addition, hybridization of cloned copies of genes for both YP1 and YP3 to total RNA from males showed that 20-HE caused the appearance of YP gene transcripts in males. Eight hours after treatment of males with 20-HE, YP gene transcript levels had increased at least 25-fold to approximately 2.7 x 10(6) copies of YP1 gene transcript per adult male fly. In normal adult females, there were 42 x 10(6) copies per fly by 24 hr. There was neither detectable YP synthesis nor translatable YP gene transcript in either normal 1- to 3-day-old males or 24-hr-old males treated with a juvenile hormone analogue. This evidence shows that 20-HE acts to regulate the levels of translatable YP mRNA in male Drosophila.

  19. Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila.

    PubMed

    Andrews, Hillary K; Giagtzoglou, Nikolaos; Yamamoto, Shinya; Schulze, Karen L; Bellen, Hugo J

    2009-06-01

    The adult Drosophila external sensory organ (ESO), comprising the hair, socket, neuron, sheath and glia cells, arises through the asymmetric division of sensory organ precursor cells (SOPs). In a mosaic screen designed to identify new components in ESO development, we isolated mutations in sequoia, which encodes a putative zinc-finger transcription factor that has previously been shown to have a role in dendritogenesis. Here, we show that adult clones mutant for seq exhibit a loss of hair cells and a gain of socket cells. We propose that the seq mutant phenotype arises, in part, owing to the loss of several crucial transcription factors known to be important in peripheral nervous system development such as D-Pax2, Prospero and Hamlet. Thus, Sequoia is a new upstream regulator of genes that orchestrates cell fate specification during development of the adult ESO lineage.

  20. In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining.

    PubMed

    Chen, Zhe; Xu, Hong

    2016-10-15

    The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2´-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues. Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

  1. In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

    PubMed Central

    Chen, Zhe; Xu, Hong

    2016-01-01

    The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2´-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues. Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations. PMID:27805603

  2. Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila

    PubMed Central

    Yu, Yue; Huang, Rui; Ye, Jie; Zhang, Vivian; Wu, Chao; Cheng, Guo; Jia, Junling; Wang, Liming

    2016-01-01

    Starvation induces sustained increase in locomotion, which facilitates food localization and acquisition and hence composes an important aspect of food-seeking behavior. We investigated how nutritional states modulated starvation-induced hyperactivity in adult Drosophila. The receptor of the adipokinetic hormone (AKHR), the insect analog of glucagon, was required for starvation-induced hyperactivity. AKHR was expressed in a small group of octopaminergic neurons in the brain. Silencing AKHR+ neurons and blocking octopamine signaling in these neurons eliminated starvation-induced hyperactivity, whereas activation of these neurons accelerated the onset of hyperactivity upon starvation. Neither AKHR nor AKHR+ neurons were involved in increased food consumption upon starvation, suggesting that starvation-induced hyperactivity and food consumption are independently regulated. Single cell analysis of AKHR+ neurons identified the co-expression of Drosophila insulin-like receptor (dInR), which imposed suppressive effect on starvation-induced hyperactivity. Therefore, insulin and glucagon signaling exert opposite effects on starvation-induced hyperactivity via a common neural target in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.15693.001 PMID:27612383

  3. Thermal nociception in adult Drosophila: behavioral characterization and the role of the painless gene.

    PubMed

    Xu, S Y; Cang, C L; Liu, X F; Peng, Y Q; Ye, Y Z; Zhao, Z Q; Guo, A K

    2006-11-01

    Nociception, warning of injury that should be avoided, serves an important protective function in animals. In this study, we show that adult Drosophila avoids noxious heat by a jump response. To quantitatively analyze this nociceptive behavior, we developed two assays. In the CO2 laser beam assay, flies exhibit this behavior when a laser beam heats their abdomens. The consistency of the jump latency in this assay meets an important criterion for a good nociceptive assay. In the hot plate assay, flies jump quickly to escape from a hot copper plate (>45 degrees C). Our results demonstrate that, as in mammals, the latency of the jump response is inversely related to stimulus intensity, and innoxious thermosensation does not elicit this nociceptive behavior. To explore the genetic mechanisms of nociception, we examined several mutants in both assays. Abnormal nociceptive behavior of a mutant, painless, indicates that painless, a gene essential for nociception in Drosophila larvae, is also required for thermal nociception in adult flies. painless is expressed in certain neurons of the peripheral nervous system and thoracic ganglia, as well as in the definite brain structures, the mushroom bodies. However, chemical or genetic insults to the mushroom bodies do not influence the nociceptive behavior, suggesting that different painless-expressing neurons play diverse roles in thermal nociception. Additionally, no-bridge(KS49), a mutant that has a structural defect in the protocerebral bridge, shows defective response to noxious heat. Thus, our results validate adult Drosophila as a useful model to study the genetic mechanisms of thermal nociception.

  4. The functional organisation of glia in the adult brain of Drosophila and other insects

    PubMed Central

    Edwards, Tara N.; Meinertzhagen, Ian A.

    2010-01-01

    This review annotates and categorises the glia of adult Drosophila and other model insects and describes the developmental origins of these in the Drosophila optic lobe. The functions of glia in the adult vary depending upon their sub-type and location in the brain. The task of annotating glia is essentially complete only for the glia of the fly's lamina, which comprise: two types of surface glia - the pseudocartridge and fenestrated glia; two types of cortex glia - the distal and proximal satellite glia; and two types of neuropile glia - the epithelial and marginal glia. We advocate that the term subretinal glia, as used to refer to both pseudocartridge and fenestrated glia, be abandoned. Other neuropiles contain similar glial subtypes, but other than the antennal lobes these have not been described in detail. Surface glia form the blood brain barrier, regulating the flow of substances into and out of the nervous system, both for the brain as a whole and the optic neuropiles in particular. Cortex glia provide a second level of barrier, wrapping axon fascicles and isolating neuronal cell bodies both from neighbouring brain regions and from their underlying neuropiles. Neuropile glia can be generated in the adult and a subtype, ensheathing glia, are responsible for cleaning up cellular debris during Wallerian degeneration. Both the neuropile ensheathing and astrocyte-like glia may be involved in clearing neurotransmitters from the extracellular space, thus modifying the levels of histamine, glutamate and possibly dopamine at the synapse to ultimately affect behaviour. PMID:20109517

  5. Midgut morphological changes and autophagy during metamorphosis in sand flies.

    PubMed

    Malta, Juliana; Heerman, Matthew; Weng, Ju Lin; Fernandes, Kenner M; Martins, Gustavo Ferreira; Ramalho-Ortigão, Marcelo

    2017-03-11

    During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector.

  6. Small-molecule screen in adult Drosophila identifies VMAT as a regulator of sleep.

    PubMed

    Nall, Aleksandra H; Sehgal, Amita

    2013-05-08

    Sleep is an important physiological state, but its function and regulation remain elusive. In Drosophila melanogaster, a useful model organism for studying sleep, forward genetic screens have identified important sleep-modulating genes and pathways; however, the results of such screens may be limited by developmental abnormalities or lethality associated with mutation of certain genes. To circumvent these limitations, we used a small-molecule screen to identify sleep-modulating genes and pathways. We administered 1280 pharmacologically active small molecules to adult flies and monitored their sleep. We found that administration of reserpine, a small-molecule inhibitor of the vesicular monoamine transporter (VMAT) that repackages monoamines into presynaptic vesicles, resulted in an increase in sleep. Supporting the idea that VMAT is the sleep-relevant target of reserpine, we found that VMAT-null mutants have an increased sleep phenotype, as well as an increased arousal threshold and resistance to the effects of reserpine. However, although the VMAT mutants are consistently resistant to reserpine, other aspects of their sleep phenotype are dependent on genetic background. These findings indicate that small-molecule screens can be used effectively to identify sleep-modulating genes whose phenotypes may be suppressed in traditional genetic screens. Mutations affecting single monoamine pathways did not affect reserpine sensitivity, suggesting that effects of VMAT/reserpine on sleep are mediated by multiple monoamines. Overall, we identify VMAT as an important regulator of sleep in Drosophila and demonstrate that small-molecule screens provide an effective approach to identify genes and pathways that impact adult Drosophila behavior.

  7. The influence of Adh function on ethanol preference and tolerance in adult Drosophila melanogaster.

    PubMed

    Ogueta, Maite; Cibik, Osman; Eltrop, Rouven; Schneider, Andrea; Scholz, Henrike

    2010-11-01

    Preference determines behavioral choices such as choosing among food sources and mates. One preference-affecting chemical is ethanol, which guides insects to fermenting fruits or leaves. Here, we show that adult Drosophila melanogaster prefer food containing up to 5% ethanol over food without ethanol and avoid food with high levels (23%) of ethanol. Although female and male flies behaved differently at ethanol-containing food sources, there was no sexual dimorphism in the preference for food containing modest ethanol levels. We also investigated whether Drosophila preference, sensitivity and tolerance to ethanol was related to the activity of alcohol dehydrogenase (Adh), the primary ethanol-metabolizing enzyme in D. melanogaster. Impaired Adh function reduced ethanol preference in both D. melanogaster and a related species, D. sechellia. Adh-impaired flies also displayed reduced aversion to high ethanol concentrations, increased sensitivity to the effects of ethanol on postural control, and negative tolerance/sensitization (i.e., a reduction of the increased resistance to ethanol's effects that normally occurs upon repeated exposure). These data strongly indicate a linkage between ethanol-induced behavior and ethanol metabolism in adult fruit flies: Adh deficiency resulted in reduced preference to low ethanol concentrations and reduced aversion to high ones, despite recovery from ethanol being strongly impaired.

  8. Utility of the CT Scan in Diagnosing Midgut Volvulus in Patients with Chronic Abdominal Pain

    PubMed Central

    Morshedi, Mehdi; Baradaran Jamili, Mohammad; Shafizadeh Barmi, Fatemeh

    2017-01-01

    Symptomatic intestinal malrotation first presenting in the adults is rare. Midgut volvulus is the most common complication of malrotation in the adults. Because of more differential diagnosis, Computed Tomography (CT) scan can play an important role in the evaluation of patients with this abnormality. The whirl pattern around the superior mesenteric artery found on CT scan in patients with midgut volvulus is pathognomonic and diagnostic. We describe a case of intestinal malrotation complicated by midgut volvulus in an adult patient. The preoperative CT findings were pathognomonic. PMID:28182093

  9. Appendiceal mucocoele with midgut malrotation

    PubMed Central

    Hassall, J; Williams, GL; McKain, ES

    2016-01-01

    Introduction Malrotation of the midgut and appendiceal mucocoele are both extremely rare pathological conditions in adults. To our knowledge, there are only two reported cases in the English literature with a combination of both conditions. Case History A 65-year-old man presented with a 10-day history of upper abdominal pain associated with abdominal bloating and weight loss. He was otherwise fit and healthy with no significant past medical history. On examination, his abdomen was soft with tenderness and palpable fullness over the left upper quadrant. The initial blood test, chest x-ray and abdominal x-ray demonstrated no significant abnormality. Computed tomography showed a 17cm x 8cm x 6cm elongated cystic mass with possible malrotation of the intestines. Histopathology showed a low grade mucinous tumour of the appendix. At 12 months following surgery, there was no evidence of recurrence or postoperative complications and the patient was discharged from the care of the colorectal team. Conclusions We report a patient with a combination of two rare conditions. This case illustrates how a combination of pathologies can present a challenge to the unwary general surgeon. PMID:27269433

  10. Evolution of increased adult longevity in Drosophila melanogaster populations selected for adaptation to larval crowding.

    PubMed

    Shenoi, V N; Ali, S Z; Prasad, N G

    2016-02-01

    In holometabolous animals such as Drosophila melanogaster, larval crowding can affect a wide range of larval and adult traits. Adults emerging from high larval density cultures have smaller body size and increased mean life span compared to flies emerging from low larval density cultures. Therefore, adaptation to larval crowding could potentially affect adult longevity as a correlated response. We addressed this issue by studying a set of large, outbred populations of D. melanogaster, experimentally evolved for adaptation to larval crowding for 83 generations. We assayed longevity of adult flies from both selected (MCUs) and control populations (MBs) after growing them at different larval densities. We found that MCUs have evolved increased mean longevity compared to MBs at all larval densities. The interaction between selection regime and larval density was not significant, indicating that the density dependence of mean longevity had not evolved in the MCU populations. The increase in longevity in MCUs can be partially attributed to their lower rates of ageing. It is also noteworthy that reaction norm of dry body weight, a trait probably under direct selection in our populations, has indeed evolved in MCU populations. To the best of our knowledge, this is the first report of the evolution of adult longevity as a correlated response of adaptation to larval crowding.

  11. The involvement of several enzymes in methanol detoxification in Drosophila melanogaster adults.

    PubMed

    Wang, Shu-Ping; Hu, Xing-Xing; Meng, Qing-Wei; Muhammad, Shahid Arain; Chen, Rui-Rui; Li, Fei; Li, Guo-Qing

    2013-09-01

    Methanol is among the most common short-chain alcohols in fermenting fruits, the natural food and oviposition sites of the fruit fly Drosophila melanogaster. Our previous results showed that cytochrome P450 monooxygenases (CYPs) were associated with methanol detoxification in the larvae. Catalases, alcohol dehydrogenases (ADHs), esterases (ESTs) and glutathione S-transferases (GSTs) were specifically inhibited by 3-amino-1,2,4-triazole (3-AT), 4-methylpyrazole (4-MP), triphenyl phosphate (TPP) and diethylmeleate (DEM), respectively. CYPs were inhibited by piperonyl butoxide (PBO) and 1-aminobenzotriazole (1-ABT). In the present paper, the involvements of these enzymes in methanol metabolism were investigated in female and male adults by determining the combination indices of methanol and their corresponding inhibitors. When PBO, 1-ABT, 3-AT, 4-MP and TPP were individually mixed with methanol, they exhibited significant synergism to the mortality of the adults after 72h of dietary exposure. In contrast, the DEM and methanol mixture showed additive effects. Moreover, methanol exposure dramatically increased CYP activity and up-regulated mRNA expression levels of several Cyp genes. Bioassays using different strains revealed that the variation in ADH activity and RNAi-mediated knockdown of α-Est7 significantly changed LC50 values for methanol. These results suggest that CYPs, catalases, ADHs and ESTs are partially responsible for methanol elimination in adults. It seems that there are some differences in methanol metabolism between larvae and adults, but not between female and male adults.

  12. From Embryo to Adult: piRNA-Mediated Silencing throughout Germline Development in Drosophila

    PubMed Central

    Marie, Pauline P.; Ronsseray, Stéphane; Boivin, Antoine

    2016-01-01

    In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development has not been addressed. Here, we show that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, we detect rhino-dependent piRNAs indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the “ping-pong” amplification step. Moreover, we show that Heterochromatin Protein 1a is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal, and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development. PMID:27932388

  13. Integration of complex larval chemosensory organs into the adult nervous system of Drosophila.

    PubMed

    Gendre, Nanaë; Lüer, Karin; Friche, Sandrine; Grillenzoni, Nicola; Ramaekers, Ariane; Technau, Gerhard M; Stocker, Reinhard F

    2004-01-01

    The sense organs of adult Drosophila, and holometabolous insects in general, derive essentially from imaginal discs and hence are adult specific. Experimental evidence presented here, however, suggests a different developmental design for the three largely gustatory sense organs located along the pharynx. In a comprehensive cellular analysis, we show that the posteriormost of the three organs derives directly from a similar larval organ and that the two other organs arise by splitting of a second larval organ. Interestingly, these two larval organs persist despite extensive reorganization of the pharynx. Thus, most of the neurons of the three adult organs are surviving larval neurons. However, the anterior organ includes some sensilla that are generated during pupal stages. Also, we observe apoptosis in a third larval pharyngeal organ. Hence, our experimental data show for the first time the integration of complex, fully differentiated larval sense organs into the nervous system of the adult fly and demonstrate the embryonic origin of their neurons. Moreover, they identify metamorphosis of this sensory system as a complex process involving neuronal persistence, generation of additional neurons and neuronal death. Our conclusions are based on combined analysis of reporter expression from P[GAL4] driver lines, horseradish peroxidase injections into blastoderm stage embryos, cell labeling via heat-shock-induced flip-out in the embryo, bromodeoxyuridine birth dating and staining for programmed cell death. They challenge the general view that sense organs are replaced during metamorphosis.

  14. Functional genomics identifies regulators of the phototransduction machinery in the Drosophila larval eye and adult ocelli.

    PubMed

    Mishra, Abhishek Kumar; Bargmann, Bastiaan O R; Tsachaki, Maria; Fritsch, Cornelia; Sprecher, Simon G

    2016-02-15

    Sensory perception of light is mediated by specialized Photoreceptor neurons (PRs) in the eye. During development all PRs are genetically determined to express a specific Rhodopsin (Rh) gene and genes mediating a functional phototransduction pathway. While the genetic and molecular mechanisms of PR development is well described in the adult compound eye, it remains unclear how the expression of Rhodopsins and the phototransduction cascade is regulated in other visual organs in Drosophila, such as the larval eye and adult ocelli. Using transcriptome analysis of larval PR-subtypes and ocellar PRs we identify and study new regulators required during PR differentiation or necessary for the expression of specific signaling molecules of the functional phototransduction pathway. We found that the transcription factor Krüppel (Kr) is enriched in the larval eye and controls PR differentiation by promoting Rh5 and Rh6 expression. We also identified Camta, Lola, Dve and Hazy as key genes acting during ocellar PR differentiation. Further we show that these transcriptional regulators control gene expression of the phototransduction cascade in both larval eye and adult ocelli. Our results show that PR cell type-specific transcriptome profiling is a powerful tool to identify key transcriptional regulators involved during several aspects of PR development and differentiation. Our findings greatly contribute to the understanding of how combinatorial action of key transcriptional regulators control PR development and the regulation of a functional phototransduction pathway in both larval eye and adult ocelli.

  15. From Embryo to Adult: piRNA-Mediated Silencing throughout Germline Development in Drosophila.

    PubMed

    Marie, Pauline P; Ronsseray, Stéphane; Boivin, Antoine

    2017-02-09

    In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development has not been addressed. Here, we show that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, we detect rhino-dependent piRNAs indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the "ping-pong" amplification step. Moreover, we show that Heterochromatin Protein 1a is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal, and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development.

  16. Lin-28 promotes symmetric stem cell division and drives adaptive growth in the adult Drosophila intestine.

    PubMed

    Chen, Ching-Huan; Luhur, Arthur; Sokol, Nicholas

    2015-10-15

    Stem cells switch between asymmetric and symmetric division to expand in number as tissues grow during development and in response to environmental changes. The stem cell intrinsic proteins controlling this switch are largely unknown, but one candidate is the Lin-28 pluripotency factor. A conserved RNA-binding protein that is downregulated in most animals as they develop from embryos to adults, Lin-28 persists in populations of adult stem cells. Its function in these cells has not been previously characterized. Here, we report that Lin-28 is highly enriched in adult intestinal stem cells in the Drosophila intestine. lin-28 null mutants are homozygous viable but display defects in this population of cells, which fail to undergo a characteristic food-triggered expansion in number and have reduced rates of symmetric division as well as reduced insulin signaling. Immunoprecipitation of Lin-28-bound mRNAs identified Insulin-like Receptor (InR), forced expression of which completely rescues lin-28-associated defects in intestinal stem cell number and division pattern. Furthermore, this stem cell activity of lin-28 is independent of one well-known lin-28 target, the microRNA let-7, which has limited expression in the intestinal epithelium. These results identify Lin-28 as a stem cell intrinsic factor that boosts insulin signaling in intestinal progenitor cells and promotes their symmetric division in response to nutrients, defining a mechanism through which Lin-28 controls the adult stem cell division patterns that underlie tissue homeostasis and regeneration.

  17. A simple method for imaging axonal transport in ageing neurons using the adult Drosophila wing

    PubMed Central

    Vagnoni, Alessio; Bullock, Simon L.

    2016-01-01

    There is growing interest in the link between axonal cargo transport and age-associated neuronal dysfunction. Studying axonal transport in neurons of adult animals requires intravital or ex vivo imaging approaches, which are laborious and expensive in vertebrate models. We describe simple, non-invasive procedures for imaging cargo motility within axons using sensory neurons of the translucent Drosophila wing. A key aspect is a method for mounting the intact fly that allows detailed imaging of transport in wing neurons. Coupled with existing genetic tools in Drosophila, this is a tractable system for studying axonal transport over the lifespan of an animal and thus for characterising the relationship between cargo dynamics, neuronal ageing and disease. Preparation of a sample for imaging takes approximately 5 minutes, with transport typically filmed for 2–3 minutes per wing. We also document procedures for quantifying transport parameters from the acquired images and describe how the protocol can be adapted to study other cell biological processes in ageing neurons. PMID:27560175

  18. Functional redundancy and nonredundancy between two Troponin C isoforms in Drosophila adult muscles.

    PubMed

    Chechenova, Maria B; Maes, Sara; Oas, Sandy T; Nelson, Cloyce; Kiani, Kaveh G; Bryantsev, Anton L; Cripps, Richard M

    2017-03-15

    We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, Drosophila melanogaster: TpnC4 is predominantly expressed in the indirect flight muscles (IFMs), whereas TpnC41C is the main isoform in the tergal depressor of the trochanter muscle (TDT; jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of TpnC41C and compared its phenotype to a line lacking functional TpnC4 We found that the removal of either of these genes leads to expression of the other isoform in both muscle types. The switching between isoforms occurs at the transcriptional level and involves minimal enhancers located upstream of the transcription start points of each gene. Functionally, the two TpnC isoforms were not equal. Although ectopic TpnC4 in TDT muscles was able to maintain jumping ability, TpnC41C in IFMs could not effectively support flying. Simultaneous functional disruption of both TpnC genes resulted in jump-defective and flightless phenotypes of the survivors, as well as abnormal sarcomere organization. These results indicated that TpnC is required for myofibril assembly, and that there is functional specialization among TpnC isoforms in Drosophila.

  19. A simple method for imaging axonal transport in aging neurons using the adult Drosophila wing.

    PubMed

    Vagnoni, Alessio; Bullock, Simon L

    2016-09-01

    There is growing interest in the link between axonal cargo transport and age-associated neuronal dysfunction. The study of axonal transport in neurons of adult animals requires intravital or ex vivo imaging approaches, which are laborious and expensive in vertebrate models. We describe simple, noninvasive procedures for imaging cargo motility within axons using sensory neurons of the translucent Drosophila wing. A key aspect is a method for mounting the intact fly that allows detailed imaging of transport in wing neurons. Coupled with existing genetic tools in Drosophila, this is a tractable system for studying axonal transport over the life span of an animal and thus for characterization of the relationship between cargo dynamics, neuronal aging and disease. Preparation of a sample for imaging takes ∼5 min, with transport typically filmed for 2-3 min per wing. We also document procedures for the quantification of transport parameters from the acquired images and describe how the protocol can be adapted to study other cell biological processes in aging neurons.

  20. Degeneration and cell regeneration in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae) during post-embryonic development.

    PubMed

    Teixeira, Aparecida das Dores; Fialho, Maria do Carmo Queiroz; Zanuncio, José Cola; Ramalho, Francisco de Souza; Serrão, José Eduardo

    2013-05-01

    Cell death, proliferation, and differentiation in some developmental stages of insects have been studied in the midgut of ametabolous, which undergo only continuous growth, and holometabolous, which undergo complete metamorphosis. However, in hemimetabolous insects, evolutionarily intermediate between ametabolous and holometabolous, midgut reorganization during the post-embryonic development has been poorly studied. The present study evaluates the post-embryonic development of the midgut of a hemimetabolous insect, Podisus nigrispinus, to test the hypothesis that these insects have programmed cell death and proliferation followed by differentiation of regenerative cells during midgut growth from nymphs to adult. The morphometrical data showed a 6-fold increase in midgut length from the first instar nymph to the adult, which did not result from an increase in the size of the midgut cells, suggesting that the growth of the midgut occurs by an increase in cell number. Cell death was rarely found in the midgut, whereas proliferation of regenerative cells occurred quite frequently. The growth of the midgut of P. nigrispinus appears to result from the proliferation of regenerative cells present in the epithelium; unlike ametabolous and holometabolous insects, the midgut of P. nigrispinus does not undergo extensive remodeling, as shown by the low frequency of digestive cell death.

  1. 454-Pyrosequencing survey of microbiota in adult Spotted Wing Drosophila (SWD) corroborates a core microbiome and additional symbiotic and entomopathogenic bacterial associates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Complete surveys of insect endosymbionts including species of economic importance have until recently been hampered by a lack of high-throughput genetic assays. We used 454-pyrosequencing of the 16S rRNA gene amplicon of adult spotted wing Drosophila (SWD) Drosophila suzukii (Matsumura) from souther...

  2. Aging Drosophila melanogaster display altered pre- and postsynaptic ultrastructure at adult neuromuscular junctions.

    PubMed

    Wagner, Nicole; Laugks, Ulrike; Heckmann, Manfred; Asan, Esther; Neuser, Kirsa

    2015-11-01

    Although age-related changes in synaptic plasticity are an important focus within neuroscience, little is known about ultrastructural changes of synaptic morphology during aging. Here we report how aging affects synaptic ultrastructure by using fluorescence and electron microscopy at the adult Drosophila neuromuscular junction (NMJ) of ventral abdominal muscles. Mainly four striking morphological changes of aging NMJs were revealed. 1) Bouton size increases with proportionally rising number of active zones (AZs). 2) Synaptic vesicle density at AZs is increased in old flies. 3) Late endosomes, cisternae, and multivesicular bodies accumulate in the presynaptic terminal, and vesicles accumulate between membranes of the terminal bouton and the subsynaptic reticulum. 4) The electron-dense pre- and postsynaptic apposition is expanded in aging NMJs, which is accompanied by an expansion of the postsynaptic glutamate receptor fields. These findings suggest that aging is possibly accompanied by impaired synaptic vesicle release and recycling and a potentially compensatory expansion of AZs and postsynaptic densities.

  3. Muscle organizers in Drosophila: the role of persistent larval fibers in adult flight muscle development

    NASA Technical Reports Server (NTRS)

    Farrell, E. R.; Fernandes, J.; Keshishian, H.

    1996-01-01

    In many organisms muscle formation depends on specialized cells that prefigure the pattern of the musculature and serve as templates for myoblast organization and fusion. These include muscle pioneers in insects and muscle organizing cells in leech. In Drosophila, muscle founder cells have been proposed to play a similar role in organizing larval muscle development during embryogenesis. During metamorphosis in Drosophila, following histolysis of most of the larval musculature, there is a second round of myogenesis that gives rise to the adult muscles. It is not known whether muscle founder cells organize the development of these muscles. However, in the thorax specific larval muscle fibers do not histolyze at the onset of metamorphosis, but instead serve as templates for the formation of a subset of adult muscles, the dorsal longitudinal flight muscles (DLMs). Because these persistent larval muscle fibers appear to be functioning in many respects like muscle founder cells, we investigated whether they were necessary for DLM development by using a microbeam laser to ablate them singly and in combination. We found that, in the absence of the larval muscle fibers, DLMs nonetheless develop. Our results show that the persistent larval muscle fibers are not required to initiate myoblast fusion, to determine DLM identity, to locate the DLMs in the thorax, or to specify the total DLM fiber volume. However, they are required to regulate the number of DLM fibers generated. Thus, while the persistent larval muscle fibers are not obligatory for DLM fiber formation and differentiation, they are necessary to ensure the development of the correct number of fibers.

  4. Reducing Lissencephaly-1 levels augments mitochondrial transport and has a protective effect in adult Drosophila neurons

    PubMed Central

    Vagnoni, Alessio; Hoffmann, Patrick C.; Bullock, Simon L.

    2016-01-01

    ABSTRACT Defective transport of mitochondria in axons is implicated in the pathogenesis of several age-associated neurodegenerative diseases. However, the regulation and function of axonal mitochondrial motility during normal ageing is poorly understood. Here, we use novel imaging procedures to characterise axonal transport of these organelles in the adult Drosophila wing nerve. During early adult life there is a boost and progressive decline in the proportion of mitochondria that are motile, which is not due to general changes in cargo transport. Experimental inhibition of the mitochondrial transport machinery specifically in adulthood accelerates the appearance of focal protein accumulations in ageing axons, which is suggestive of defects in protein homeostasis. Unexpectedly, lowering levels of Lissencephaly-1 (Lis1), a dynein motor co-factor, augments axonal mitochondrial transport in ageing wing neurons. Lis1 mutations suppress focal protein accumulations in ageing neurons, including those caused by interfering with the mitochondrial transport machinery. Our data provide new insights into the dynamics of mitochondrial motility in adult neurons in vivo, identify Lis1 as a negative regulator of transport of these organelles, and provide evidence of a link between mitochondrial movement and neuronal protein homeostasis. PMID:26598558

  5. A Systematic Analysis of Drosophila Regulatory Peptide Expression in Enteroendocrine Cells.

    PubMed

    Chen, Ji; Kim, Seol-Min; Kwon, Jae Young

    2016-04-30

    The digestive system is gaining interest as a major regulator of various functions including immune defense, nutrient accumulation, and regulation of feeding behavior, aside from its conventional function as a digestive organ. The Drosophila midgut epithelium is completely renewed every 1-2 weeks due to differentiation of pluripotent intestinal stem cells in the midgut. Intestinal stem cells constantly divide and differentiate into enterocytes that secrete digestive enzymes and absorb nutrients, or enteroendocrine cells that secrete regulatory peptides. Regulatory peptides have important roles in development and metabolism, but study has mainly focused on expression and functions in the nervous system, and not much is known about the roles in endocrine functions of enteroendocrine cells. We systemically examined the expression of 45 regulatory peptide genes in the Drosophila midgut, and verified that at least 10 genes are expressed in the midgut enteroendocrine cells through RT-PCR, in situ hybridization, antisera, and 25 regulatory peptide-GAL transgenes. The Drosophila midgut is highly compartmentalized, and individual peptides in enteroendocrine cells were observed to express in specific regions of the midgut. We also confirmed that some peptides expressed in the same region of the midgut are expressed in mutually exclusive enteroendocrine cells. These results indicate that the midgut enteroendocrine cells are functionally differentiated into different subgroups. Through this study, we have established a basis to study regulatory peptide functions in enteroendocrine cells as well as the complex organization of enteroendocrine cells in the Drosophila midgut.

  6. An essential role for the Drosophila Pax2 homolog in the differentiation of adult sensory organs.

    PubMed

    Kavaler, J; Fu, W; Duan, H; Noll, M; Posakony, J W

    1999-05-01

    The adult peripheral nervous system of Drosophila includes a complex array of mechanosensory organs (bristles) that cover much of the body surface of the fly. The four cells (shaft, socket, sheath, and neuron) which compose each of these organs adopt distinct fates as a result of cell-cell signaling via the Notch (N) pathway. However, the specific mechanisms by which these cells execute their conferred fates are not well understood. Here we show that D-Pax2, the Drosophila homolog of the vertebrate Pax2 gene, has an essential role in the differentiation of the shaft cell. In flies bearing strong loss-of-function mutations in the shaven function of D-Pax2, shaft structures specifically fail to develop. Consistent with this, we find that D-Pax2 protein is expressed in all cells of the bristle lineage during the mitotic (cell fate specification) phase of bristle development, but becomes sharply restricted to the shaft and sheath cells in the post-mitotic (differentiative) phase. Two lines of evidence described here indicate that D-Pax2 expression and function is at least in part downstream of cell fate specification mechanisms such as N signaling. First, we find that the lack of late D-Pax2 expression in the socket cell (the sister of the shaft cell) is controlled by N pathway activity; second, we find that loss of D-Pax2 function is epistatic to the socket-to-shaft cell fate transformation caused by reduced N signaling. Finally, we show that misexpression of D-Pax2 is sufficient to induce the production of ectopic shaft structures. From these results, we propose that D-Pax2 is a high-level transcriptional regulator of the shaft cell differentiation program, and acts downstream of the N signaling pathway as a specific link between cell fate determination and cell differentiation in the bristle lineage.

  7. Targeted Manipulation of Serotonergic Neurotransmission Affects the Escalation of Aggression in Adult Male Drosophila melanogaster

    PubMed Central

    Alekseyenko, Olga V.; Lee, Carol; Kravitz, Edward A.

    2010-01-01

    Dopamine (DA) and serotonin (5HT) are reported to serve important roles in aggression in a wide variety of animals. Previous investigations of 5HT function in adult Drosophila behavior have relied on pharmacological manipulations, or on combinations of genetic tools that simultaneously target both DA and 5HT neurons. Here, we generated a transgenic line that allows selective, direct manipulation of serotonergic neurons and asked whether DA and 5HT have separable effects on aggression. Quantitative morphological examination demonstrated that our newly generated tryptophan hydroxylase (TRH)-Gal4 driver line was highly selective for 5HT-containing neurons. This line was used in conjunction with already available Gal4 driver lines that target DA or both DA and 5HT neurons to acutely alter the function of aminergic systems. First, we showed that acute impairment of DA and 5HT neurotransmission using expression of a temperature sensitive form of dynamin completely abolished mid- and high-level aggression. These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships. We showed next that manipulation of either 5HT or DA neurotransmission failed to duplicate this phenotype. Selective disruption of 5HT neurotransmission yielded flies that fought, but with reduced ability to escalate fights, leading to fewer dominance relationships. Acute activation of 5HT neurons using temperature sensitive dTrpA1 channel expression, in contrast, resulted in flies that escalated fights faster and that fought at higher intensities. Finally, acute disruption of DA neurotransmission produced hyperactive flies that moved faster than controls, and rarely engaged in any social interactions. By separately manipulating 5HT- and DA- neuron systems, we collected evidence demonstrating a direct role for 5HT in the escalation of aggression in Drosophila. PMID:20520823

  8. Development of diet-induced insulin resistance in adult Drosophila melanogaster.

    PubMed

    Morris, Siti Nur Sarah; Coogan, Claire; Chamseddin, Khalil; Fernandez-Kim, Sun Ok; Kolli, Santharam; Keller, Jeffrey N; Bauer, Johannes H

    2012-08-01

    The fruit fly Drosophila melanogaster is increasingly utilized as an alternative to costly rodent models to study human diseases. Fly models exist for a wide variety of human conditions, such as Alzheimer's and Parkinson's Disease, or cardiac function. Advantages of the fly system are its rapid generation time and its low cost. However, the greatest strength of the fly system are the powerful genetic tools that allow for rapid dissection of molecular disease mechanisms. Here, we describe the diet-dependent development of metabolic phenotypes in adult fruit flies. Depending on the specific type of nutrient, as well as its relative quantity in the diet, flies show weight gain and changes in the levels of storage macromolecules. Furthermore, the activity of insulin-signaling in the major metabolic organ of the fly, the fat body, decreases upon overfeeding. This decrease in insulin-signaling activity in overfed flies is moreover observed when flies are challenged with an acute food stimulus, suggesting that overfeeding leads to insulin resistance. Similar changes were observed in aging flies, with the development of the insulin resistance-like phenotype beginning at early middle ages. Taken together, these data demonstrate that imbalanced diet disrupts metabolic homeostasis in adult D. melanogaster and promotes insulin-resistant phenotypes. Therefore, the fly system may be a useful alternative tool in the investigation of molecular mechanisms of insulin resistance and the development of pharmacologic treatment options.

  9. Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system

    PubMed Central

    Lacin, Haluk; Truman, James W

    2016-01-01

    Neurogenesis in Drosophila occurs in two phases, embryonic and post-embryonic, in which the same set of neuroblasts give rise to the distinct larval and adult nervous systems, respectively. Here, we identified the embryonic neuroblast origin of the adult neuronal lineages in the ventral nervous system via lineage-specific GAL4 lines and molecular markers. Our lineage mapping revealed that neurons born late in the embryonic phase show axonal morphology and transcription factor profiles that are similar to the neurons born post-embryonically from the same neuroblast. Moreover, we identified three thorax-specific neuroblasts not previously characterized and show that HOX genes confine them to the thoracic segments. Two of these, NB2-3 and NB3-4, generate leg motor neurons. The other neuroblast is novel and appears to have arisen recently during insect evolution. Our findings provide a comprehensive view of neurogenesis and show how proliferation of individual neuroblasts is dictated by temporal and spatial cues. DOI: http://dx.doi.org/10.7554/eLife.13399.001 PMID:26975248

  10. Correlated changes in circadian clocks in response to selection for faster pre-adult development in fruit flies Drosophila melanogaster.

    PubMed

    Yadav, Pankaj; Sharma, Vijay Kumar

    2013-04-01

    Although, circadian clocks are believed to be involved in the regulation of life-history traits such as pre-adult development time and lifespan in fruit flies Drosophila melanogaster, there is very little unequivocal evidence either to support or refute this. Here we report the results of a long-term study aimed at examining the role of circadian clocks in the temporal regulation of pre-adult development in D. melanogaster. We employed laboratory selection protocol for faster pre-adult development on four large, outbred, random mating populations of Drosophila. We assayed pre-adult development time and circadian period of locomotor activity rhythm of these flies at regular intervals of 5-10 generations. After 50 generations of selection, the overall egg-to-adult duration in the selected stocks was reduced by ~29 h (~12.5%) relative to controls, with the selected populations showing a concurrent reduction in time taken to hatching, pupation and wing pigmentation, by ~2, ~16, and ~25.2 h, respectively. Furthermore, selected populations showed a concomitant reduction in the circadian period of locomotor activity rhythm, implying that circadian clocks and development time are correlated. Thus, our study provides the first ever unequivocal evidence for the evolution of circadian clocks as a correlated response to selection for faster pre-adult development, suggesting that circadian clocks and development are linked in fruit flies D. melanogaster.

  11. Developmental effects of exposing Drosophila embryos to ether vapour.

    PubMed

    Bownes, M; Seiler, M

    1977-01-01

    Drosophila embryos at precise developmental stages were exposed to ether vapour. The defects in the resulting embryos and adults were observed. Ether disrupted embroygenesis in specific ways, causing defects primarily at the anterior of the embryo and disorganizing the arrangement of the segments. Adults showed deficiencies and duplications of many imaginal disc and histoblast derivatives. Phenocopies of the bithorax mutation which transforms metathorax to mesothorax were observed. They were first induced at the syncytial blastoderm stage, had their peak of production at the cellular blastoderm, and were no longer observed after the anterior and posterior midgut were partially invaginated. It was observed that not only are the halter/wing transformations confined to the anterior compartment, but also leg 3 to leg 2 transformations only occurred in the anterior leg compartment.

  12. Steroid hormone inactivation is required during the juvenile-adult transition in Drosophila.

    PubMed

    Rewitz, Kim F; Yamanaka, Naoki; O'Connor, Michael B

    2010-12-14

    Steroid hormones are systemic signaling molecules that regulate juvenile-adult transitions in both insects and mammals. In insects, pulses of the steroid hormone 20-hydroxyecdysone (20E) are generated by increased biosynthesis followed by inactivation/clearance. Although mechanisms that control 20E synthesis have received considerable recent attention, the physiological significance of 20E inactivation remains largely unknown. We show that the cytochrome P450 Cyp18a1 lowers 20E titer during the Drosophila prepupal to pupal transition. Furthermore, this reduction of 20E levels is a prerequisite to induce βFTZ-F1, a key factor in the genetic hierarchy that controls early metamorphosis. Resupplying βFTZ-F1 rescues Cyp18a1-deficient prepupae. Because Cyp18a1 is 20E-inducible, it appears that the increased production of steroid is responsible for its eventual decline, thereby generating the regulatory pulse required for proper temporal progression of metamorphosis. The coupling of hormone clearance to βFTZ-F1 expression suggests a general mechanism by which transient signaling drives unidirectional progression through a multistep process.

  13. The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila.

    PubMed

    Singh, Shree Ram; Zeng, Xiankun; Zhao, Jiangsha; Liu, Ying; Hou, Gerald; Liu, Hanhan; Hou, Steven X

    2016-10-06

    Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. Here we investigated stem-cell death in the digestive system of adult Drosophila melanogaster. We found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively killed normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spared differentiated cells. The dying stem cells were engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduced CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers.

  14. Why Adult Stem Cell Functionality Declines with Age? Studies from the Fruit Fly Drosophila Melanogaster Model Organism

    PubMed Central

    Gonen, Oren; Toledano, Hila

    2014-01-01

    Highly regenerative adult tissues are supported by rare populations of stem cells that continuously divide to self-renew and generate differentiated progeny. This process is tightly regulated by signals emanating from surrounding cells to fulfill the dynamic demands of the tissue. One of the hallmarks of aging is slow and aberrant tissue regeneration due to deteriorated function of stem and supporting cells. Several Drosophila regenerative tissues are unique in that they provide exact identification of stem and neighboring cells in whole-tissue anatomy. This allows for precise tracking of age-related changes as well as their targeted manipulation within the tissue. In this review we present the stem cell niche of Drosophila testis, ovary and intestine and describe the major changes and phenotypes that occur in the course of aging. Specifically we discuss changes in both intrinsic properties of stem cells and their microenvironment that contribute to the decline in tissue functionality. Understanding these mechanisms in adult Drosophila tissues will likely provide new paradigms in the field of aging. PMID:24955030

  15. The Drosophila BTB Domain Protein Jim Lovell Has Roles in Multiple Larval and Adult Behaviors

    PubMed Central

    Bjorum, Sonia M.; Simonette, Rebecca A.; Alanis, Raul; Wang, Jennifer E.; Lewis, Benjamin M.; Trejo, Michael H.; Hanson, Keith A.; Beckingham, Kathleen M.

    2013-01-01

    Innate behaviors have their origins in the specification of neural fates during development. Within Drosophila, BTB (Bric-a-brac,Tramtrack, Broad) domain proteins such as Fruitless are known to play key roles in the neural differentiation underlying such responses. We previously identified a gene, which we have termed jim lovell (lov), encoding a BTB protein with a role in gravity responses. To understand more fully the behavioral roles of this gene we have investigated its function through several approaches. Transcript and protein expression patterns have been examined and behavioral phenotypes of new lov mutations have been characterized. Lov is a nuclear protein, suggesting a role as a transcriptional regulator, as for other BTB proteins. In late embryogenesis, Lov is expressed in many CNS and PNS neurons. An examination of the PNS expression indicates that lov functions in the late specification of several classes of sensory neurons. In particular, only two of the five abdominal lateral chordotonal neurons express Lov, predicting functional variation within this highly similar group. Surprisingly, Lov is also expressed very early in embryogenesis in ways that suggests roles in morphogenetic movements, amnioserosa function and head neurogenesis. The phenotypes of two new lov mutations that delete adjacent non-coding DNA regions are strikingly different suggesting removal of different regulatory elements. In lov47, Lov expression is lost in many embryonic neurons including the two lateral chordotonal neurons. lov47 mutant larvae show feeding and locomotor defects including spontaneous backward movement. Adult lov47 males perform aberrant courtship behavior distinguished by courtship displays that are not directed at the female. lov47 adults also show more defective negative gravitaxis than the previously isolated lov91Y mutant. In contrast, lov66 produces largely normal behavior but severe female sterility associated with ectopic lov expression in the ovary. We

  16. Adult plasticity of cold tolerance in a continental-temperate population of Drosophila suzukii.

    PubMed

    Jakobs, Ruth; Gariepy, Tara D; Sinclair, Brent J

    2015-08-01

    Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a worldwide emerging pest of soft fruits, but its cold tolerance has not been thoroughly explored. We determined the cold tolerance strategy, low temperature thermal limits, and plasticity of cold tolerance in both male and female adult D. suzukii. We reared flies under common conditions (long days, 21°C; control) and induced plasticity by rapid cold-hardening (RCH, 1h at 0°C followed by 1h recovery), cold acclimation (CA, 5 days at 6°C) or acclimation under fluctuating temperatures (FA). D. suzukii had supercooling points (SCPs) between -16 and -23°C, and were chill-susceptible. 80% of control flies were killed after 1h at -7.2°C (males) or -7.5°C (females); CA and FA improved survival of this temperature in both sexes, but RCH did not. 80% of control flies were killed after 70 h (male) or 92 h (female) at 0°C, and FA shifted this to 112 h (males) and 165 h (females). FA flies entered chill coma (CTmin) at approximately -1.7°C, which was ca. 0.5°C colder than control flies; RCH and CA increased the CTmin compared to controls. Control and RCH flies exposed to 0°C for 8h took 30-40 min to recover movement, but this was reduced to <10 min in CA and FA. Flies placed outside in a field cage in London, Ontario, were all killed by a transient cold snap in December. We conclude that adult phenotypic plasticity is not sufficient to allow D. suzukii to overwinter in temperate habitats, and suggest that flies could overwinter in association with built structures, or that there may be additional cold tolerance imparted by developmental plasticity.

  17. Serratia odorifera a Midgut Inhabitant of Aedes aegypti Mosquito Enhances Its Susceptibility to Dengue-2 Virus

    PubMed Central

    Apte-Deshpande, Anjali; Paingankar, Mandar; Gokhale, Mangesh D.; Deobagkar, Dileep N.

    2012-01-01

    Mosquito midgut plays a crucial role in its vector susceptibility and pathogen interaction. Identification of the sustainable microflora of the midgut environment can therefore help in evaluating its contribution in mosquito-pathogen interaction and in turn vector competence. To understand the bacterial diversity in the midgut of Aedes aegypti mosquitoes, we conducted a screening study of the gut microbes of these mosquitoes which were either collected from fields or reared in the laboratory “culture-dependent” approach. This work demonstrated that the microbial flora of larvae and adult Ae. aegypti midgut is complex and is dominated by Gram negative proteobacteria. Serratia odorifera was found to be stably associated in the midguts of field collected and laboratory reared larvae and adult females. The potential influence of this sustainable gut microbe on DENV-2 susceptibility of this vector was evaluated by co-feeding S. odorifera with DENV-2 to adult Ae. aegypti females (free of gut flora). The observations revealed that the viral susceptibility of these Aedes females enhanced significantly as compared to solely dengue-2 fed and another gut inhabitant, Microbacterium oxydans co-fed females. Based on the results of this study we proposed that the enhancement in the DENV-2 susceptibility of Ae. aegypti females was due to blocking of prohibitin molecule present on the midgut surface of these females by the polypeptide of gut inhabitant S. odorifera. PMID:22848375

  18. Preparation of adult Drosophila eyes for thin sectioning and microscopic analysis.

    PubMed

    Jenny, Andreas

    2011-08-27

    Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes. The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g.). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior

  19. Roles for Drosophila melanogaster Myosin IB in Maintenance of Enterocyte Brush-Border Structure and Resistance to the Bacterial Pathogen Pseudomonas entomophila

    PubMed Central

    Mermall, Valerie; Tilney, Lewis G.; Mooseker, Mark S.

    2007-01-01

    Drosophila myosin IB (Myo1B) is one of two class I myosins in the Drosophila genome. In the larval and adult midgut enterocyte, Myo1B is present within the microvillus (MV) of the apical brush border (BB) where it forms lateral tethers between the MV membrane and underlying actin filament core. Expression of green fluorescent protein-Myo1B tail domain in the larval gut showed that the tail domain is sufficient for localization of Myo1B to the BB. A Myo1B deletion mutation exhibited normal larval gut physiology with respect to food uptake, clearance, and pH regulation. However, there is a threefold increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive enterocyte nuclei in the Myo1B mutant. Ultrastructural analysis of mutant midgut revealed many perturbations in the BB, including membrane tethering defects, MV vesiculation, and membrane shedding. The apical localization of both singed (fascin) and Dmoesin is impaired. BBs isolated from mutant and control midgut revealed that the loss of Myo1B causes the BB membrane and underlying cytoskeleton to become destabilized. Myo1B mutant larvae also exhibit enhanced sensitivity to oral infection by the bacterial pathogen Pseudomonas entomophila, and severe cytoskeletal defects are observed in the BB of proximal midgut epithelial cells soon after infection. Resistance to P. entomophila infection is restored in Myo1B mutant larvae expressing a Myo1B transgene. These results indicate that Myo1B may play a role in the local midgut response pathway of the Imd innate immune response to Gram-negative bacterial infection. PMID:17855510

  20. Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction.

    PubMed

    Kiss, Márton; Kiss, András A; Radics, Monika; Popovics, Nikoletta; Hermesz, Edit; Csiszár, Katalin; Mink, Mátyás

    2016-01-01

    The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that

  1. Interaction of light regimes and circadian clocks modulate timing of pre-adult developmental events in Drosophila

    PubMed Central

    2014-01-01

    Background Circadian clocks have been postulated to regulate development time in several species of insects including fruit flies Drosophila melanogaster. Previously we have reported that selection for faster pre-adult development reduces development time (by ~19 h or ~11%) and clock period (by ~0.5 h), suggesting a role of circadian clocks in the regulation of development time in D. melanogaster. We reasoned that these faster developing flies could serve as a model to study stage-specific interaction of circadian clocks and developmental events with the environmental light/dark (LD) conditions. We assayed the duration of three pre-adult stages in the faster developing (FD) and control (BD) populations under a variety of light regimes that are known to modulate circadian clocks and pre-adult development time of Drosophila to examine the role of circadian clocks in the timing of pre-adult developmental stages. Results We find that the duration of pre-adult stages was shorter under constant light (LL) and short period light (L)/dark (D) cycles (L:D = 10:10 h; T20) compared to the standard 24 h day (L:D = 12:12 h; T24), long LD cycles (L:D = 14:14 h; T28) and constant darkness (DD). The difference in the duration of pre-adult stages between the FD and BD populations was significantly smaller under the three LD cycles and LL compared to DD, possibly due to the fact that clocks of both FD and BD flies are driven at the same pace in the three LD regimes owing to circadian entrainment, or are rendered dysfunctional under LL. Conclusions These results suggest that interaction between light regimes and circadian clocks regulate the duration of pre-adult developmental stages in fruit flies D. melanogaster. PMID:24885932

  2. Transient Dysregulation of Dopamine Signaling in a Developing Drosophila Arousal Circuit Permanently Impairs Behavioral Responsiveness in Adults

    PubMed Central

    Ferguson, Lachlan; Petty, Alice; Rohrscheib, Chelsie; Troup, Michael; Kirszenblat, Leonie; Eyles, Darryl W.; van Swinderen, Bruno

    2017-01-01

    The dopamine ontogeny hypothesis for schizophrenia proposes that transient dysregulation of the dopaminergic system during brain development increases the likelihood of this disorder in adulthood. To test this hypothesis in a high-throughput animal model, we have transiently manipulated dopamine signaling in the developing fruit fly Drosophila melanogaster and examined behavioral responsiveness in adult flies. We found that either a transient increase of dopamine neuron activity or a transient decrease of dopamine receptor expression during fly brain development permanently impairs behavioral responsiveness in adults. A screen for impaired responsiveness revealed sleep-promoting neurons in the central brain as likely postsynaptic dopamine targets modulating these behavioral effects. Transient dopamine receptor knockdown during development in a restricted set of ~20 sleep-promoting neurons recapitulated the dopamine ontogeny phenotype, by permanently reducing responsiveness in adult animals. This suggests that disorders involving impaired behavioral responsiveness might result from defective ontogeny of sleep/wake circuits. PMID:28243212

  3. Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death

    PubMed Central

    Shimono, Kohei; Fujimoto, Azusa; Tsuyama, Taiichi; Yamamoto-Kochi, Misato; Sato, Motohiko; Hattori, Yukako; Sugimura, Kaoru; Usui, Tadao; Kimura, Ken-ichi; Uemura, Tadashi

    2009-01-01

    Background For the establishment of functional neural circuits that support a wide range of animal behaviors, initial circuits formed in early development have to be reorganized. One way to achieve this is local remodeling of the circuitry hardwiring. To genetically investigate the underlying mechanisms of this remodeling, one model system employs a major group of Drosophila multidendritic sensory neurons - the dendritic arborization (da) neurons - which exhibit dramatic dendritic pruning and subsequent growth during metamorphosis. The 15 da neurons are identified in each larval abdominal hemisegment and are classified into four categories - classes I to IV - in order of increasing size of their receptive fields and/or arbor complexity at the mature larval stage. Our knowledge regarding the anatomy and developmental basis of adult da neurons is still fragmentary. Results We identified multidendritic neurons in the adult Drosophila abdomen, visualized the dendritic arbors of the individual neurons, and traced the origins of those cells back to the larval stage. There were six da neurons in abdominal hemisegment 3 or 4 (A3/4) of the pharate adult and the adult just after eclosion, five of which were persistent larval da neurons. We quantitatively analyzed dendritic arbors of three of the six adult neurons and examined expression in the pharate adult of key transcription factors that result in the larval class-selective dendritic morphologies. The 'baseline design' of A3/4 in the adult was further modified in a segment-dependent and age-dependent manner. One of our notable findings is that a larval class I neuron, ddaE, completed dendritic remodeling in A2 to A4 and then underwent caspase-dependent cell death within 1 week after eclosion, while homologous neurons in A5 and in more posterior segments degenerated at pupal stages. Another finding is that the dendritic arbor of a class IV neuron, v'ada, was immediately reshaped during post-eclosion growth. It exhibited

  4. Population Genetics of Drosophila Amylase. II. Geographic Patterns in D. PSEUDOOBSCURA

    PubMed Central

    Powell, Jeffrey R.

    1979-01-01

    Morph frequencies of three related polymorphisms were determined in ten natural populations of Drosophila pseudoobscura. They are the well-known inversion polymorphism of the third chromosome and the polymorphism for α-amylase produced by the structural gene Amy (which resides on the third chromosome). The third polymorphism was for tissue-specific expression of Amy in adult midguts; a total of 13 different patterns of activity have been observed. The preceding paper (Powell and Lichtenfels 1979) reports evidence that the variation in Amy expression is under polygenic control. Here we show that the polymorphism for midgut activity patterns occurs in natural populations and is not an artifact of laboratory rearing.—From population to population, Amy allele freuencies and frequencies of inversions belonging to different phylads vary coordinately. The geographic variation in α-amylase midgut activity patterns is uncorrelated with that for the other two types of polymorphisms. Furthermore, no correlation was detected between activity pattern(s) and Amy genotype(s) when both were assayed in the same individual.—These results imply that whatever the evolutionary-ecological forces are that control frequencies of the structural gene variants, they are not the same factors that control the frequencies of polymorphic genetic factors responsible for the tissue-specific expression of the enzyme. PMID:488707

  5. Gene-environment interplay in Drosophila melanogaster: chronic nutritional deprivation in larval life affects adult fecal output.

    PubMed

    Urquhart-Cronish, Mackenzie; Sokolowski, Marla B

    2014-10-01

    Life history consequences of stress in early life are varied and known to have lasting impacts on the fitness of an organism. Gene-environment interactions play a large role in how phenotypic differences are mediated by stressful conditions during development. Here we use natural allelic 'rover/sitter' variants of the foraging (for) gene and chronic early life nutrient deprivation to investigate gene-environment interactions on excretion phenotypes. Excretion assay analysis and a fully factorial nutritional regimen encompassing the larval and adult life cycle of Drosophila melanogaster were used to assess the effects of larval and adult nutritional stress on adult excretion phenotypes. Natural allelic variants of for exhibited differences in the number of fecal spots when they were nutritionally deprived as larvae and well fed as adults. for mediates the excretion response to chronic early-life nutritional stress in mated female, virgin female, and male rovers and sitters. Transgenic manipulations of for in a sitter genetic background under larval but not adult food deprivation increases the number of fecal spots. Our study shows that food deprivation early in life affects adult excretion phenotypes and these excretion differences are mediated by for.

  6. Gene-environment interplay in Drosophila melanogaster: chronic food deprivation in early life affects adult exploratory and fitness traits.

    PubMed

    Burns, James Geoffrey; Svetec, Nicolas; Rowe, Locke; Mery, Frederic; Dolan, Michael J; Boyce, W Thomas; Sokolowski, Marla B

    2012-10-16

    Early life adversity has known impacts on adult health and behavior, yet little is known about the gene-environment interactions (GEIs) that underlie these consequences. We used the fruit fly Drosophila melanogaster to show that chronic early nutritional adversity interacts with rover and sitter allelic variants of foraging (for) to affect adult exploratory behavior, a phenotype that is critical for foraging, and reproductive fitness. Chronic nutritional adversity during adulthood did not affect rover or sitter adult exploratory behavior; however, early nutritional adversity in the larval period increased sitter but not rover adult exploratory behavior. Increasing for gene expression in the mushroom bodies, an important center of integration in the fly brain, changed the amount of exploratory behavior exhibited by sitter adults when they did not experience early nutritional adversity but had no effect in sitters that experienced early nutritional adversity. Manipulation of the larval nutritional environment also affected adult reproductive output of sitters but not rovers, indicating GEIs on fitness itself. The natural for variants are an excellent model to examine how GEIs underlie the biological embedding of early experience.

  7. Non-Nutritive Polyol Sweeteners Differ in Insecticidal Activity When Ingested by Adult Drosophila melanogaster (Diptera: Drosophilidae)

    PubMed Central

    O’Donnell, Sean; Baudier, Kaitlin; Marenda, Daniel R.

    2016-01-01

    Previous work showed the non-nutritive polyol sweetener Erythritol was toxic when ingested by Drosophila melanogaster (Meigen, 1930). This study assessed whether insect toxicity is a general property of polyols. Among tested compounds, toxicity was highest for erythritol. Adult fruit flies (D. melanogaster) fed erythritol had reduced longevity relative to controls. Other polyols did not reduce longevity; the only exception was a weaker but significant reduction of female (but not male) longevity when flies were fed D-mannitol. We conclude at least some non-nutritive polyols are not toxic to adult D. melanogaster when ingested for 17 days. The longer time course (relative to erythritol) and female specificity of D-mannitol mortality suggests different mechanisms for D-mannitol and erythritol toxicity to D. melanogaster. PMID:27271968

  8. Midgut of the non-hematophagous mosquito Toxorhynchites theobaldi (Diptera, Culicidae).

    PubMed

    Godoy, Raquel S M; Fernandes, Kenner M; Martins, Gustavo F

    2015-10-30

    In most mosquito species, the females require a blood-feeding for complete egg development. However, in Toxorhynchites mosquitoes, the eggs develop without blood-feeding, and both females and males exclusively feed on sugary diets. The midgut is a well-understood organ in blood-feeding mosquitoes, but little is known about it in non-blood-feeding ones. In the present study, the detailed morphology of the midgut of Toxorhynchites theobaldi were investigated using histochemical and ultrastructural methods. The midgut of female and male T. theobaldi adults consists of a long, slender anterior midgut (AMG), and a short, dilated posterior midgut (PMG). The AMG is subdivided into AMG1 (short, with folds) and AMG2 (long, without folds). Nerve branches and enteroendocrine cells are present in AMG and PMG, respectively. Compared with the PMG of blood-feeding female mosquitoes, the PMG of T. theobaldi is smaller; however, in both mosquitoes, PMG seems be the main region of food digestion and absorption, and protein secretion. The epithelial folds present in the AMG of T. theobaldi have not been reported in other mosquitoes; however, the midgut muscle organization and endocrine control of the digestion process are conserved in both T. theobaldi and blood-feeding mosquitoes.

  9. Midgut of the non-hematophagous mosquito Toxorhynchites theobaldi (Diptera, Culicidae)

    PubMed Central

    Godoy, Raquel S. M.; Fernandes, Kenner M.; Martins, Gustavo F.

    2015-01-01

    In most mosquito species, the females require a blood-feeding for complete egg development. However, in Toxorhynchites mosquitoes, the eggs develop without blood-feeding, and both females and males exclusively feed on sugary diets. The midgut is a well-understood organ in blood-feeding mosquitoes, but little is known about it in non-blood-feeding ones. In the present study, the detailed morphology of the midgut of Toxorhynchites theobaldi were investigated using histochemical and ultrastructural methods. The midgut of female and male T. theobaldi adults consists of a long, slender anterior midgut (AMG), and a short, dilated posterior midgut (PMG). The AMG is subdivided into AMG1 (short, with folds) and AMG2 (long, without folds). Nerve branches and enteroendocrine cells are present in AMG and PMG, respectively. Compared with the PMG of blood-feeding female mosquitoes, the PMG of T. theobaldi is smaller; however, in both mosquitoes, PMG seems be the main region of food digestion and absorption, and protein secretion. The epithelial folds present in the AMG of T. theobaldi have not been reported in other mosquitoes; however, the midgut muscle organization and endocrine control of the digestion process are conserved in both T. theobaldi and blood-feeding mosquitoes. PMID:26514271

  10. DNA damage in haemocytes and midgut gland cells of Steatoda grossa (Theridiidae) spiders exposed to food contaminated with cadmium.

    PubMed

    Stalmach, Monika; Wilczek, Grażyna; Wilczek, Piotr; Skowronek, Magdalena; Mędrzak, Monika

    2015-03-01

    The aim of this study was to assess the genotoxic effects of Cd on haemocytes and midgut gland cells of web-building spiders, Steatoda grossa (Theridiidae), exposed to the metal under laboratory conditions. Analyzes were conducted on adult females and males, fed for four weeks with cadmium-contaminated Drosophila hydei flies, grown on a medium suplemented with 0.25 mM CdCl2. The comet assay, providing a quantitative measure of DNA strand breaks, was used to evaluate the DNA damage caused by the metal. Cadmium content was measured in whole spider bodies by the AAS method. Metal body burden was significantly lower in females (0.25 µgg(-1) dry weight) than in males (3.03 µgg(-1) dry weight), suggesting that females may have more effective mechanisms controlling the uptake of metal, via the digestive tract, or its elimination from the body. Irrespectively of sex, spiders fed prey contaminated with cadmium showed significantly higher values of comet parameters: tail DNA (TDNA), tail length (TL) and olive tail moment (OTM), in comparison with the control. In midgut gland cells, the level of DNA damage was higher for males than females, while in haemocytes the genotoxic effect of cadmium was greater in females. The obtained results indicate that in spiders cadmium displays strong genotoxic effects and may cause DNA damage even at low concentrations, however the severity of damage seems to be sex- and internal organ-dependent. The comet assay can be considered a sensitive tool for measuring the deleterious effect of cadmium on DNA integrity in spiders.

  11. The Jak-STAT target Chinmo prevents sex transformation of adult stem cells in the Drosophila testis niche

    PubMed Central

    Ma, Qing; Wawersik, Matthew; Matunis, Erika L.

    2014-01-01

    Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). Here, we find that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor DoublesexM (DsxM) within CySCs and their progeny, and ectopic expression of DsxM in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of DsxM. The Dsx homologue DMRT1 prevents the male-to female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. Our work indicates that sex maintenance occurs in adult somatic stem cells, and that this highly conserved process is governed by effectors of niche signals. PMID:25453558

  12. Stable Host Gene Expression in the Gut of Adult Drosophila melanogaster with Different Bacterial Mono-Associations

    PubMed Central

    Zhang, Vivian; Ludington, William B.; Eisen, Michael B.

    2016-01-01

    There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but we still do not understand how they accomplish this. Here, we evaluated how different microbial species commonly associated with laboratory-reared Drosophila melanogaster impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. We observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, we found that the gut transcriptional profiles of animals reared in the presence of the yeast Saccharomyces cerevisiae alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, we found whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. Our data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body. PMID:27898741

  13. Larval Population Density Alters Adult Sleep in Wild-Type Drosophila melanogaster but Not in Amnesiac Mutant Flies.

    PubMed

    Chi, Michael W; Griffith, Leslie C; Vecsey, Christopher G

    2014-08-11

    Sleep has many important biological functions, but how sleep is regulated remains poorly understood. In humans, social isolation and other stressors early in life can disrupt adult sleep. In fruit flies housed at different population densities during early adulthood, social enrichment was shown to increase subsequent sleep, but it is unknown if population density during early development can also influence adult sleep. To answer this question, we maintained Drosophila larvae at a range of population densities throughout larval development, kept them isolated during early adulthood, and then tested their sleep patterns. Our findings reveal that flies that had been isolated as larvae had more fragmented sleep than those that had been raised at higher population densities. This effect was more prominent in females than in males. Larval population density did not affect sleep in female flies that were mutant for amnesiac, which has been shown to be required for normal memory consolidation, adult sleep regulation, and brain development. In contrast, larval population density effects on sleep persisted in female flies lacking the olfactory receptor or83b, suggesting that olfactory signals are not required for the effects of larval population density on adult sleep. These findings show that population density during early development can alter sleep behavior in adulthood, suggesting that genetic and/or structural changes are induced by this developmental manipulation that persist through metamorphosis.

  14. A High-Resolution Whole-Genome Map of Key Chromatin Modifications in the Adult Drosophila melanogaster

    PubMed Central

    Yin, Hang; Sweeney, Sarah; Raha, Debasish; Snyder, Michael; Lin, Haifan

    2011-01-01

    Epigenetic research has been focused on cell-type-specific regulation; less is known about common features of epigenetic programming shared by diverse cell types within an organism. Here, we report a modified method for chromatin immunoprecipitation and deep sequencing (ChIP–Seq) and its use to construct a high-resolution map of the Drosophila melanogaster key histone marks, heterochromatin protein 1a (HP1a) and RNA polymerase II (polII). These factors are mapped at 50-bp resolution genome-wide and at 5-bp resolution for regulatory sequences of genes, which reveals fundamental features of chromatin modification landscape shared by major adult Drosophila cell types: the enrichment of both heterochromatic and euchromatic marks in transposons and repetitive sequences, the accumulation of HP1a at transcription start sites with stalled polII, the signatures of histone code and polII level/position around the transcriptional start sites that predict both the mRNA level and functionality of genes, and the enrichment of elongating polII within exons at splicing junctions. These features, likely conserved among diverse epigenomes, reveal general strategies for chromatin modifications. PMID:22194694

  15. UPRT, a suicide-gene therapy candidate in higher eukaryotes, is required for Drosophila larval growth and normal adult lifespan

    PubMed Central

    Ghosh, Arpan C.; Shimell, MaryJane; Leof, Emma R.; Haley, Macy J.; O’Connor, Michael B.

    2015-01-01

    Uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage pathway enzyme that catalyzes the conversion of uracil to uridine monophosphate (UMP). The enzyme is highly conserved from prokaryotes to humans and yet phylogenetic evidence suggests that UPRT homologues from higher-eukaryotes, including Drosophila, are incapable of binding uracil. Purified human UPRT also do not show any enzymatic activity in vitro, making microbial UPRT an attractive candidate for anti-microbial drug development, suicide-gene therapy, and cell-specific mRNA labeling techniques. Nevertheless, the enzymatic site of UPRT remains conserved across the animal kingdom indicating an in vivo role for the enzyme. We find that the Drosophila UPRT homologue, krishah (kri), codes for an enzyme that is required for larval growth, pre-pupal/pupal viability and long-term adult lifespan. Our findings suggest that UPRT from all higher eukaryotes is likely enzymatically active in vivo and challenges the previous notion that the enzyme is non-essential in higher eukaryotes and cautions against targeting the enzyme for therapeutic purposes. Our findings also suggest that expression of the endogenous UPRT gene will likely cause background incorporation when using microbial UPRT as a cell-specific mRNA labeling reagent in higher eukaryotes. PMID:26271729

  16. A high-resolution whole-genome map of key chromatin modifications in the adult Drosophila melanogaster.

    PubMed

    Yin, Hang; Sweeney, Sarah; Raha, Debasish; Snyder, Michael; Lin, Haifan

    2011-12-01

    Epigenetic research has been focused on cell-type-specific regulation; less is known about common features of epigenetic programming shared by diverse cell types within an organism. Here, we report a modified method for chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and its use to construct a high-resolution map of the Drosophila melanogaster key histone marks, heterochromatin protein 1a (HP1a) and RNA polymerase II (polII). These factors are mapped at 50-bp resolution genome-wide and at 5-bp resolution for regulatory sequences of genes, which reveals fundamental features of chromatin modification landscape shared by major adult Drosophila cell types: the enrichment of both heterochromatic and euchromatic marks in transposons and repetitive sequences, the accumulation of HP1a at transcription start sites with stalled polII, the signatures of histone code and polII level/position around the transcriptional start sites that predict both the mRNA level and functionality of genes, and the enrichment of elongating polII within exons at splicing junctions. These features, likely conserved among diverse epigenomes, reveal general strategies for chromatin modifications.

  17. The Tolkin Gene Is a Tolloid/Bmp-1 Homologue That Is Essential for Drosophila Development

    PubMed Central

    Finelli, A. L.; Xie, T.; Bossie, C. A.; Blackman, R. K.; Padgett, R. W.

    1995-01-01

    The Drosophila decapentaplegic (dpp) gene, a member of the tranforming growth factor β superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interaction between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp. PMID:8536976

  18. Contribution of ethyl methanesulfonate vapors to the yield of mutations detected in Drosophila melanogaster when the adult feeding technique is used

    SciTech Connect

    Munoz, E.R.

    1987-01-01

    Ethyl methanesulfonate (EMS) is an alkylating agent widely used in mutation research. In experiments with adult Drosophila melanogaster, EMS is either injected or fed to the flies using different feeding methods that essentially consist of placing the flies in bottles or vials with a piece of tissue paper moistened with a sucrose solution containing the desired concentration of EMS. To determine the extent to which vapors contribute to the mutagenic effect detected in Drosophila when the feeding technique is used, 7-day-old wild-type Samarkand males were fed EMS or were exposed only to its vapors.

  19. Exploring the midgut proteome of partially fed female cattle tick (Rhipicephalus (Boophilus) microplus).

    PubMed

    Kongsuwan, Kritaya; Josh, Peter; Zhu, Ying; Pearson, Roger; Gough, Joanne; Colgrave, Michelle L

    2010-02-01

    The continued development of effective anti-tick vaccines remains the most promising prospect for the control of the cattle tick, Rhipicephalus (Boophilus) microplus. A vaccine based on midgut proteins could interfere with successful tick feeding and additionally interfere with midgut developmental stages of Babesia parasites, providing opportunities for the control of both the tick and the pathogens it transmits. Midgut proteins from partially fed adult female cattle ticks were analysed using a combination of 2-DE and gel-free LC-MS/MS. Analysis of the urea-soluble protein fraction resulted in the confident identification of 105 gut proteins, while the PBS-soluble fraction yielded an additional 37 R. microplus proteins. The results show an abundance of proteins involved in mitochondrial ATP synthesis, electron transport chain, protein synthesis, chaperone, antioxidant and protein folding and transport activities in midgut tissues of adult female ticks. Among the novel products identified were clathrin-adaptor protein, which is involved in the assembly of clathrin-coated vesicles, and membrane-associated trafficking proteins such as syntaxin 6 and surfeit 4. The observations allow the formulation of hypotheses regarding midgut physiology and will serve as a basis for future vaccine development and tick-host interaction research.

  20. Antagonism of EGFR and notch signalling in the reiterative recruitment of Drosophila adult chordotonal sense organ precursors.

    PubMed

    zur Lage, P; Jarman, A P

    1999-06-01

    The selection of Drosophila melanogaster sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. Here we show that for one large adult chordotonal SOP array, clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor-receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signalling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs.

  1. A protein related to p21-activated kinase (PAK) that is involved in neurogenesis in the Drosophila adult central nervous system.

    PubMed

    Melzig, J; Rein, K H; Schäfer, U; Pfister, H; Jäckle, H; Heisenberg, M; Raabe, T

    1998-11-05

    Brains are organized by the developmental processes generating them. The embryonic neurogenic phase of Drosophila melanogaster has been studied in detail at the genetic, cellular and molecular level. In contrast, much of what is known of postembryonic brain development has been gathered by neuroanatomical and gene expression studies. The molecular mechanisms underlying cellular diversity and structural organisation in the adult brain, such as the establishment of the correct neuroblast number, the spatial and temporal control of neuroblast proliferation, cell fate determination, and the generation of the precise pattern of neuronal connectivity, are largely unknown. In a screen for viable mutations affecting adult central brain structures, we isolated the mushroom bodies tiny (mbt) gene of Drosophila, which encodes a protein related to p21-activated kinase (PAK). We show that mutations in mbt primarily interfere with the generation or survival of the intrinsic cells (Kenyon cells) of the mushroom body, a paired neuropil structure in the adult brain involved in learning and memory.

  2. Anopheles Midgut FREP1 Mediates Plasmodium Invasion*

    PubMed Central

    Zhang, Genwei; Niu, Guodong; Franca, Caio M.; Dong, Yuemei; Wang, Xiaohong; Butler, Noah S.; Dimopoulos, George; Li, Jun

    2015-01-01

    Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae, and FREP1 is important for Plasmodium berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a blood meal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito-expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission. PMID:25991725

  3. TRAP-seq Profiling and RNAi-Based Genetic Screens Identify Conserved Glial Genes Required for Adult Drosophila Behavior

    PubMed Central

    Ng, Fanny S.; Sengupta, Sukanya; Huang, Yanmei; Yu, Amy M.; You, Samantha; Roberts, Mary A.; Iyer, Lakshmanan K.; Yang, Yongjie; Jackson, F. Rob

    2016-01-01

    Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens to identify glial factors that regulate behavior. Importantly, our studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia—different classes of glial cells—have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, 1 of the relevant genes encodes a vesicle recycling factor, 4 encode secreted proteins and 3 encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior. PMID:28066175

  4. Maintenance of Glia in the Optic Lamina Is Mediated by EGFR Signaling by Photoreceptors in Adult Drosophila

    PubMed Central

    Lee, Yuan-Ming; Sun, Y. Henry

    2015-01-01

    The late onset of neurodegeneration in humans indicates that the survival and function of cells in the nervous system must be maintained throughout adulthood. In the optic lamina of the adult Drosophila, the photoreceptor axons are surrounded by multiple types of glia. We demonstrated that the adult photoreceptors actively contribute to glia maintenance in their target field within the optic lamina. This effect is dependent on the epidermal growth factor receptor (EGFR) ligands produced by the R1-6 photoreceptors and transported to the optic lamina to act on EGFR in the lamina glia. EGFR signaling is necessary and sufficient to act in a cell-autonomous manner in the lamina glia. Our results suggest that EGFR signaling is required for the trafficking of the autophagosome/endosome to the lysosome. The loss of EGFR signaling results in cell degeneration most likely because of the accumulation of autophagosomes. Our findings provide in vivo evidence for the role of adult neurons in the maintenance of glia and a novel role for EGFR signaling in the autophagic flux. PMID:25909451

  5. Timing of Expression of a Gene in the Adult Drosophila Is Regulated by Mechanisms Independent of Temperature and Metabolic Rate

    PubMed Central

    Rogina, B.; Helfand, S. L.

    1996-01-01

    The examination of β-galactosidase (β-gal) expression in the third segment of the antenna of the 2216 enhancer trap line in Drosophila melanogaster reveals two distinct spatial and temporal regulatory patterns of expression during adult life. Type I expression is characterized by a decline in the level of β-gal expression with increasing age. Starting from a maximal level of expression at the time of adult emergence, there is a decrease in the number of cells that express β-gal so that by 40-50 days of adult life few cells express β-gal. Varying the ambient temperature and using hyperactivity mutants (Hyperkinetic(1), Shaker(5)) demonstrates that the rate of this decline is independent of temperature and metabolic rate. Type II expression is distinctly different in spatial distribution and temporal regulation from the first pattern. Type II expression is restricted in the antenna to a small (<20-30) set of cells whose level of expression changes in a periodic manner with time. The regulation of this periodicity appears to be linked to ambient temperature. PMID:8844152

  6. Differential Toxicity of Carbon Nanomaterials in Drosophila: Larval Dietary Uptake Is Benign, but Adult Exposure Causes Locomotor Impairment and Mortality

    PubMed Central

    LIU, XINYUAN; VINSON, DANIEL; ABT, DAWN; HURT, ROBERT H.; RAND, DAVID M.

    2011-01-01

    Rapid growth in nanomaterial manufacturing is raising concerns about potential adverse effects on the environment. Nanoparticle contact with intact organisms in the wild may lead to different biological responses than those observed in laboratory cell-based toxicity assays. In nature, the scale and chemistry of nanoparticles coupled with the surface properties, texture, and behaviors of the organisms will influence biologically significant exposure and ultimate toxicity. We used larval and adult Drosophila melanogaster to study the effects of carbon nanomaterial exposure under several different scenarios. Dietary uptake of fullerene C60, carbon black (CB), or single-walled or multiwalled nanotubes (SWNTs, MWNTs) delivered through the food to the larval stage had no detectable effect on egg to adult survivorship, despite evidence that the nanomaterials are taken up and become sequestered in tissue. However, when these same nanocarbons were exposed in dry form to adults, some materials (CB, SWNTs) adhered extensively to fly surfaces, overwhelmed natural grooming mechanisms, and led to impaired locomotor function and mortality. Others (C60, MWNT arrays) adhered weakly, could be removed by grooming, and did not reduce locomotor function or survivorship. Evidence is presented that these differences are primarily due to differences in nanomaterial superstructure, or aggregation state, and that the combination of adhesion and grooming can lead to active fly borne nanoparticle transport. PMID:19746737

  7. A gain-of-function screen to identify genes that reduce lifespan in the adult of Drosophila melanogaster

    PubMed Central

    2014-01-01

    Background Several lines of evidence associate misregulated genetic expression with risk factors for diabetes, Alzheimer’s, and other diseases that sporadically develop in healthy adults with no background of hereditary disorders. Thus, we are interested in genes that may be expressed normally through parts of an individual’s life, but can cause physiological defects and disease when misexpressed in adulthood. Results We attempted to identify these genes in a model organism by arbitrarily misexpressing specific genes in adult Drosophila melanogaster, using 14,133 Gene Search lines. We identified 39 “reduced-lifespan genes” that, when misexpressed in adulthood, shortened the flies’ lifespan to less than 30% of that of control flies. About half of these genes have human orthologs that are known to be involved in human diseases. For about one-fourth of the reduced-lifespan genes, suppressing apoptosis restored the lifespan shortened by their misexpression. We determined the organs responsible for reduced lifespan when these genes were misexpressed specifically in adulthood, and found that while some genes induced reduced lifespan only when misexpressed in specific adult organs, others could induce reduced lifespan when misexpressed in various organs. This finding suggests that tissue-specific dysfunction may be involved in reduced lifespan related to gene misexpression. Gene ontology analysis showed that reduced-lifespan genes are biased toward genes related to development. Conclusions We identified 39 genes that, when misexpressed in adulthood, shortened the lifespan of adult flies. Suppressing apoptosis rescued this shortened lifespan for only a subset of the reduced-lifespan genes. The adult tissues in which gene misexpression caused early death differed among the reduced-lifespan genes. These results suggest that the cause of reduced lifespan upon misexpression differed among the genes. PMID:24739137

  8. Postembryonic lineages of the Drosophila ventral nervous system: Neuroglian expression reveals the adult hemilineage associated fiber tracts in the adult thoracic neuromeres

    PubMed Central

    Harris, Robin; Williams, Darren W.; Truman, James W.

    2016-01-01

    During larval life most of the thoracic neuroblasts (NBs) in Drosophila undergo a second phase of neurogenesis to generate adult‐specific neurons that remain in an immature, developmentally stalled state until pupation. Using a combination of MARCM and immunostaining with a neurotactin antibody, Truman et al. (2004; Development 131:5167–5184) identified 24 adult‐specific NB lineages within each thoracic hemineuromere of the larval ventral nervous system (VNS), but because of the neurotactin labeling of lineage tracts disappearing early in metamorphosis, they were unable extend the identification of these lineages into the adult. Here we show that immunostaining with an antibody against the cell adhesion molecule neuroglian reveals the same larval secondary lineage projections through metamorphosis and bfy identifying each neuroglian‐positive tract at selected stages we have traced the larval hemilineage tracts for all three thoracic neuromeres through metamorphosis into the adult. To validate tract identifications we used the genetic toolkit developed by Harris et al. (2015; Elife 4) to preserve hemilineage‐specific GAL4 expression patterns from larval into the adult stage. The immortalized expression proved a powerful confirmation of the analysis of the neuroglian scaffold. This work has enabled us to directly link the secondary, larval NB lineages to their adult counterparts. The data provide an anatomical framework that 1) makes it possible to assign most neurons to their parent lineage and 2) allows more precise definitions of the neuronal organization of the adult VNS based in developmental units/rules. J. Comp. Neurol. 524:2677–2695, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26878258

  9. Transient adult microbiota, gut homeostasis and longevity: novel insights from the Drosophila model.

    PubMed

    Erkosar, Berra; Leulier, François

    2014-11-17

    In the last decade, Drosophila has emerged as a useful model to study host-microbiota interactions, creating an active research field with prolific publications. In the last 2 years, several studies contributed to a better understanding of the dynamic nature of microbiota composition and its impact on gut immunity and intestinal tissue homeostasis. These studies depicted the mechanisms by which microbiota regulates gut homeostasis to modulate host fitness and lifespan. Moreover, the latest findings demonstrating that the gut is a physiologically and histologically compartmentalized organ brought fresh perspectives to study the region-specific nature of the interactions between the commensal microbes and the intestinal tissue, and consequences of these interactions on overall host biology.

  10. Inhibition of GSK-3 Ameliorates Aβ Pathology in an Adult-Onset Drosophila Model of Alzheimer's Disease

    PubMed Central

    Killick, Richard; Augustin, Hrvoje; Gandy, Carina; Allen, Marcus J.; Hardy, John; Lovestone, Simon; Partridge, Linda

    2010-01-01

    Aβ peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimer's disease (AD), with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3) is increasingly implicated as playing a pivotal role in this amyloid cascade. We have developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Aβ42 specifically in adult neurons, to avoid developmental effects. Aβ42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Aβ42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment) rescued Aβ42 toxicity. Aβ42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Aβ42. The GSK-3–mediated effects on Aβ42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Aβ42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Aβ42 peptide level, in the absence of APP processing. Our study points to the need both to identify the mechanisms by which GSK-3 modulates Aβ42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD. PMID:20824130

  11. Intestinal obstruction from midgut volvulus after laparoscopic appendectomy.

    PubMed

    Cuadra, S A; Khalife, M E; Char, D J; Wax, M R; Halpern, D

    2002-01-01

    We present the case of a 30-year-old man who developed a small bowel obstruction from an acute midgut volvulus 8 days after undergoing a laparoscopic appendectomy. There was no evidence of congenital malrotation or midgut volvulus on the initial computed tomography (CT) scan or at laparoscopy. Subsequently, a midgut volvulus developed in the absence of congenital malrotation.

  12. RNA-seq analyses of the midgut from blood- and serum-fed Ixodes ricinus ticks

    PubMed Central

    Perner, Jan; Provazník, Jan; Schrenková, Jana; Urbanová, Veronika; Ribeiro, José M. C.; Kopáček, Petr

    2016-01-01

    Adult females of the genus Ixodes imbibe blood meals exceeding about 100 times their own weight within 7‒9 days. During this period, ticks internalise components of host blood by endocytic digest cells that line the tick midgut epithelium. Using RNA-seq, we aimed to characterise the midgut transcriptome composition in adult Ixodes ricinus females during early and late phase of engorgement. To address specific adaptations to the haemoglobin-rich diet, we compared the midgut transcriptomes of genetically homogenous female siblings fed either bovine blood or haemoglobin-depleted serum. We noted that tick gut transcriptomes are subject to substantial temporal-dependent expression changes between day 3 and day 8 of feeding. In contrast, the number of transcripts significantly affected by the presence or absence of host red blood cells was low. Transcripts relevant to the processes associated with blood-meal digestion were analysed and involvement of selected encoded proteins in the tick midgut physiology discussed. A total of 7215 novel sequences from I. ricinus were deposited in public databases as an additional outcome of this study. Our results broaden the current knowledge of tick digestive system and may lead to the discovery of potential molecular targets for efficient tick control. PMID:27824139

  13. Brain-midgut short neuropeptide F mechanism that inhibits digestive activity of the American cockroach, Periplaneta americana upon starvation.

    PubMed

    Mikani, Azam; Wang, Qiu-Shi; Takeda, Makio

    2012-03-01

    Immunohistochemical reactivity against short neuropeptide F (sNPF) was observed in the brain-corpus cardiacum and midgut paraneurons of the American cockroach, Periplaneta americana. Four weeks of starvation increased the number of sNPF-ir cells in the midgut epithelium but the refeeding decreased the number in 3h. Dramatic rises in sNPF contents in the midgut epithelium and hemolymph of roaches starved for 4 weeks were confirmed by ELISA. Starvation for 4 weeks reduced α-amylase, protease and lipase activities in the midgut of P. americana but refeeding restored these to high levels. Co-incubation of dissected midgut with sNPF at physiological concentrations inhibited α-amylase, protease and lipase activities. sNPF injection into the hemocoel led to a decrease in α-amylase, protease and lipase activities, whereas PBS injection had no effects. The injection of d-(+)-trehalose and l-proline into the hemocoel of decapitated adult male cockroaches that had been starved for 4 weeks had no effect on these digestive enzymes. However, injection into the hemocoel of head-intact starved cockroaches stimulated digestive activity. Injection of d-(+)-trehalose and l-proline into the lumen of decapitated cockroaches that had been starved for 4 weeks increased enzymes activities and suppressed sNPF in the midgut. Our data indicate that sNPF from the midgut paraneurons suppresses α-amylase, protease and lipase activities during starvation. Injection of d-(+)-trehalose/l-proline into the hemocoel of head-intact starved cockroach decreased the hemolymph sNPF content, which suggests that sNPF could be one of the brain factors, demonstrating brain-midgut interplay in the regulation of digestive activities and possibly nutrition-associated behavioral modifications.

  14. Discovery of midgut genes for the RNA interference control of corn rootworm

    PubMed Central

    Hu, Xu; Richtman, Nina M.; Zhao, Jian-Zhou; Duncan, Keith E.; Niu, Xiping; Procyk, Lisa A.; Oneal, Meghan A.; Kernodle, Bliss M.; Steimel, Joseph P.; Crane, Virginia C.; Sandahl, Gary; Ritland, Julie L.; Howard, Richard J.; Presnail, James K.; Lu, Albert L.; Wu, Gusui

    2016-01-01

    RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by “blebbing” of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality. PMID:27464714

  15. Midgut and salivary gland transcriptomes of the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae).

    PubMed

    Campbell, C L; Vandyke, K A; Letchworth, G J; Drolet, B S; Hanekamp, T; Wilson, W C

    2005-04-01

    Numerous Culicoides spp. are important vectors of livestock or human disease pathogens. Transcriptome information from midguts and salivary glands of adult female Culicoides sonorensis provides new insight into vector biology. Of 1719 expressed sequence tags (ESTs) from adult serum-fed female midguts harvested within 5 h of feeding, twenty-eight clusters of serine proteases were derived. Four clusters encode putative iron binding proteins (FER1, FERL, PXDL1, PXDL2), and two clusters encode metalloendopeptidases (MDP6C, MDP6D) that probably function in bloodmeal catabolism. In addition, a diverse variety of housekeeping cDNAs were identified. Selected midgut protease transcripts were analysed by quantitative real-time PCR (q-PCR): TRY1_115 and MDP6C mRNAs were induced in adult female midguts upon feeding, whereas TRY1_156 and CHYM1 were abundant in midguts both before and immediately after feeding. Of 708 salivary gland ESTs analysed, clusters representing two new classes of protein families were identified: a new class of D7 proteins and a new class of Kunitz-type protease inhibitors. Additional cDNAs representing putative immunomodulatory proteins were also identified: 5' nucleotidases, antigen 5-related proteins, a hyaluronidase, a platelet-activating factor acetylhydrolase, mucins and several immune response cDNAs. Analysis by q-PCR showed that all D7 and Kunitz domain transcripts tested were highly enriched in female heads compared with other tissues and were generally absent from males. The mRNAs of two additional protease inhibitors, TFPI1 and TFPI2, were detected in salivary glands of paraffin-embedded females by in situ hybridization.

  16. Assessing Basal and Acute Autophagic Responses in the Adult Drosophila Nervous System: The Impact of Gender, Genetics and Diet on Endogenous Pathway Profiles

    PubMed Central

    Molina, Brandon; Mauntz, Ruth E.; Gonzalez, Arysa; Barekat, Ayeh; El-Mecharrafie, Nadja; Garza, Shannon; Gurney, Michael A.; Achal, Madhulika; Linton, Phyllis-Jean; Harris, Greg L.; Finley, Kim D.

    2016-01-01

    The autophagy pathway is critical for the long-term homeostasis of cells and adult organisms and is often activated during periods of stress. Reduced pathway efficacy plays a central role in several progressive neurological disorders that are associated with the accumulation of cytotoxic peptides and protein aggregates. Previous studies have shown that genetic and transgenic alterations to the autophagy pathway impacts longevity and neural aggregate profiles of adult Drosophila. In this study, we have identified methods to measure the acute in vivo induction of the autophagy pathway in the adult fly CNS. Our findings indicate that the genotype, age, and gender of adult flies can influence pathway responses. Further, we demonstrate that middle-aged male flies exposed to intermittent fasting (IF) had improved neuronal autophagic profiles. IF-treated flies also had lower neural aggregate profiles, maintained more youthful behaviors and longer lifespans, when compared to ad libitum controls. In summary, we present methodology to detect dynamic in vivo changes that occur to the autophagic profiles in the adult Drosophila CNS and that a novel IF-treatment protocol improves pathway response in the aging nervous system. PMID:27711219

  17. Genetic correlation between the pre-adult developmental period and locomotor activity rhythm in Drosophila melanogaster.

    PubMed

    Takahashi, K H; Teramura, K; Muraoka, S; Okada, Y; Miyatake, T

    2013-04-01

    Biological clocks regulate various behavioural and physiological traits; slower circadian clocks are expected to slow down the development, suggesting a potential genetic correlation between the developmental period and circadian rhythm. However, a correlation between natural genetic variations in the developmental period and circadian rhythm has only been found in Bactrocera cucurbitae. The number of genetic factors that contribute to this genetic correlation is largely unclear. In this study, to examine whether natural genetic variations in the developmental period and circadian rhythm are correlated in Drosophila melanogaster, we performed an artificial disruptive selection on the developmental periods using wild-type strains and evaluated the circadian rhythms of the selected lines. To investigate whether multiple genetic factors mediate the genetic correlation, we reanalyzed previously published genome-wide deficiency screening data based on DrosDel isogenic deficiency strains and evaluated the effect of 438 genomic deficiencies on the developmental periods. We then randomly selected 32 genomic deficiencies with significant effects on the developmental periods and tested their effects on circadian rhythms. As a result, we found a significant response to selection for longer developmental periods and their correlated effects on circadian rhythms of the selected lines. We also found that 18 genomic regions had significant effects on the developmental periods and circadian rhythms, indicating their potential for mediating the genetic correlation between the developmental period and circadian rhythm. The novel findings of our study might lead to a better understanding of how this correlation is regulated genetically in broader taxonomic groups.

  18. White - cGMP Interaction Promotes Fast Locomotor Recovery from Anoxia in Adult Drosophila

    PubMed Central

    2017-01-01

    Increasing evidence indicates that the white (w) gene in Drosophila possesses extra-retinal functions in addition to its classical role in eye pigmentation. We have previously shown that w+ promotes fast and consistent locomotor recovery from anoxia, but how w+ modulates locomotor recovery is largely unknown. Here we show that in the absence of w+, several PDE mutants, especially cyclic guanosine monophosphate (cGMP)-specific PDE mutants, display wildtype-like fast locomotor recovery from anoxia, and that during the night time, locomotor recovery was light-sensitive in white-eyed mutant w1118, and light-insensitive in PDE mutants under w1118 background. Data indicate the involvement of cGMP in the modulation of recovery timing and presumably, light-evoked cGMP fluctuation is associated with light sensitivity of locomotor recovery. This was further supported by the observations that w-RNAi-induced delay of locomotor recovery was completely eliminated by upregulation of cGMP through multiple approaches, including PDE mutation, simultaneous overexpression of an atypical soluble guanylyl cyclase Gyc88E, or sildenafil feeding. Lastly, prolonged sildenafil feeding promoted fast locomotor recovery from anoxia in w1118. Taken together, these data suggest that a White-cGMP interaction modulates the timing of locomotor recovery from anoxia. PMID:28060942

  19. The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults

    PubMed Central

    Barry, William E; Thummel, Carl S

    2016-01-01

    Although mutations in HNF4A were identified as the cause of Maturity Onset Diabetes of the Young 1 (MODY1) two decades ago, the mechanisms by which this nuclear receptor regulates glucose homeostasis remain unclear. Here we report that loss of Drosophila HNF4 recapitulates hallmark symptoms of MODY1, including adult-onset hyperglycemia, glucose intolerance and impaired glucose-stimulated insulin secretion (GSIS). These defects are linked to a role for dHNF4 in promoting mitochondrial function as well as the expression of Hex-C, a homolog of the MODY2 gene Glucokinase. dHNF4 is required in the fat body and insulin-producing cells to maintain glucose homeostasis by supporting a developmental switch toward oxidative phosphorylation and GSIS at the transition to adulthood. These findings establish an animal model for MODY1 and define a developmental reprogramming of metabolism to support the energetic needs of the mature animal. DOI: http://dx.doi.org/10.7554/eLife.11183.001 PMID:27185732

  20. Lamellipodia-based migrations of larval epithelial cells are required for normal closure of the adult epidermis of Drosophila

    PubMed Central

    Bischoff, Marcus

    2012-01-01

    Cell migrations are an important feature of animal development. They are, furthermore, essential to wound healing and tumour progression. Despite recent progress, it is still mysterious how cell migration is spatially and temporally regulated during morphogenesis and how cell migration is coordinated with other cellular behaviours to shape tissues and organs. The formation of the abdominal epithelium of Drosophila during metamorphosis provides an attractive system to study morphogenesis. Here, the diploid adult histoblasts replace the polyploid larval epithelial cells (LECs). Using in vivo 4D microscopy, I show that, besides apical constriction and apoptosis, the LECs undergo extensive coordinated migrations. The migrations follow a transition from a stationary (epithelial) to a migratory mode. The migratory behaviour is stimulated by autocrine Dpp signalling. Directed apical lamellipodia-like protrusions propel the cells. Initially, planar cell polarity determines the orientation of LEC migration. While LECs are migrating they also constrict apically, and changes in activity of the small GTPase Rho1 can favour one behaviour over the other. This study shows that the LECs play a more active role in morphogenesis than previously thought, with their migrations contributing to abdominal closure. It furthermore provides insights into how the migratory behaviour of cells is regulated during morphogenesis. PMID:22230614

  1. Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells

    PubMed Central

    Ramirez, Juanma; Martinez, Aitor; Lectez, Benoit; Lee, So Young; Franco, Maribel; Barrio, Rosa; Dittmar, Gunnar; Mayor, Ugo

    2015-01-01

    Background Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. Methodology/Principal Findings Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. Conclusions/Significance These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system. PMID:26460970

  2. Seasonality and Locality Affect the Diversity of Anopheles gambiae and Anopheles coluzzii Midgut Microbiota from Ghana

    PubMed Central

    Gendrin, Mathilde; Pels, Nana Adjoa P.; Yeboah-Manu, Dorothy; Christophides, George K.; Wilson, Michael D.

    2016-01-01

    Symbiotic bacteria can have important implications in the development and competence of disease vectors. In Anopheles mosquitoes, the composition of the midgut microbiota is largely influenced by the larval breeding site, but the exact factors shaping this composition are currently unknown. Here, we examined whether the proximity to urban areas and seasons have an impact on the midgut microbial community of the two major malaria vectors in Africa, An. coluzzii and An. gambiae. Larvae and pupae were collected from selected habitats in two districts of Ghana during the dry and rainy season periods. The midgut microbiota of adults that emerged from these collections was determined by 454-pyrosequencing of the 16S ribosomal DNA. We show that in both mosquito species, Shewanellaceae constituted on average of 54% and 73% of the midgut microbiota from each site in the dry and rainy season, respectively. Enterobacteriaceae was found in comparatively low abundance below 1% in 22/30 samples in the dry season, and in 25/38 samples in the rainy season. Our data indicate that seasonality and locality significantly affect both the diversity of microbiota and the relative abundance of bacterial families with a positive impact of dry season and peri-urban settings. PMID:27322614

  3. Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila

    PubMed Central

    Auer, Jasmin S.; Nagel, Anja C.; Schulz, Adriana; Wahl, Vanessa; Preiss, Anette

    2015-01-01

    In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H)MAPK-ko and a phospho-mimetic Su(H)MAPK-ac isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vgBE-lacZ). In general, Su(H)MAPK-ko induced a stronger response than wild-type Su(H), whereas the response to Su(H)MAPK-ac was very weak. Notch target genes cut, wingless and vgBE-lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DERact) or the MAPK (rlSEM) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones. PMID:26702412

  4. Adult Drosophila melanogaster evolved for antibacterial defense invest in infection-induced expression of both humoral and cellular immunity genes

    PubMed Central

    2011-01-01

    Background While the transcription of innate immunity genes in response to bacterial infection has been well-characterised in the Drosophila model, we recently demonstrated the capacity for such transcription to evolve in flies selected for improved antibacterial defense. Here we use this experimental system to examine how insects invest in constitutive versus infection-induced transcription of immunity genes. These two strategies carry with them different consequences with respect to energetic and pleiotropic costs and may be more or less effective in improving defense depending on whether the genes contribute to humoral or cellular aspects of immunity. Findings Contrary to expectation we show that selection preferentially increased the infection-induced expression of both cellular and humoral immunity genes. Given their functional roles, infection induced increases in expression were expected for the humoral genes, while increases in constitutive expression were expected for the cellular genes. We also report a restricted ability to improve transcription of immunity genes that is on the order of 2-3 fold regardless of total transcription level of the gene. Conclusions The evolved increases in infection-induced expression of the cellular genes may result from specific cross talk with humoral pathways or from generalised strategies for enhancing immunity gene transcription. A failure to see improvements in constitutive expression of the cellular genes suggests either that increases might come at too great a cost or that patterns of expression in adults are decoupled from the larval phase where increases would be most effective. The similarity in fold change increase across all immunity genes may suggest a shared mechanism for the evolution of increased transcription in small, discrete units such as duplication of cis-regulatory elements. PMID:21859495

  5. Enhancer of polycomb coordinates multiple signaling pathways to promote both cyst and germline stem cell differentiation in the Drosophila adult testis.

    PubMed

    Feng, Lijuan; Shi, Zhen; Chen, Xin

    2017-02-01

    Stem cells reside in a particular microenvironment known as a niche. The interaction between extrinsic cues originating from the niche and intrinsic factors in stem cells determines their identity and activity. Maintenance of stem cell identity and stem cell self-renewal are known to be controlled by chromatin factors. Herein, we use the Drosophila adult testis which has two adult stem cell lineages, the germline stem cell (GSC) lineage and the cyst stem cell (CySC) lineage, to study how chromatin factors regulate stem cell differentiation. We find that the chromatin factor Enhancer of Polycomb [E(Pc)] acts in the CySC lineage to negatively control transcription of genes associated with multiple signaling pathways, including JAK-STAT and EGF, to promote cellular differentiation in the CySC lineage. E(Pc) also has a non-cell-autonomous role in regulating GSC lineage differentiation. When E(Pc) is specifically inactivated in the CySC lineage, defects occur in both germ cell differentiation and maintenance of germline identity. Furthermore, compromising Tip60 histone acetyltransferase activity in the CySC lineage recapitulates loss-of-function phenotypes of E(Pc), suggesting that Tip60 and E(Pc) act together, consistent with published biochemical data. In summary, our results demonstrate that E(Pc) plays a central role in coordinating differentiation between the two adult stem cell lineages in Drosophila testes.

  6. Enhancer of polycomb coordinates multiple signaling pathways to promote both cyst and germline stem cell differentiation in the Drosophila adult testis

    PubMed Central

    Feng, Lijuan; Shi, Zhen; Chen, Xin

    2017-01-01

    Stem cells reside in a particular microenvironment known as a niche. The interaction between extrinsic cues originating from the niche and intrinsic factors in stem cells determines their identity and activity. Maintenance of stem cell identity and stem cell self-renewal are known to be controlled by chromatin factors. Herein, we use the Drosophila adult testis which has two adult stem cell lineages, the germline stem cell (GSC) lineage and the cyst stem cell (CySC) lineage, to study how chromatin factors regulate stem cell differentiation. We find that the chromatin factor Enhancer of Polycomb [E(Pc)] acts in the CySC lineage to negatively control transcription of genes associated with multiple signaling pathways, including JAK-STAT and EGF, to promote cellular differentiation in the CySC lineage. E(Pc) also has a non-cell-autonomous role in regulating GSC lineage differentiation. When E(Pc) is specifically inactivated in the CySC lineage, defects occur in both germ cell differentiation and maintenance of germline identity. Furthermore, compromising Tip60 histone acetyltransferase activity in the CySC lineage recapitulates loss-of-function phenotypes of E(Pc), suggesting that Tip60 and E(Pc) act together, consistent with published biochemical data. In summary, our results demonstrate that E(Pc) plays a central role in coordinating differentiation between the two adult stem cell lineages in Drosophila testes. PMID:28196077

  7. Implications for the functions of the four known midgut differentiation factors: An immunohistologic study of Heliothis virescens midgut.

    PubMed

    Loeb, Marcia J; Coronel, Nicholas; Natsukawa, Dai; Takeda, Makio

    2004-05-01

    Antibodies to the peptides that induce differentiation of midgut larval stem cells, the midgut differentiating factors MDF-2, MDF-3, and MDF-4, bind to columnar cells in midgut cultures and in intact midgut of Heliothis virescens, in manners similar to the binding of anti- MDF-1 to those tissues. Antibodies to MDF-2 and MDF-3 also stained droplets in the midgut lumen, suggesting that columnar cells may also release MDF-2- and MDF-3-like cytokines to the lumen. Antibody to MDF-4 exhibited similar staining patterns but also recognized stem and differentiating cells, the presumed targets of peptides that regulate stem cell differentiation. Antibody to MDF-4 also bound to one type of endocrine cell in midgut cultures and in sections of midgut, as well as to the endocrine secretion released both to the midgut lumen and the hemolymph. Antibodies to the MDFs 1, 2, and 3, incubated with cultures of midgut cells, did not appear to prevent differentiation of the stem cells in the cultures but affected viability of mature cells, reflected in increased apoptosis and doubling of the number of differentiating cells compared to controls. Only antibody to MDF-4 induced temporary necrosis and inhibition of population recovery, indicating that MDF4 may be the true differentiation factor. The other MDFs may have additional functions beyond regulation of midgut stem cell differentiation in vivo.

  8. Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, Aedes albopictus from Tezpur, India.

    PubMed

    Yadav, Kamlesh K; Datta, Sibnarayan; Naglot, Ashok; Bora, Ajitabh; Hmuaka, Vanlal; Bhagyawant, Sameer; Gogoi, Hemanta K; Veer, Vijay; Raju, P Srinivas

    2016-01-01

    Aedes aegypti and Ae. albopictus are among the most important vectors of arboviral diseases, worldwide. Recent studies indicate that diverse midgut microbiota of mosquitoes significantly affect development, digestion, metabolism, and immunity of their hosts. Midgut microbiota has also been suggested to modulate the competency of mosquitoes to transmit arboviruses, malaria parasites etc. Interestingly, the midgut microbial flora is dynamic and the diversity changes with the development of vectors, in addition to other factors such as species, sex, life-stage, feeding behavior and geographical origin. The aim of the present study was to investigate the midgut bacterial diversity among larva, adult male, sugar fed female and blood fed female Ae. albopictus collected from Tezpur, Northeastern India. Based on colony morphological characteristics, we selected 113 cultivable bacterial isolates for 16S rRNA gene sequence based molecular identification. Of the 113 isolates, we could identify 35 bacterial species belonging to 18 distinct genera under four major phyla, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Phyla Proteobacteria and Firmicutes accounted for majority (80%) of the species, while phylum Actinobacteria constituted 17% of the species. Bacteroidetes was the least represented phylum, characterized by a single species- Chryseobacterium rhizoplanae, isolated from blood fed individuals. Dissection of midgut microbiota diversity at different developmental stages of Ae. albopictus will be helpful in better understanding mosquito-borne diseases, and for designing effective strategies to manage mosquito-borne diseases.

  9. Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, Aedes albopictus from Tezpur, India

    PubMed Central

    Datta, Sibnarayan; Naglot, Ashok; Bora, Ajitabh; Hmuaka, Vanlal; Bhagyawant, Sameer; Gogoi, Hemanta K.; Veer, Vijay; Raju, P. Srinivas

    2016-01-01

    Aedes aegypti and Ae. albopictus are among the most important vectors of arboviral diseases, worldwide. Recent studies indicate that diverse midgut microbiota of mosquitoes significantly affect development, digestion, metabolism, and immunity of their hosts. Midgut microbiota has also been suggested to modulate the competency of mosquitoes to transmit arboviruses, malaria parasites etc. Interestingly, the midgut microbial flora is dynamic and the diversity changes with the development of vectors, in addition to other factors such as species, sex, life-stage, feeding behavior and geographical origin. The aim of the present study was to investigate the midgut bacterial diversity among larva, adult male, sugar fed female and blood fed female Ae. albopictus collected from Tezpur, Northeastern India. Based on colony morphological characteristics, we selected 113 cultivable bacterial isolates for 16S rRNA gene sequence based molecular identification. Of the 113 isolates, we could identify 35 bacterial species belonging to 18 distinct genera under four major phyla, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Phyla Proteobacteria and Firmicutes accounted for majority (80%) of the species, while phylum Actinobacteria constituted 17% of the species. Bacteroidetes was the least represented phylum, characterized by a single species- Chryseobacterium rhizoplanae, isolated from blood fed individuals. Dissection of midgut microbiota diversity at different developmental stages of Ae. albopictus will be helpful in better understanding mosquito-borne diseases, and for designing effective strategies to manage mosquito-borne diseases. PMID:27941985

  10. Cytotoxic effects of neem oil in the midgut of the predator Ceraeochrysa claveri.

    PubMed

    Scudeler, Elton Luiz; Garcia, Ana Silvia Gimenes; Padovani, Carlos Roberto; Pinheiro, Patricia Fernanda Felipe; dos Santos, Daniela Carvalho

    2016-01-01

    Studies of morphological and ultrastructural alterations in target organs have been useful for evaluating the sublethal effects of biopesticides regarded as safe for non-target organisms in ecotoxicological analyses. One of the most widely used biopesticides is neem oil, and its safety and compatibility with natural enemies have been further clarified through bioassays performed to analyze the effects of indirect exposure by the intake of poisoned prey. Thus, this study examined the cellular response of midgut epithelial cells of the adult lacewing, Ceraeochrysa claveri, to neem oil exposure via intake of neem oil-contaminated prey during the larval stage. C. claveri larvae were fed Diatraea saccharalis eggs treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval stage. The adult females obtained from these treatments were used at two ages (newly emerged and at the start of oviposition) in morphological and ultrastructural analyses. Neem oil was found to cause pronounced cytotoxic effects in the adult midgut, such as cell dilation, emission of cytoplasmic protrusions, cell lysis, loss of integrity of the cell cortex, dilation of cisternae of the rough endoplasmic reticulum, swollen mitochondria, vesiculated appearance of the Golgi complex and dilated invaginations of the basal labyrinth. Epithelial cells responded to those injuries with various cytoprotective and detoxification mechanisms, including increases in cell proliferation, the number of calcium-containing cytoplasmic granules, and HSP 70 expression, autophagic processes and the development of smooth endoplasmic reticulum, but these mechanisms were insufficient for recovery from all of the cellular damage to the midgut. This study demonstrates that neem oil exposure impairs the midgut by causing sublethal effects that may affect the physiological functions of this organ, indicating the importance of studies of different life stages of this species and similar species to evaluate the

  11. Hormonal activation of let-7-C microRNAs via EcR is required for adult Drosophila melanogaster morphology and function

    PubMed Central

    Chawla, Geetanjali; Sokol, Nicholas S.

    2012-01-01

    Steroid hormones and their nuclear receptors drive developmental transitions in diverse organisms, including mammals. In this study, we show that the Drosophila steroid hormone 20-hydroxyecdysone (20E) and its nuclear receptor directly activate transcription of the evolutionarily conserved let-7-complex (let-7-C) locus, which encodes the co-transcribed microRNAs miR-100, let-7 and miR-125. These small RNAs post-transcriptionally regulate the expression of target genes, and are required for the remodeling of the Drosophila neuromusculature during the larval-to-adult transition. Deletion of three 20E responsive elements located in the let-7-C locus results in reduced levels of let-7-C microRNAs, leading to neuromuscular and behavioral defects in adults. Given the evolutionary conservation of let-7-C microRNA sequences and temporal expression profiles, these findings indicate that steroid hormone-coupled control of let-7-C microRNAs is part of an ancestral pathway controlling the transition from larval-to-reproductive animal forms. PMID:22510985

  12. Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila.

    PubMed

    Armstrong, Alissa R; Laws, Kaitlin M; Drummond-Barbosa, Daniela

    2014-12-01

    How adipocytes contribute to the physiological control of stem cells is a critical question towards understanding the link between obesity and multiple diseases, including cancers. Previous studies have revealed that adult stem cells are influenced by whole-body physiology through multiple diet-dependent factors. For example, nutrient-dependent pathways acting within the Drosophila ovary control the number and proliferation of germline stem cells (GSCs). The potential role of nutrient sensing by adipocytes in modulating stem cells in other organs, however, remains largely unexplored. Here, we report that amino acid sensing by adult adipocytes specifically modulates the maintenance of GSCs through a Target of Rapamycin-independent mechanism. Instead, reduced amino acid levels and the consequent increase in uncoupled tRNAs trigger activation of the GCN2-dependent amino acid response pathway within adipocytes, causing increased rates of GSC loss. These studies reveal a new step in adipocyte-stem cell crosstalk.

  13. Heat-stress survival in the pre-adult stage of the life cycle in an intercontinental set of recombinant inbred lines of Drosophila melanogaster.

    PubMed

    Sambucetti, P; Scannapieco, A C; Loeschcke, V; Norry, F M

    2013-08-01

    In insects, pre-adult stages of the life cycle are exposed to variation in temperature that may differ from that in adults. However, the genetic basis for adaptation to environmental temperature could be similar between the pre-adult and the adult stages of the life cycle. Here, we tested quantitative trait loci (QTL) for heat-stress survival in larvae of Drosophila melanogaster, with and without a mild-heat-stress pre-treatment. Two sets of recombinant inbred lines derived from lines artificially selected for high and low levels of knockdown resistance to high temperature in young flies were used as the mapping population. There was no apparent increase in heat-shock survival between heat-pretreated and non-pretreated larvae. There was a positive correlation between the two experimental conditions of heat-shock survival (with and without a heat pre-treatment) except for males from one set of lines. Several QTL were identified involving all three major chromosomes. Most QTL for larval thermotolerance overlapped with thermotolerance QTL identified in previous studies for adults, indicating that heat-stress resistance is not genetically independent between life cycle stages because of either linkage or pleiotropy. The sign of the effects of some QTL alleles differed both between the sexes and between life stages.

  14. Bacterial Communities and Midgut Microbiota Associated with Mosquito Populations from Waste Tires in East-Central Illinois.

    PubMed

    Kim, Chang-Hyun; Lampman, Richard L; Muturi, Ephantus J

    2015-01-01

    Mosquito-microbe interactions tend to influence larval nutrition, immunity, and development, as well as fitness and vectorial capacity of adults. Understanding the role of different bacterial species not only improves our knowledge of the physiological and ecological consequences of these interactions, but also provides the basis for developing novel strategies for controlling mosquito-borne diseases. We used culture-dependent and culture-independent techniques to characterize the bacterial composition and abundance in water and midgut samples of larval and adult females of Aedes japonicus (Theobald), Aedes triseriatus (Say), and Culex restuans (Theobald) collected from waste tires at two wooded study sites in Urbana, IL. The phylum-specific real-time quantitative polymerase chain reaction assay revealed a higher proportion of Actinobacteria and a lower proportion of gamma-Proteobacteria and Bacteroidetes in water samples and larval midguts compared to adult female midguts. Only 15 of the 57 bacterial species isolated in this study occurred in both study sites. The number of bacterial species was highest in water samples (28 species from Trelease Woods; 25 species from South Farms), intermediate in larval midguts (13 species from Ae. japonicus; 12 species from Ae. triseriatus; 8 species from Cx. restuans), and lowest in adult female midguts (2 species from Ae. japonicus; 3 species from Ae. triseriatus). These findings suggest that the composition and richness of bacterial communities varies both between habitats and among mosquito species and that the reduction in bacteria diversity during metamorphosis is more evident among bacteria detected using the culture-dependent method.

  15. Regional Cell Specific RNA Expression Profiling of FACS Isolated Drosophila Intestinal Cell Populations.

    PubMed

    Dutta, Devanjali; Buchon, Nicolas; Xiang, Jinyi; Edgar, Bruce A

    2015-08-03

    The adult Drosophila midgut is built of five distinct cell types, including stem cells, enteroblasts, enterocytes, enteroendocrine cells, and visceral muscles, and is divided into five major regions (R1 to R5), which are morphologically and functionally distinct from each other. This unit describes a protocol for the isolation of Drosophila intestinal cell populations for the purpose of cell type-specific transcriptome profiling from the five different regions. A method to select a cell type of interest labeled with green or yellow fluorescent protein (GFP, YFP) by making use of the GAL4-UAS bipartite system and fluorescent-activated cell sorting (FACS) is presented. Total RNA is isolated from the sorted cells of each region, and linear RNA amplification is used to obtain sufficient amounts of high-quality RNA for analysis by microarray, RT-PCR, or RNA sequencing. This method will be useful for quantitative transcriptome comparison across intestinal cell types in the different regions under normal and various experimental conditions.

  16. Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis.

    PubMed

    Liao, Min; Zhou, Jinlin; Gong, Haiyan; Boldbaatar, Damdinsuren; Shirafuji, Rika; Battur, Banzragch; Nishikawa, Yoshifumi; Fujisaki, Kozo

    2009-02-01

    A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.

  17. Imidacloprid impairs the post-embryonic development of the midgut in the yellow fever mosquito Stegomyia aegypti (=Aedes aegypti).

    PubMed

    Fernandes, K M; Gonzaga, W G; Pascini, T V; Miranda, F R; Tomé, H V V; Serrão, J E; Martins, G F

    2015-09-01

    The mosquito Stegomyia aegypti (=Aedes aegypti) (Diptera: Culicidae) is a vector for the dengue and yellow fever viruses. As blood digestion occurs in the midgut, this organ constitutes the route of entry of many pathogens. The effects of the insecticide imidacloprid on the survival of St. aegypti were investigated and the sub-lethal effects of the insecticide on midgut development were determined. Third instar larvae were exposed to different concentrations of imidacloprid (0.15, 1.5, 3.0, 6.0 and 15.0 p.p.m.) and survival was monitored every 24 h for 10 days. Midguts from imidacloprid-treated insects at different stages of development were dissected and processed for analyses by transmission electron microscopy, immunofluorescence microscopy and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays. Imidacloprid concentrations of 3.0 and 15.0 p.p.m. were found to affect midgut development similarly. Digestive cells of the fourth instar larvae (L4) midgut exposed to imidacloprid had more multilamellar bodies, abundantly found in the cell apex, and more electron-lucent vacuoles in the basal region compared with those from untreated insects. Moreover, imidacloprid interfered with the differentiation of regenerative cells, dramatically reducing the number of digestive and endocrine cells and leading to malformation of the midgut epithelium in adults. The data demonstrate that imidacloprid can reduce the survival of mosquitoes and thus indicate its potentially high efficacy in the control of St. aegypti populations.

  18. Female-specific specialization of a posterior end region of the midgut symbiotic organ in Plautia splendens and allied stinkbugs.

    PubMed

    Hayashi, Toshinari; Hosokawa, Takahiro; Meng, Xian-Ying; Koga, Ryuichi; Fukatsu, Takema

    2015-04-01

    Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae.

  19. Female-Specific Specialization of a Posterior End Region of the Midgut Symbiotic Organ in Plautia splendens and Allied Stinkbugs

    PubMed Central

    Hayashi, Toshinari; Hosokawa, Takahiro; Meng, Xian-Ying; Koga, Ryuichi

    2015-01-01

    Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae. PMID:25636847

  20. The posterior determinant gene nanos is required for the maintenance of the adult germline stem cells during Drosophila oogenesis.

    PubMed

    Bhat, K M

    1999-04-01

    In a variety of tissues in eukaryotes, multipotential stem cells are responsible for maintaining a germinal population and generating a differentiated progeny. The Drosophila germline is one such tissue where a continuous supply of eggs or sperm relies on the normal functioning of stem cells. Recent studies have implicated a possible role for the posterior determinant gene nanos (nos) in stem cells. Here, I report that nanos is required in the Drosophila female germline as well as in the male germline. In the female, nos is required for the functioning of stem cells. In nos mutants, while the stem cells are specified, these cells divide only a few times at the most and then degenerate. The loss of germline stem cells in nos mutant mothers appears to be due to a progressive degeneration of the plasma membrane. Furthermore, following germ cell loss, the germaria in the nos mutant mothers appear to carry on massive mitochondrial biogenesis activity. Thus, the syncytia of such germaria are filled with mitochondria. In the male germline, the male fertility assay indicates that nos appears to be also required for the maintenance of stem cells. In these mutant males, spermatogenesis is progressively affected and these males eventually become sterile. These results indicate novel requirements for nos in the Drosophila germline.

  1. Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

    PubMed Central

    Boissière, Anne; Tchioffo, Majoline T.; Bachar, Dipankar; Abate, Luc; Marie, Alexandra; Nsango, Sandrine E.; Shahbazkia, Hamid R.; Awono-Ambene, Parfait H.; Levashina, Elena A.; Christen, Richard; Morlais, Isabelle

    2012-01-01

    The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission. PMID:22693451

  2. Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis.

    PubMed

    Nakajima, Mie; Kodama, Michi; Yanase, Haruko; Iwanaga, Toshihiko; Mulenga, Albert; Ohashi, Kazuhiko; Onuma, Misao

    2003-08-14

    There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.

  3. Dynamics of Midgut Microflora and Dengue Virus Impact on Life History Traits in Aedes aegypti

    PubMed Central

    Hill, Casey L.; Sharma, Avinash; Shouche, Yogesh; Severson, David W.

    2014-01-01

    Significant morbidity and potential mortality following dengue virus infection is a re-emerging global health problem. Due to the limited effectiveness of current disease control methods, mosquito biologists have been searching for new methods of controlling dengue transmission. While much effort has concentrated on determining genetic aspects to vector competence, paratransgenetic approaches could also uncover novel vector control strategies. The interactions of mosquito midgut microflora and pathogens may play significant roles in vector biology. However, little work has been done to see how the microbiome influences the host's fitness and ultimately vector competence. Here we investigated the effects of the midgut microbial environment and dengue infection on several fitness characteristics among three strains of the primary dengue virus vector mosquito Aedes aegypti. This included comparisons of dengue infection rates of females with and without their normal midgut flora. According to our findings, few effects on fitness characteristics were evident following microbial clearance or with dengue virus infection. Adult survivorship significantly varied due to strain and in one strain varied due to antibiotic treatment. Fecundity varied in one strain due to microbial clearance by antibiotics but no variation was observed in fertility due to either treatment. We show here that fitness characteristics of Ae. aegypti vary largely between strains, including varying response to microflora presence or absence, but did not vary in response to dengue virus infection. PMID:25193134

  4. The bHLH Transcription Factor Hand Regulates the Expression of Genes Critical to Heart and Muscle Function in Drosophila melanogaster

    PubMed Central

    Hallier, Benjamin; Hoffmann, Julia; Roeder, Thomas; Tögel, Markus; Meyer, Heiko; Paululat, Achim

    2015-01-01

    Hand proteins belong to the highly conserved family of basic Helix-Loop-Helix transcription factors and are critical to distinct developmental processes, including cardiogenesis and neurogenesis in vertebrates. In Drosophila melanogaster a single orthologous hand gene is expressed with absence of the respective protein causing semilethality during early larval instars. Surviving adult animals suffer from shortened lifespan associated with a disorganized myofibrillar structure being apparent in the dorsal vessel, the wing hearts and in midgut tissue. Based on these data, the major biological significance of Hand seems to be related to muscle development, maintenance or function; however, up to now the physiological basis for Hand functionality remains elusive. Thus, the identification of genes whose expression is, directly or indirectly, regulated by Hand has considerable relevance with respect to understanding its biological functionality in flies and vertebrates. Beneficially, hand mutants are viable and exhibit affected tissues, which renders Drosophila an ideal model to investigate up- or downregulated target genes by a comparative microarray approach focusing on the respective tissues from mutant specimens. Our present work reveals for the first time that Drosophila Hand regulates the expression of numerous genes of diverse physiological relevancy, including distinct factors required for proper muscle development and function such as Zasp52 or Msp-300. These results relate Hand activity to muscle integrity and functionality and may thus be highly beneficial to the evaluation of corresponding hand phenotypes. PMID:26252215

  5. Identification of singles bar as a direct transcriptional target of Drosophila Myocyte enhancer factor-2 and a regulator of adult myoblast fusion.

    PubMed

    Brunetti, Tonya M; Fremin, Brayon J; Cripps, Richard M

    2015-05-15

    In Drosophila, myoblast fusion is a conserved process in which founder cells (FCs) and fusion competent myoblasts (FCMs) fuse to form a syncytial muscle fiber. Mutants for the myogenic regulator Myocyte enhancer factor-2 (MEF2) show a failure of myoblast fusion, indicating that MEF2 regulates the fusion process. Indeed, chromatin immunoprecipitation studies show that several genes involved in myoblast fusion are bound by MEF2 during embryogenesis. Of these, the MARVEL domain gene singles bar (sing), is down-regulated in MEF2 knockdown pupae, and has five consensus MEF2 binding sites within a 9000-bp region. To determine if MEF2 is an essential and direct regulator of sing during pupal muscle development, we identified a 315-bp myoblast enhancer of sing. This enhancer was active during myoblast fusion, and mutation of two MEF2 sites significantly decreased enhancer activity. We show that lack of sing expression resulted in adult lethality and muscle loss, due to a failure of fusion during the pupal stage. Additionally, we sought to determine if sing was required in either FCs or FCMs to support fusion. Interestingly, knockdown of sing in either population did not significantly affect fusion, however, knockdown in both FCs and FCMs resulted in muscles with significantly reduced nuclei numbers, provisionally indicating that sing function is required in either cell type, but not both. Finally, we found that MEF2 regulated sing expression at the embryonic stage through the same 315-bp enhancer, indicating that sing is a MEF2 target at both critical stages of myoblast fusion. Our studies define for the first time how MEF2 directly controls fusion at multiple stages of the life cycle, and provide further evidence that the mechanisms of fusion characterized in Drosophila embryos is also used in the formation of the more complex adult muscles.

  6. The Par complex and integrins direct asymmetric cell division in adult intestinal stem cells.

    PubMed

    Goulas, Spyros; Conder, Ryan; Knoblich, Juergen A

    2012-10-05

    The adult Drosophila midgut is maintained by intestinal stem cells (ISCs) that generate both self-renewing and differentiating daughter cells. How this asymmetry is generated is currently unclear. Here, we demonstrate that asymmetric ISC division is established by a unique combination of extracellular and intracellular polarity mechanisms. We show that Integrin-dependent adhesion to the basement membrane induces cell-intrinsic polarity and results in the asymmetric segregation of the Par proteins Par-3, Par-6, and aPKC into the apical daughter cell. Cell-specific knockdown and overexpression experiments suggest that increased activity of aPKC enhances Delta/Notch signaling in one of the two daughter cells to induce terminal differentiation. Perturbing this mechanism or altering the orientation of ISC division results in the formation of intestinal tumors. Our data indicate that mechanisms for intrinsically asymmetric cell division can be adapted to allow for the flexibility in lineage decisions that is required in adult stem cells.

  7. Chitin is a component of the Rhodnius prolixus midgut.

    PubMed

    Alvarenga, Evelyn S L; Mansur, Juliana F; Justi, Silvia A; Figueira-Mansur, Janaina; Dos Santos, Vivian M; Lopez, Sheila G; Masuda, Hatisaburo; Lara, Flavio A; Melo, Ana C A; Moreira, Monica F

    2016-02-01

    Chitin is an essential component of the peritrophic matrix (PM), which is a structure that lines the insect's gut and protects against mechanical damage and pathogens. Rhodnius prolixus (Hemiptera: Reduviidae) does not have a PM, but it has an analogous structure, the perimicrovillar membrane (PMM); chitin has not been described in this structure. Here, we show that chitin is present in the R. prolixus midgut using several techniques. The FTIR spectrum of the KOH-resistant putative chitin-material extracted from the midgut bolus showed peaks characteristic of the chitin molecule at 3500, 1675 and 1085 cm(1). Both the midgut bolus material and the standard chitin NMR spectra showed a peak at 1.88 ppm, which is certainly due to methyl protons in the acetamide a group. The percentages of radioactive N-acetylglucosamine (CPM) incorporated were 2 and 4% for the entire intestine and bolus, respectively. The KOH-resistant putative chitin-material was also extracted and purified from the N-acetylglucosamine radioactive bolus, and the radioactivity was estimated through liquid scintillation. The intestinal CHS cDNA translated sequence was the same as previously described for the R. prolixus cuticle and ovaries. Phenotypic alterations were observed in the midgut of females with a silenced CHS gene after a blood meal, such as retarded blood meal digestion; the presence of fresh blood that remained red nine days after the blood meal; and reduced trachea and hemozoin content compared with the control. Wheat germ agglutinin (a specific probe that detects chitin) labeling proximal to the intestine (crop and midgut) was much lower in females with a silenced CHS gene, especially in the midgut region, where almost no fluorescence signal was detected compared with the control groups. Midguts from females with a CHS gene silenced by dsRNA-CHS and control midguts pre-treated with chitinase showed that the chitin-derived fluorescence signal decreased in the region around the epithelium

  8. Update on management of midgut neuroendocrine tumors

    PubMed Central

    Mehrvarz Sarshekeh, Amir; Halperin, Daniel M; Dasari, Arvind

    2016-01-01

    Midgut neuroendocrine tumors are typically indolent but can be fatal when advanced. They can also cause significant morbidity due to the characteristic carcinoid syndrome. Somatostatin analogs continue to be the mainstay of treatment given their antiproliferative properties, as well as inhibitory effects on hormones that cause carcinoid syndrome. There have been several recent advances in the systemic therapy of these tumors including consolidation of somatostatin analogs as the cornerstone of therapy, completion of pivotal trials with mTOR inhibitors, and the establishment of novel approaches including peptide receptor radionuclide therapy and oral inhibitors of peripheral tryptophan hydroxylase in tumor and symptom control, respectively. In this review article, the recent advances are summarized and an updated approach to management is proposed. PMID:27347369

  9. Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. X. Age-specific dynamics of adult epicuticular hydrocarbon expression in response to different host plants.

    PubMed

    Etges, William J; de Oliveira, Cassia C

    2014-06-01

    Analysis of sexual selection and sexual isolation in Drosophila mojavensis and its relatives has revealed a pervasive role of rearing substrates on adult courtship behavior when flies were reared on fermenting cactus in preadult stages. Here, we assessed expression of contact pheromones comprised of epicuticular hydrocarbons (CHCs) from eclosion to 28 days of age in adults from two populations reared on fermenting tissues of two host cacti over the entire life cycle. Flies were never exposed to laboratory food and showed significant reductions in average CHC amounts consistent with CHCs of wild-caught flies. Overall, total hydrocarbon amounts increased from eclosion to 14-18 days, well past age at sexual maturity, and then declined in older flies. Most flies did not survive past 4 weeks. Baja California and mainland populations showed significantly different age-specific CHC profiles where Baja adults showed far less age-specific changes in CHC expression. Adults from populations reared on the host cactus typically used in nature expressed more CHCs than on the alternate host. MANCOVA with age as the covariate for the first six CHC principal components showed extensive differences in CHC composition due to age, population, cactus, sex, and age × population, age × sex, and age × cactus interactions. Thus, understanding variation in CHC composition as adult D. mojavensis age requires information about population and host plant differences, with potential influences on patterns of mate choice, sexual selection, and sexual isolation, and ultimately how these pheromones are expressed in natural populations. Studies of drosophilid aging in the wild are badly needed.

  10. Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. X. Age-specific dynamics of adult epicuticular hydrocarbon expression in response to different host plants

    PubMed Central

    Etges, William J; de Oliveira, Cassia C

    2014-01-01

    Analysis of sexual selection and sexual isolation in Drosophila mojavensis and its relatives has revealed a pervasive role of rearing substrates on adult courtship behavior when flies were reared on fermenting cactus in preadult stages. Here, we assessed expression of contact pheromones comprised of epicuticular hydrocarbons (CHCs) from eclosion to 28 days of age in adults from two populations reared on fermenting tissues of two host cacti over the entire life cycle. Flies were never exposed to laboratory food and showed significant reductions in average CHC amounts consistent with CHCs of wild-caught flies. Overall, total hydrocarbon amounts increased from eclosion to 14–18 days, well past age at sexual maturity, and then declined in older flies. Most flies did not survive past 4 weeks. Baja California and mainland populations showed significantly different age-specific CHC profiles where Baja adults showed far less age-specific changes in CHC expression. Adults from populations reared on the host cactus typically used in nature expressed more CHCs than on the alternate host. MANCOVA with age as the covariate for the first six CHC principal components showed extensive differences in CHC composition due to age, population, cactus, sex, and age × population, age × sex, and age × cactus interactions. Thus, understanding variation in CHC composition as adult D. mojavensis age requires information about population and host plant differences, with potential influences on patterns of mate choice, sexual selection, and sexual isolation, and ultimately how these pheromones are expressed in natural populations. Studies of drosophilid aging in the wild are badly needed. PMID:25360246

  11. Environmental ethanol as an ecological constraint on dietary breadth of Spotted-Wing Drosophila, Drosophila suzukii Mat. (Diptera: Drosophilidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spotted-wing Drosophila (SWD), Drosophila suzukii, is a recent fruit pest of the Americas whose destructiveness stems from its subcutaneous insertion of eggs into cultivated berries via a female’s prominent double bladed and serrated ovipositor. Atypical of most other Drosophila, D. suzukii adults a...

  12. The development of malaria parasites in the mosquito midgut

    PubMed Central

    Bennink, Sandra; Kiesow, Meike J.

    2016-01-01

    Summary The mosquito midgut stages of malaria parasites are crucial for establishing an infection in the insect vector and to thus ensure further spread of the pathogen. Parasite development in the midgut starts with the activation of the intraerythrocytic gametocytes immediately after take‐up and ends with traversal of the midgut epithelium by the invasive ookinetes less than 24 h later. During this time period, the plasmodia undergo two processes of stage conversion, from gametocytes to gametes and from zygotes to ookinetes, both accompanied by dramatic morphological changes. Further, gamete formation requires parasite egress from the enveloping erythrocytes, rendering them vulnerable to the aggressive factors of the insect gut, like components of the human blood meal. The mosquito midgut stages of malaria parasites are unprecedented objects to study a variety of cell biological aspects, including signal perception, cell conversion, parasite/host co‐adaptation and immune evasion. This review highlights recent insights into the molecules involved in gametocyte activation and gamete formation as well as in zygote‐to‐ookinete conversion and ookinete midgut exit; it further discusses factors that can harm the extracellular midgut stages as well as the measures of the parasites to protect themselves from any damage. PMID:27111866

  13. Opposite effects of 5-HT/AKH and octopamine on the crop contractions in adult Drosophila melanogaster: Evidence of a double brain-gut serotonergic circuitry

    PubMed Central

    Picciau, Lorenzo; Murru, Ludovico; Stoffolano, John G.

    2017-01-01

    This study showed that in adult Drosophila melanogaster, the type of sugar—either present within the crop lumen or in the bathing solution of the crop—had no effect on crop muscle contraction. What is important, however, is the volume within the crop lumen. Electrophysiological recordings demonstrated that exogenous applications of serotonin on crop muscles increases both the amplitude and the frequency of crop contraction rate, while adipokinetic hormone mainly enhances the crop contraction frequency. Conversely, octopamine virtually silenced the overall crop activity. The present study reports for the first time an analysis of serotonin effects along the gut-brain axis in adult D. melanogaster. Injection of serotonin into the brain between the interocellar area shows that brain applications of serotonin decrease the frequency of crop activity. Based on our results, we propose that there are two different, opposite pathways for crop motility control governed by serotonin: excitatory when added in the abdomen (i.e., directly bathing the crop) and inhibitory when supplied within the brain (i.e., by injection). Finally, our results point to a double brain-gut serotonergic circuitry suggesting that not only the brain can affect gut functions, but the gut can also affect the central nervous system. On the basis of our results, and data in the literature, a possible mechanism for these two discrete serotonergic functions is suggested. PMID:28334024

  14. Elevated extension of longevity by cyclically heat stressing a set of recombinant inbred lines of Drosophila melanogaster throughout their adult life.

    PubMed

    Gomez, Federico H; Sambucetti, Pablo; Norry, Fabian M

    2016-11-01

    An extremely high (about 100 %) increase in longevity is reported for a subset of recombinant inbred lines (RILs) of Drosophila melanogaster subjected to a cyclic heat stress throughout the adult life. Previous work showed that both longevity and heat sensitivity highly differed among RILs. The novel heat stress treatment used in this study consisted of 5 min at 38 °C applicated approximately every 125 min throughout the adult life starting at the age of 2 days. In spite of the exceptionally high increase in longevity in a set of RILs, the same heat stress treatment reduced rather than increased longevity in other RILs, suggesting that heat-induced hormesis is dependent on the genotype and/or the genetic background. Further, one quantitative trait locus (QTL) was identified for heat-induced hormesis on chromosome 2 (bands 28A1-34D2) in one RIL panel (RIL-D48) but it was not significant in its reciprocal panel (RIL-SH2). The level of heat-induced hormesis showed a sexual dimorphism, with a higher number of lines exhibiting higher hormesis effects in males than in females. The new heat stress treatment in this study suggests that longevity can be further extended than previously suggested by applying a cyclic and mild stress throughout the life, depending on the genotype.

  15. Increased abundance of frost mRNA during recovery from cold stress is not essential for cold tolerance in adult Drosophila melanogaster.

    PubMed

    Udaka, H; Percival-Smith, A; Sinclair, B J

    2013-10-01

    Frost (Fst) is a candidate gene associated with the response to cold in Drosophila melanogaster because Fst mRNA accumulation increases during recovery from low temperature exposure. We investigated the contribution of Fst expression to chill-coma recovery time, acute cold tolerance and rapid cold hardening (RCH) in adult D. melanogaster by knocking down Fst mRNA expression using GAL4/UAS-mediated RNA interference. In this experiment, four UAS-Fst and one tubulin-GAL4 lines were used. We predicted that if Fst is essential for cold tolerance phenotypes, flies with low Fst mRNA levels should be less cold tolerant than flies with normal levels of cold-induced Fst mRNA. Cold-induced Fst abundance and recovery time from chill-coma were not negatively correlated in male or female flies. Survival of 2 h exposures to sub-zero temperatures in Fst knockdown lines was not lower than that in a control line. Moreover, a low temperature pretreatment increased survival of severe cold exposure in flies regardless of Fst abundance level during recovery from cold stress, suggesting that Fst expression is not essential for RCH. Thus, cold-induced Fst accumulation is not essential for cold tolerance measured as chill-coma recovery time, survival to acute cold stress and RCH response in adult D. melanogaster.

  16. The Drosophila melanogaster importin alpha3 locus encodes an essential gene required for the development of both larval and adult tissues.

    PubMed Central

    Mason, D Adam; Máthé, Endre; Fleming, Robert J; Goldfarb, David S

    2003-01-01

    The nuclear transport of classical nuclear localization signal (cNLS)-containing proteins is mediated by the cNLS receptor importin alpha. The conventional importin alpha gene family in metazoan animals is composed of three clades that are conserved between flies and mammals and are referred to here as alpha1, alpha2, and alpha3. In contrast, plants and fungi contain only alpha1 genes. In this study we report that Drosophila importin alpha3 is required for the development of both larval and adult tissues. Importin alpha3 mutant flies die around the transition from first to second instar larvae, and homozygous importin alpha3 mutant eyes are defective. The transition to second instar larvae was rescued with importin alpha1, alpha2, or alpha3 transgenes, indicating that Importin alpha3 is normally required at this stage for an activity shared by all three importin alpha's. In contrast, an alpha3-specific biochemical activity(s) of Importin alpha3 is probably required for development to adults and photoreceptor cell development, since only an importin alpha3 transgene rescued these processes. These results are consistent with the view that the importin alpha's have both overlapping and distinct functions and that their role in animal development involves the spatial and temporal control of their expression. PMID:14704178

  17. EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells.

    PubMed

    Wang, Chenhui; Guo, Xingting; Xi, Rongwen

    2014-05-01

    Quiescent, multipotent gastric stem cells (GSSCs) in the copper cell region of adult Drosophila midgut can produce all epithelial cell lineages found in the region, including acid-secreting copper cells, interstitial cells and enteroendocrine cells, but mechanisms controlling their quiescence and the ternary lineage differentiation are unknown. By using cell ablation or damage-induced regeneration assays combined with cell lineage tracing and genetic analysis, here we demonstrate that Delta (Dl)-expressing cells in the copper cell region are the authentic GSSCs that can self-renew and continuously regenerate the gastric epithelium after a sustained damage. Lineage tracing analysis reveals that the committed GSSC daughter with activated Notch will invariably differentiate into either a copper cell or an interstitial cell, but not the enteroendocrine cell lineage, and loss-of-function and gain-of-function studies revealed that Notch signaling is both necessary and sufficient for copper cell/interstitial cell differentiation. We also demonstrate that elevated epidermal growth factor receptor (EGFR) signaling, which is achieved by the activation of ligand Vein from the surrounding muscle cells and ligand Spitz from progenitor cells, mediates the regenerative proliferation of GSSCs following damage. Taken together, we demonstrate that Dl is a specific marker for Drosophila GSSCs, whose cell cycle status is dependent on the levels of EGFR signaling activity, and the Notch signaling has a central role in controlling cell lineage differentiation from GSSCs by separating copper/interstitial cell lineage from enteroendocrine cell lineage.

  18. Ttk69 acts as a master repressor of enteroendocrine cell specification in Drosophila intestinal stem cell lineages.

    PubMed

    Wang, Chenhui; Guo, Xingting; Dou, Kun; Chen, Hongyan; Xi, Rongwen

    2015-10-01

    In adult Drosophila midgut, intestinal stem cells (ISCs) periodically produce progenitor cells that undergo a binary fate choice determined primarily by the levels of Notch activity that they receive, before terminally differentiating into enterocytes (ECs) or enteroendocrine (EE) cells. Here we identified Ttk69, a BTB domain-containing transcriptional repressor, as a master repressor of EE cell specification in the ISC lineages. Depletion of ttk69 in progenitor cells induced ISC proliferation and caused all committed progenitor cells to adopt EE fate, leading to the production of supernumerary EE cells in the intestinal epithelium. Conversely, forced expression of Ttk69 in progenitor cells was sufficient to prevent EE cell specification. The expression of Ttk69 was not regulated by Notch signaling, and forced activation of Notch, which is sufficient to induce EC specification of normal progenitor cells, failed to prevent EE cell specification of Ttk69-depleted progenitors. Loss of Ttk69 led to derepression of the acheate-scute complex (AS-C) genes scute and asense, which then induced prospero expression to promote EE cell specification. These studies suggest that Ttk69 functions in parallel with Notch signaling and acts as a master repressor of EE cell specification in Drosophila ISC lineages primarily by suppressing AS-C genes.

  19. The effect of developmental nutrition on life span and fecundity depends on the adult reproductive environment in Drosophila melanogaster

    PubMed Central

    May, Christina M; Doroszuk, Agnieszka; Zwaan, Bas J

    2015-01-01

    Both developmental nutrition and adult nutrition affect life-history traits; however, little is known about whether the effect of developmental nutrition depends on the adult environment experienced. We used the fruit fly to determine whether life-history traits, particularly life span and fecundity, are affected by developmental nutrition, and whether this depends on the extent to which the adult environment allows females to realize their full reproductive potential. We raised flies on three different developmental food levels containing increasing amounts of yeast and sugar: poor, control, and rich. We found that development on poor or rich larval food resulted in several life-history phenotypes indicative of suboptimal conditions, including increased developmental time, and, for poor food, decreased adult weight. However, development on poor larval food actually increased adult virgin life span. In addition, we manipulated the reproductive potential of the adult environment by adding yeast or yeast and a male. This manipulation interacted with larval food to determine adult fecundity. Specifically, under two adult conditions, flies raised on poor larval food had higher reproduction at certain ages – when singly mated this occurred early in life and when continuously mated with yeast this occurred during midlife. We show that poor larval food is not necessarily detrimental to key adult life-history traits, but does exert an adult environment-dependent effect, especially by affecting virgin life span and altering adult patterns of reproductive investment. Our findings are relevant because (1) they may explain differences between published studies on nutritional effects on life-history traits; (2) they indicate that optimal nutritional conditions are likely to be different for larvae and adults, potentially reflecting evolutionary history; and (3) they urge for the incorporation of developmental nutritional conditions into the central life-history concept of

  20. Monitoring the developmental impact of copper and silver nanoparticle exposure in Drosophila and their microbiomes.

    PubMed

    Han, Xu; Geller, Brennen; Moniz, Kristy; Das, Pranab; Chippindale, Adam K; Walker, Virginia K

    2014-07-15

    There is concern that waste waters containing manufactured metal nanoparticles (NPs) originating from consumer goods, will find their way into streams and larger water bodies. Aquatic invertebrates could be vulnerable to such pollution, and here we have used fruit flies, Drosophila melanogaster, as a model invertebrate, to test for the effect of NPs on fitness. Both copper NP and microparticle (MP)-containing medium slowed development, reduced adult longevity and decreased sperm competition. In contrast, ingestion of silver resulted in a significant reduction in developmental success only if the metal particles were nanosized. Ag NP-treatments resulted in reduced developmental success as assessed by larval and pupal survival as well as larval climbing ability, but there was no impact of silver on adult longevity and little effect on reproductive success. However, Cu NPs generally appeared to be no more toxic to this invertebrate model than the bulk counterpart. The impact of silver ingestion in larvae was further investigated by 454 pyrosequencing of the 16S rRNA genes of the midgut flora. There was a striking reduction in the diversity of the gut microbiota of Ag NP-treated larvae with a rise in the predominance of Lactobacillus brevis and a decrease in Acetobacter compared to control or Ag MP-treatment groups. Importantly, these experiments show that perturbation of the microbial assemblage within a metazoan model may contribute to Ag NP-mediated toxicity. These observations have implications for impact assessments of nanoparticles as emerging contaminants.

  1. Cav2-type calcium channels encoded by cac regulate AP-independent neurotransmitter release at cholinergic synapses in adult Drosophila brain.

    PubMed

    Gu, Huaiyu; Jiang, Shaojuan Amy; Campusano, Jorge M; Iniguez, Jorge; Su, Hailing; Hoang, Andy An; Lavian, Monica; Sun, Xicui; O'Dowd, Diane K

    2009-01-01

    Voltage-gated calcium channels containing alpha1 subunits encoded by Ca(v)2 family genes are critical in regulating release of neurotransmitter at chemical synapses. In Drosophila, cac is the only Ca(v)2-type gene. Cacophony (CAC) channels are localized in motor neuron terminals where they have been shown to mediate evoked, but not AP-independent, release of glutamate at the larval neuromuscular junction (NMJ). Cultured embryonic neurons also express CAC channels, but there is no information about the properties of CAC-mediated currents in adult brain nor how these channels regulate transmission in central neural circuits where fast excitatory synaptic transmission is predominantly cholinergic. Here we report that wild-type neurons cultured from late stage pupal brains and antennal lobe projection neurons (PNs) examined in adult brains, express calcium currents with two components: a slow-inactivating current sensitive to the spider toxin Plectreurys toxin II (PLTXII) and a fast-inactivating PLTXII-resistant component. CAC channels are the major contributors to the slow-inactivating PLTXII-sensitive current based on selective reduction of this component in hypomorphic cac mutants (NT27 and TS3). Another characteristic of cac mutant neurons both in culture and in whole brain recordings is a reduced cholinergic miniature excitatory postsynaptic current frequency that is mimicked in wild-type neurons by acute application of PLTXII. These data demonstrate that cac encoded Ca(v)2-type calcium channels regulate action potential (AP)-independent release of neurotransmitter at excitatory cholinergic synapses in the adult brain, a function not predicted from studies at the larval NMJ.

  2. Plasmodium ookinetes coopt mammalian plasminogen to invade the mosquito midgut

    PubMed Central

    Ghosh, Anil K.; Coppens, Isabelle; Gårdsvoll, Henrik; Ploug, Michael; Jacobs-Lorena, Marcelo

    2011-01-01

    Ookinete invasion of the mosquito midgut is an essential step for the development of the malaria parasite in the mosquito. Invasion involves recognition between a presumed mosquito midgut receptor and an ookinete ligand. Here, we show that enolase lines the ookinete surface. An antienolase antibody inhibits oocyst development of both Plasmodium berghei and Plasmodium falciparum, suggesting that enolase may act as an invasion ligand. Importantly, we demonstrate that surface enolase captures plasminogen from the mammalian blood meal via its lysine motif (DKSLVK) and that this interaction is essential for midgut invasion, because plasminogen depletion leads to a strong inhibition of oocyst formation. Although addition of recombinant WT plasminogen to depleted serum rescues oocyst formation, recombinant inactive plasminogen does not, thus emphasizing the importance of plasmin proteolytic activity for ookinete invasion. The results support the hypothesis that enolase on the surface of Plasmodium ookinetes plays a dual role in midgut invasion: by acting as a ligand that interacts with the midgut epithelium and, further, by capturing plasminogen, whose conversion to active plasmin promotes the invasion process. PMID:21949403

  3. Identification of the essential protein domains for Mib2 function during the development of the Drosophila larval musculature and adult flight muscles

    PubMed Central

    Domsch, Katrin; Acs, Andreas; Obermeier, Claudia; Nguyen, Hanh T.

    2017-01-01

    The proper differentiation and maintenance of myofibers is fundamental to a functional musculature. Disruption of numerous mostly structural factors leads to perturbations of these processes. Among the limited number of known regulatory factors for these processes is Mind bomb2 (Mib2), a muscle-associated E3 ubiquitin ligase, which was previously established to be required for maintaining the integrity of larval muscles. In this study, we have examined the mechanistic aspects of Mib2 function by performing a detailed functional dissection of the Mib2 protein. We show that the ankyrin repeats, in its entirety, and the hitherto uncharacterized Mib-specific domains (MIB), are important for the major function of Mib2 in skeletal and visceral muscles in the Drosophila embryo. Furthermore, we characterize novel mib2 alleles that have arisen from a forward genetic screen aimed at identifying regulators of myogenesis. Two of these alleles are viable, but flightless hypomorphic mib2 mutants, and harbor missense mutations in the MIB domain and RING finger, respectively. Functional analysis of these new alleles, including in vivo imaging, demonstrates that Mib2 plays an additional important role in the development of adult thorax muscles, particularly in maintaining the larval templates for the dorsal longitudinal indirect flight muscles during metamorphosis. PMID:28282454

  4. Expression of the Troponin C at 41C Gene in Adult Drosophila Tubular Muscles Depends upon Both Positive and Negative Regulatory Inputs.

    PubMed

    Chechenova, Maria B; Maes, Sara; Cripps, Richard M

    2015-01-01

    Most animals express multiple isoforms of structural muscle proteins to produce tissues with different physiological properties. In Drosophila, the adult muscles include tubular-type muscles and the fibrillar indirect flight muscles. Regulatory processes specifying tubular muscle fate remain incompletely understood, therefore we chose to analyze the transcriptional regulation of TpnC41C, a Troponin C gene expressed in the tubular jump muscles, but not in the fibrillar flight muscles. We identified a 300-bp promoter fragment of TpnC41C sufficient for the fiber-specific reporter expression. Through an analysis of this regulatory element, we identified two sites necessary for the activation of the enhancer. Mutations in each of these sites resulted in 70% reduction of enhancer activity. One site was characterized as a binding site for Myocyte Enhancer Factor-2. In addition, we identified a repressive element that prevents activation of the enhancer in other muscle fiber types. Mutation of this site increased jump muscle-specific expression of the reporter, but more importantly reporter expression expanded into the indirect flight muscles. Our findings demonstrate that expression of the TpnC41C gene in jump muscles requires integration of multiple positive and negative transcriptional inputs. Identification of the transcriptional regulators binding the cis-elements that we identified will reveal the regulatory pathways controlling muscle fiber differentiation.

  5. New functions of the Drosophila rhomboid gene during embryonic and adult development are revealed by a novel genetic method, enhancer piracy.

    PubMed

    Noll, R; Sturtevant, M A; Gollapudi, R R; Bier, E

    1994-08-01

    Localized expression of the Drosophila rhomboid (rho) gene has been proposed to hyperactivate EGF-Receptor signaling in specific cells during development of the embryo and adult. In this report we use a novel transposon based genetic method, enhancer piracy, to drive ectopic expression of a rho cDNA transgene by endogenous genomic enhancers. Many enhancer piracy transposon-rho insertions cause dominant phenotypes, over half of which cannot be duplicated by ubiquitous expression of rho. Genetic interactions between various dominant enhancer piracy alleles and mutations in the EGF-R/RAS signaling pathway indicate that many of these novel phenotypes result from ectopic activation of EGF-R signaling. Patterned mis-expression of the rho cDNA transgene correlates in several cases with localized dominant enhancer piracy phenotypes. Enhancer piracy lines reveal an unanticipated role for rho in imaginal disc formation and provide the first evidence that mis-expression of rho is sufficient for converting entire intervein sectors into veins. Enhancer piracy may prove to be a general strategy for obtaining dominant alleles of a gene of interest in diverse insects, worms, plants, and potentially in vertebrates such as mice and fish.

  6. Laminin and the malaria parasite's journey through the mosquito midgut.

    PubMed

    Arrighi, Romanico B G; Lycett, Gareth; Mahairaki, Vassiliki; Siden-Kiamos, Inga; Louis, Christos

    2005-07-01

    During the invasion of the mosquito midgut epithelium, Plasmodium ookinetes come to rest on the basal lamina, where they transform into the sporozoite-producing oocysts. Laminin, one of the basal lamina's major components, has previously been shown to bind several surface proteins of Plasmodium ookinetes. Here, using the recently developed RNAi technique in mosquitoes, we used a specific dsRNA construct targeted against the LANB2 gene (laminin gamma1) of Anopheles gambiae to reduce its mRNA levels, leading to a substantial reduction in the number of successfully developed oocysts in the mosquito midgut. Moreover, this molecular relationship is corroborated by the intimate association of developing P. berghei parasites and laminin in the gut, as observed using confocal microscopy. Our data support the notion of laminin playing a functional role in the development of the malaria parasite within the mosquito midgut.

  7. Schinus terebinthifolius Leaf Extract Causes Midgut Damage, Interfering with Survival and Development of Aedes aegypti Larvae

    PubMed Central

    Procópio, Thamara Figueiredo; Fernandes, Kenner Morais; Pontual, Emmanuel Viana; Ximenes, Rafael Matos; de Oliveira, Aline Rafaella Cardoso; Souza, Carolina de Santana; Melo, Ana Maria Mendonça de Albuquerque; Navarro, Daniela Maria do Amaral Ferraz; Paiva, Patrícia Maria Guedes; Martins, Gustavo Ferreira; Napoleão, Thiago Henrique

    2015-01-01

    In this study, a leaf extract from Schinus terebinthifolius was evaluated for effects on survival, development, and midgut of A. aegypti fourth instar larvae (L4), as well as for toxic effect on Artemia salina. Leaf extract was obtained using 0.15 M NaCl and evaluated for phytochemical composition and lectin activity. Early L4 larvae were incubated with the extract (0.3–1.35%, w/v) for 8 days, in presence or absence of food. Polymeric proanthocyanidins, hydrolysable tannins, heterosid and aglycone flavonoids, cinnamic acid derivatives, traces of steroids, and lectin activity were detected in the extract, which killed the larvae at an LC50 of 0.62% (unfed larvae) and 1.03% (fed larvae). Further, the larvae incubated with the extract reacted by eliminating the gut content. No larvae reached the pupal stage in treatments at concentrations between 0.5% and 1.35%, while in the control (fed larvae), 61.7% of individuals emerged as adults. The extract (1.0%) promoted intense disorganization of larval midgut epithelium, including deformation and hypertrophy of cells, disruption of microvilli, and vacuolization of cytoplasms, affecting digestive, enteroendocrine, regenerative, and proliferating cells. In addition, cells with fragmented DNA were observed. Separation of extract components by solid phase extraction revealed that cinnamic acid derivatives and flavonoids are involved in larvicidal effect of the extract, being the first most efficient in a short time after larvae treatment. The lectin present in the extract was isolated, but did not show deleterious effects on larvae. The extract and cinnamic acid derivatives were toxic to A. salina nauplii, while the flavonoids showed low toxicity. S. terebinthifolius leaf extract caused damage to the midgut of A. aegypti larvae, interfering with survival and development. The larvicidal effect of the extract can be attributed to cinnamic acid derivatives and flavonoids. The data obtained using A. salina indicates that caution

  8. Effects of combining a cryptochrome mutation with other visual-system variants on entrainment of locomotor and adult-emergence rhythms in Drosophila.

    PubMed

    Mealey-Ferrara, Marion L; Montalvo, Alexandra G; Hall, Jeffrey C

    2003-01-01

    "For every behavioral observation, there is an equal and opposite observation." S. Benzer Photoreception is an important component of rhythm systems and is involved in adjusting circadian clocks to photic features of daily cycles. In Drosophila, it has been suggested that there are three light input pathways to the clock that underlie rhythms of adult behavior: One involves the eyes; the other two extraocular photoreception through a structure called the Hofbauer-Buchner (H-B) eyelet and light reception carried out by pacemaker neurons themselves, mediated by a substance called cryptochrome. All photoreceptor cells including the H-B eyelet have been surmised to be removed by glass-null mutations. Mutations in the no-receptor-potential-A (norpA) gene cause the compound eyes and ocelli to be non-functional and may also affect the eyelet's function. The one cryptochrome mutant known (cryb) harbors an amino-acid substitution in the blue-light absorbing protein encoded by this gene. With regard to adult locomotor rhythms, all single mutants (gl60j, norpAP41, and cryb) re-entrained to altered light:dark (LD) cycles in which the L phase involved relatively intense light. Dropping light levels ca. 10 or ca. 30-fold permitted small percentages of doubly-mutant gl60j cryb flies clearly to re-synchronize their behavior. The marginal re-entrainability in the lowest-light situation nevertheless involved superior responsiveness of the gl60j cryb type, compared with that observed previously using a different re-entrainment protocol. Furthermore, transgenic types in which rhodopsin-expressing cells within the H-B eyelet were ablated or suffered from the effects of tetanus-toxin also entrained with behavior similar or superior to that of gl60j cryb at a low light level. Light inputs that are necessary to synchronize periodic adult emergence can be inferred (from previous studies) to involve a cry-dependent pathway and perhaps also a norpA-dependent one, so that combining mutations

  9. Control of lipid metabolism by Tachykinin in Drosophila

    PubMed Central

    Song, Wei; Veenstra, Jan A.; Perrimon, Norbert

    2015-01-01

    Summary The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. We demonstrate that Tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila, and that TKs repress lipogenesis in enterocytes (ECs) associated with the TKR99D receptor and PKA signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions. PMID:25263556

  10. Cytoplasmic myosin from Drosophila melanogaster

    PubMed Central

    1986-01-01

    Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized. PMID

  11. Damage-Induced Cell Regeneration in the Midgut of Aedes albopictus Mosquitoes

    PubMed Central

    Janeh, Maria; Osman, Dani; Kambris, Zakaria

    2017-01-01

    Mosquito-transmitted diseases cause over one million deaths every year. A better characterization of the vector’s physiology and immunity should provide valuable knowledge for the elaboration of control strategies. Mosquitoes depend on their innate immunity to defend themselves against pathogens. These pathogens are acquired mainly through the oral route, which places the insects’ gut at the front line of the battle. Indeed, the epithelium of the mosquito gut plays important roles against invading pathogens acting as a physical barrier, activating local defenses and triggering the systemic immune response. Therefore, the gut is constantly confronted to stress and often suffers cellular damage. In this study, we show that dividing cells exist in the digestive tract of adult A. albopictus and that these cells proliferate in the midgut after bacterial or chemical damage. An increased transcription of signaling molecules that regulate the EGFR and JAK/STAT pathways was also observed, suggesting a possible involvement of these pathways in the regeneration of damaged guts. This work provides evidence for the presence of regenerative cells in the mosquito guts, and paves the way towards a molecular and cellular characterization of the processes required to maintain mosquito’s midgut homeostasis in both normal and infectious conditions. PMID:28300181

  12. Mitochondria in the midgut epithelial cells of sugarcane borer parasitized by Cotesia flavipes (Cameron, 1891).

    PubMed

    Pinheiro, D O; Silva, M D; Gregório, E A

    2010-02-01

    The sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae) has been controlled by Cotesia flavipes (Hymenoptera: Braconidae); however, very little is known about the effect of the parasitism in the host organs, including the midgut. This work aims to verify mitochondrial alteration in the different midgut epithelial cells of D. saccharalis parasitized by C. flavipes. Midgut fragments (anterior and posterior region) of both non-parasitized and parasitized larvae were processed for transmission electron microscopy. The mitochondria of midgut epithelial cell in the parasitized larvae exhibit morphological alteration, represented by matrix rarefaction and vacuolisation. These mitochondrial alterations are more pronounced in the anterior midgut region during the parasitism process, mainly in the columnar cell.

  13. Role of cathepsins D in the midgut of Dysdercus peruvianus.

    PubMed

    Pimentel, André C; Fuzita, Felipe J; Palmisano, Giuseppe; Ferreira, Clélia; Terra, Walter R

    2017-02-01

    Hemipteran ancestors probably lost their digestive serine peptidases on adapting to a plant sap diet. On returning to protein ingestion, these insects start using cathepsin (lysosomal) peptidases as digestive enzymes, from which the less known is cathepsin D. Nine of the ten cathepsin D transcribing genes found in Dysdercus peruvianus midgut are expressed exclusively in this tissue and only DpCatD10 is also expressed in other tissues. The main action of cathepsins D is in the first (V1) (from three, V1-3) midgut regions, where 40% of the total proteolytic activity was assigned to aspartic peptidases with an optimum pH of 3.5. The most expressed cathepsins D were identified in the midgut luminal contents by proteomics. The data indicate that D. peruvianus have kept a lysosomal gene expressed in all tissues and evolved another set of genes with a digestive function restricted to midgut. Digestive cathepsins D apparently complement the action of digestive cathepsin L and they are arguably responsible for the hydrolysis of cysteine peptidase inhibitors known to be present in the cotton seeds eaten by the insect, before they meet cathepsin L.

  14. A regulatory network controls nephrocan expression and midgut patterning

    PubMed Central

    Hou, Juan; Wei, Wei; Saund, Ranajeet S.; Xiang, Ping; Cunningham, Thomas J.; Yi, Yuyin; Alder, Olivia; Lu, Daphne Y. D.; Savory, Joanne G. A.; Krentz, Nicole A. J.; Montpetit, Rachel; Cullum, Rebecca; Hofs, Nicole; Lohnes, David; Humphries, R. Keith; Yamanaka, Yojiro; Duester, Gregg; Saijoh, Yukio; Hoodless, Pamela A.

    2014-01-01

    Although many regulatory networks involved in defining definitive endoderm have been identified, the mechanisms through which these networks interact to pattern the endoderm are less well understood. To explore the mechanisms involved in midgut patterning, we dissected the transcriptional regulatory elements of nephrocan (Nepn), the earliest known midgut specific gene in mice. We observed that Nepn expression is dramatically reduced in Sox17−/− and Raldh2−/− embryos compared with wild-type embryos. We further show that Nepn is directly regulated by Sox17 and the retinoic acid (RA) receptor via two enhancer elements located upstream of the gene. Moreover, Nepn expression is modulated by Activin signaling, with high levels inhibiting and low levels enhancing RA-dependent expression. In Foxh1−/− embryos in which Nodal signaling is reduced, the Nepn expression domain is expanded into the anterior gut region, confirming that Nodal signaling can modulate its expression in vivo. Together, Sox17 is required for Nepn expression in the definitive endoderm, while RA signaling restricts expression to the midgut region. A balance of Nodal/Activin signaling regulates the anterior boundary of the midgut expression domain. PMID:25209250

  15. Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

    PubMed Central

    Caccia, Silvia; Di Lelio, Ilaria; La Storia, Antonietta; Marinelli, Adriana; Varricchio, Paola; Franzetti, Eleonora; Banyuls, Núria; Tettamanti, Gianluca; Casartelli, Morena; Giordana, Barbara; Ferré, Juan; Gigliotti, Silvia; Pennacchio, Francesco

    2016-01-01

    Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host. PMID:27506800

  16. Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism.

    PubMed

    Caccia, Silvia; Di Lelio, Ilaria; La Storia, Antonietta; Marinelli, Adriana; Varricchio, Paola; Franzetti, Eleonora; Banyuls, Núria; Tettamanti, Gianluca; Casartelli, Morena; Giordana, Barbara; Ferré, Juan; Gigliotti, Silvia; Ercolini, Danilo; Pennacchio, Francesco

    2016-08-23

    Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host.

  17. Aarskog's syndrome with Hirschsprung's disease, midgut malrotation, and dental anomalies.

    PubMed Central

    Hassinger, D D; Mulvihill, J J; Chandler, J B

    1980-01-01

    A 23-year-old man with Aarskog's syndrome had Hirschspring's disease, midgut malrotation, a renal cyst, a cartilaginous projection of the pinna, geographic tongue, and dental anomalies. The family history, negative for these features, including several malignancies. Any or all of these features could be considered part of Aarskog's syndrome and may represent anomalies of neural crest development. Images PMID:7401138

  18. Mamestra configurata nucleopolyhedrovirus-A transcriptome from infected host midgut.

    PubMed

    Donly, B Cameron; Theilmann, David A; Hegedus, Dwayne D; Baldwin, Douglas; Erlandson, Martin A

    2014-02-01

    Infection of an insect by a baculovirus occurs in two distinct phases, an initial infection of host midgut by occlusion-derived virions (ODVs) and subsequent systemic infection of other tissues by budded virions (BV). A vast majority of investigations of the infection process have been restricted to cell culture studies using BV that emulate the systemic phase of infection. This is one of the first studies to investigate baculovirus gene expression in ODV infected midgut cells. We have focused on the critical first phase of in vivo infection by Mamestra configurata nucleopolyhedrovirus-A in M. configurata larvae, using qPCR and RNAseq mass sequencing to measure virus gene expression in midgut cells. The earliest genes detected by each method had significant overlap, including known early genes as well as genes unique to MacoNPV-A and genes of unknown function. The RNAseq data also revealed a large range of expression levels across all ORFs, which could not be measured using qPCR. This dataset provides a first whole genome transcriptomic analysis of viral genes required for virus infection in vivo and will provide the basis for functionally analyzing specific genes that may be critical elements in baculovirus midgut infectivity and host range.

  19. Anopheles gambiae collagen IV genes: cloning, phylogeny and midgut expression associated with blood feeding and Plasmodium infection.

    PubMed

    Gare, D C; Piertney, S B; Billingsley, P F

    2003-07-01

    A prerequisite for understanding the role that mosquito midgut extracellular matrix molecules play in malaria parasite development is proper isolation and characterisation of the genes coding for components of the basal lamina. Here we have identified genes coding for alpha1 and alpha2 chains of collagen IV from the major malaria vector, Anopheles gambiae. Conserved sequences in the terminal NC1 domain were used to obtain partial gene sequences of this functional region, and full sequence was isolated from a pupal cDNA library. In a DNA-derived phylogeny, the alpha1 and alpha2 chains cluster with dipteran orthologs, and the alpha2 is ancestral. The expression of collagen alpha1(IV) peaked during the pupal stage of mosquito development, and was expressed continuously in the adult female following a blood meal with a further rise detected in older mosquitoes. Collagen alpha1(IV) is also upregulated when the early oocyst of Plasmodium yoelii was developing within the mosquito midgut and may contribute to a larger wound healing response. A model describing the expression of basal lamina proteins during oocyst development is presented, and we hypothesise that the development of new basal lamina between the oocyst and midgut epithelium is akin to a wound healing process.

  20. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

    PubMed Central

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIA-BLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

  1. Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells

    PubMed Central

    Korzelius, Jerome; Naumann, Svenja K; Loza-Coll, Mariano A; Chan, Jessica SK; Dutta, Devanjali; Oberheim, Jessica; Gläßer, Christine; Southall, Tony D; Brand, Andrea H; Jones, D Leanne; Edgar, Bruce A

    2014-01-01

    Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). We demonstrate here that loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1. PMID:25298397

  2. Brazilian Pampa Biome Honey Protects Against Mortality, Locomotor Deficits and Oxidative Stress Induced by Hypoxia/Reperfusion in Adult Drosophila melanogaster.

    PubMed

    Cruz, L C; Ecker, A; Dias, R S; Seeger, R L; Braga, M M; Boligon, A A; Martins, I K; Costa-Silva, D G; Barbosa, N V; Cañedo, A D; Posser, T; Franco, J L

    2016-02-01

    We aimed to investigate the potential beneficial effects of the Brazilian Pampa biome honey in a Drosophila-based hypoxia model. Adult flies were reared in standard medium in the presence or absence of honey (at a final concentration of 10 % in medium). Then, control flies (4 % sucrose in medium) and honey-treated flies were submitted to hypoxia. Subsequently, flies were analyzed for mortality, neurolocomotor behavior (negative geotaxis), mitochondrial/oxidative stress parameters and expression of hypoxia/stress related genes by RT-qPCR. The HPLC analysis revealed the presence of phenolics and flavonoids in the studied honey. Caffeic acid was the major compound followed by p-coumaric acid and kaempferol. The presence of such compounds was correlated with a substantial antioxidant activity in vitro. Flies subjected to hypoxia presented marked mortality, locomotor deficits and changes in oxidative stress and mitochondrial activity parameters. Honey treatment was able to completely block mortality and locomotor phenotypes. In addition, honey was able to reverse ROS production and hypoxia-induced changes in mitochondrial complex I and II activity. Hypoxia also induced an up-regulation in mRNA expression of Sima (HIF-1), NFκβ, NRF2, HOX, AKT-1, InR, dILP2, dILP5 and HSP27. Honey treatment was not able to modulate changes in the tested genes, indicating that its protective effects involve additional mechanisms other than transcriptional activity of hypoxia-driven adaptive responses in flies. Our results demonstrated, for the first time, the beneficial effects of honey against the deleterious effects of hypoxia/reperfusion processes in a complex organism.

  3. Midgut Microbial Community of Culex quinquefasciatus Mosquito Populations from India

    PubMed Central

    Chandel, Kshitij; Mendki, Murlidhar J.; Parikh, Rasesh Y.; Kulkarni, Girish; Tikar, Sachin N.; Sukumaran, Devanathan; Prakash, Shri; Parashar, Brahma D.; Shouche, Yogesh S.; Veer, Vijay

    2013-01-01

    The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a

  4. The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit.

    PubMed

    Melnattur, Krishna V; Berdnik, Daniela; Rusan, Zeid; Ferreira, Christopher J; Nambu, John R

    2013-02-01

    In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete-expressing local interneurons in development of the adult olfactory circuitry.

  5. Cloning of PaAtg8 and roles of autophagy in adaptation to starvation with respect to the fat body and midgut of the Americana cockroach, Periplaneta americana.

    PubMed

    Park, Moon Soo; Takeda, Makio

    2014-05-01

    Starvation, in particular amino acid deprivation, induces autophagy in trophocytes (adipocytes), the major component of the fat body cell types, in the larvae of Drosophila melanogaster. However, the fat body of cockroach has two additional cell types: urocytes depositing uric acid in urate vacuoles as a nitrogen resource and mycetocytes harboring an endosymbiont, Blattabacterium cuenoti, which can synthesize amino acids from the metabolites of the stored uric acid. These cells might complement the roles of autophagy in recycling amino acids in the fat body or other organs of cockroaches under starvation. We investigate the presence of autophagy in tissues such as the fat body and midgut of the American cockroach, Periplaneta americana, under starvation by immunoblotting with antibody against Atg8, a ubiquitin-like protein required for the formation of autophagosomes and by electron microscopy. Corresponding changes in acid phosphatase activity were also investigated as representing lysosome activity. Starvation increased the level of an autophagic marker, Atg8-II, in both the tissues, extensively stimulating the formation of autophagic compartments in trophocytes of the fat body and columnar cells of the midgut for over 2 weeks. Acid phosphatase showed no significant increase in the fat body of the starved cockroaches but was higher in the midgut of the continuously fed animals. Thus, a distinct autophagic mechanism operates in these tissues under starvation of 2 weeks and longer. The late induction of autophagy implies exhaustion of the stored uric acid in the fat body. High activity of acid phosphatase in the midgut of the fed cockroaches might represent enhanced assimilation and not an autophagy-related function.

  6. Side Effects of Neem Oil on the Midgut Endocrine Cells of the Green Lacewing Ceraeochrysa claveri (Navás) (Neuroptera: Chrysopidae).

    PubMed

    Scudeler, E L; Santos, D C

    2014-04-01

    We described the ultrastructure of Ceraeochrysa claveri (Navás) midgut endocrine cells in larva, pupa, and adult, and evaluated the side effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree (Azadirachta indica), on these cells. During the larval period, C. claveri were fed (ad libitum) Diatraea saccharalis (F.) eggs treated with neem oil at concentrations of 0.5%, 1%, or 2%. Transmission electron microscopy showed that two subtypes of endocrine cells, namely granular and vesicular, occurred in the midgut epithelium during the three stages of the life cycle. Both cell types did not reach the midgut lumen and were positioned basally in the epithelium. The endocrine cells did not show extensive infoldings of the basal plasma membrane, and there were numerous secretory granules in the basal region of the cytoplasm. In the granular endocrine cells, the granules were completely filled with a dense matrix. In the vesicular endocrine cells, the main secretory products consisted of haloed vesicles. Ultrastructural examination indicated that only the granular endocrine cells exhibited signs of morphologic changes of cell injury present in all life cycle stages after the larvae were chronically exposed to neem oil by ingestion. The major cellular damage consisted of dilatation and vesiculation of the rough endoplasmic reticulum and the development of smooth endoplasmic reticulum and mitochondrial swelling. Our data suggest that cytotoxic effects on midgut endocrine cells can contribute to a generalized disruption of the physiological processes in this organ due to a general alteration of endocrine function.

  7. Drosophila spermiogenesis

    PubMed Central

    Fabian, Lacramioara; Brill, Julie A.

    2012-01-01

    Drosophila melanogaster spermatids undergo dramatic morphological changes as they differentiate from small round cells approximately 12 μm in diameter into highly polarized, 1.8 mm long, motile sperm capable of participating in fertilization. During spermiogenesis, syncytial cysts of 64 haploid spermatids undergo synchronous differentiation. Numerous changes occur at a subcellular level, including remodeling of existing organelles (mitochondria, nuclei), formation of new organelles (flagellar axonemes, acrosomes), polarization of elongating cysts and plasma membrane addition. At the end of spermatid morphogenesis, organelles, mitochondrial DNA and cytoplasmic components not needed in mature sperm are stripped away in a caspase-dependent process called individualization that results in formation of individual sperm. Here, we review the stages of Drosophila spermiogenesis and examine our current understanding of the cellular and molecular mechanisms involved in shaping male germ cell-specific organelles and forming mature, fertile sperm. PMID:23087837

  8. Drosophila sensory cilia lacking MKS proteins exhibit striking defects in development but only subtle defects in adults

    PubMed Central

    Titlow, Joshua S.; Davis, Ilan; Barker, Amy R.; Dawe, Helen R.

    2016-01-01

    ABSTRACT Cilia are conserved organelles that have important motility, sensory and signalling roles. The transition zone (TZ) at the base of the cilium is crucial for cilia function, and defects in several TZ proteins are associated with human congenital ciliopathies such as nephronophthisis (NPHP) and Meckel–Gruber syndrome (MKS). In several species, MKS and NPHP proteins form separate complexes that cooperate with Cep290 to assemble the TZ, but flies seem to lack core components of the NPHP module. We show that MKS proteins in flies are spatially separated from Cep290 at the TZ, and that flies mutant for individual MKS genes fail to recruit other MKS proteins to the TZ, whereas Cep290 seems to be recruited normally. Although there are abnormalities in microtubule and membrane organisation in developing MKS mutant cilia, these defects are less apparent in adults, where sensory cilia and sperm flagella seem to function quite normally. Thus, localising MKS proteins to the cilium or flagellum is not essential for viability or fertility in flies. PMID:27577095

  9. Heme crystallization in the midgut of triatomine insects.

    PubMed

    Oliveira, Marcus F; Gandara, Ana Caroline P; Braga, Cláudia M S; Silva, José R; Mury, Flavia B; Dansa-Petretski, Marílvia; Menezes, Diego; Vannier-Santos, Marcos A; Oliveira, Pedro L

    2007-01-01

    Hemozoin (Hz) is a heme crystal produced by several blood-feeding organisms in order to detoxify free heme released upon hemoglobin (Hb) digestion. Here we show that heme crystallization also occurs in three species of triatomine insects. Ultraviolet-visible and infrared light absorption spectra of insoluble pigments isolated from the midgut of three triatomine species Triatoma infestans, Dipetalogaster maximus and Panstrongylus megistus indicated that all produce Hz. Morphological analysis of T. infestans and D. maximus midguts revealed the close association of Hz crystals to perimicrovillar membranes and also as multicrystalline assemblies, forming nearly spherical structures. Heme crystallization was promoted by isolated perimicrovillar membranes from all three species of triatomine bugs in vitro in heat-sensitive reactions. In conclusion, the data presented here indicate that Hz formation is an ancestral adaptation of Triatominae to a blood-sucking habit and that the presence of perimicrovillar membranes plays a central role in this process.

  10. Insulin receptor in Drosophila melanogaster

    SciTech Connect

    Petruzzelli, L.; Herrera, R.; Rosen, O.

    1986-05-01

    A specific, high affinity insulin receptor is present in both adult Drosophila and in Drosophila embryos. Wheat germ lectin-enriched extracts of detergent-solubilized membranes from embryos and adults bind insulin with a K/sub d/ of 15 nM. Binding is specific for insulin; micromolar concentrations of proinsulin, IGFI, and IGFII are required to displace bound /sup 125/I-insulin. Insulin-dependent protein tyrosine kinase activity appears during embryogenesis. It is evident between 6 and 12 hours of development, peaks between 12 and 18 hours and falls in the adult. During 0-6 hours of embryogenesis, and in the adult, a specific protein band (Mr = 135,000) is crosslinked to /sup 125/I-insulin. During 6-12 and 12-18 hours of embryogenesis stages in which insulin-dependent protein tyrosine kinase is high, an additional band (Mr = 100,000) becomes crosslinked to /sup 125/I-insulin. Isolation and DNA sequence analysis of genomic clones encoding the Drosophila insulin receptor will be presented as will the characterization of insulin receptor mRNA's during development.

  11. The beta subunit of the Drosophila melanogaster ATP synthase: cDNA cloning, amino acid analysis and identification of the protein in adult flies.

    PubMed

    Peña, P; Garesse, R

    1993-09-15

    The cDNA encoding the Drosophila melanogaster beta subunit of H+ ATP synthase has been cloned and sequenced. The predicted mature protein is highly homologous to the equivalent beta subunits of other organisms and is preceded by a signal peptide of 31 amino acids, that although not conserved at primary sequence level has the characteristics of leader peptides present in other mitochondrial proteins. We have raised polyclonal antibodies that specifically recognize the beta H+ ATP synthase subunit present in Drosophila melanogaster protein extracts. This is the first time that a gene of the ATP synthase complex has been characterized in the invertebrate phyla.

  12. Drosophila suzukii population response to environment and management strategies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spotted wing drosophila, Drosophila suzukii, quickly emerged as a devastating invasive pest of small and stone fruits in the Americas and Europe. To better understand the population dynamics of D. suzukii, we reviewed recent work on juvenile development, adult reproduction, and seasonal variation in...

  13. Differential expression of chemosensory-protein genes in midguts in response to diet of Spodoptera litura.

    PubMed

    Yi, Xin; Qi, Jiangwei; Zhou, Xiaofan; Hu, Mei Ying; Zhong, Guo Hua

    2017-03-22

    While it has been well characterized that chemosensory receptors in guts of mammals have great influence on food preference, much remains elusive in insects. Insect chemosensory proteins (CSPs) are soluble proteins that could deliver chemicals to olfactory and gustatory receptors. Recent studies have identified a number of CSPs expressed in midgut in Lepidoptera insects, which started to reveal their roles in chemical recognition and stimulating appetite in midgut. In this study, we examined expression patterns in midgut of 21 Spodoptera litura CSPs (SlitCSPs) characterized from a previously reported transcriptome, and three CSPs were identified to be expressed highly in midgut. The orthologous relationships between midgut expressed CSPs in S. litura and those in Bombyx mori and Plutella xylostella also suggest a conserved pattern of CSP expression in midgut. We further demonstrated that the expression of midgut-CSPs may change in response to different host plants, and SlitCSPs could bind typical chemicals from host plant in vitro. Overall, our results suggested midgut expressed SlitCSPs may have functional roles, likely contributing to specialization and adaption to different ecosystems. Better knowledge of this critical component of the chemsensation signaling pathways in midguts may improve our understanding of food preference processes in a new perspective.

  14. Genetic characterization and cloning of Mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster

    SciTech Connect

    Sekelsky, J.J.; Newfeld, S.J.; Raftery, L.A.; Chartoff, E.H.; Gelbart, W.M.

    1995-03-01

    The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a growth factor that belongs to the transforming growth factor-{beta} (TGF-{beta}) superfamily and that plays a central role in multiple cell-cell signaling events throughout development. Through genetic screens we are seeking to identify other functions that act upstream, downstream or in concert with dpp to mediate its signaling role. We report the genetic characterization and cloning of Mothers against dpp (Mad), a gene identified in two such screens. Mad loss-of-function mutations interact with dpp alleles to enhance embryonic dorsal-ventral patterning defects, as well as adult appendage defects, suggesting a role for Mad in mediating some aspect of dpp function. In support of this, homozygous Mad mutant animals exhibit defects in midgut morphogenesis, imaginal disk development and embryonic dorsal-ventral patterning that are very reminiscent of dpp mutant phenotypes. We cloned the Mad region and identified the Mad transcription unit through germline transformation rescue. We sequenced a Mad cDNA and identified three Mad point mutations that alter the coding information. The predicted MAD polypeptide lacks known protein motifs, but has strong sequence similarity to three polypeptides predicted from genomic sequence from the nematode Caenorhabiditis elegans. Hence, MAD is a member of a novel, highly conserved protein family. 60 refs., 8 figs., 3 tabs.

  15. Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development.

    PubMed Central

    Dornan, S; Jackson, A P; Gay, N J

    1997-01-01

    A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to

  16. Cryobiological preservation of Drosophila embryos

    SciTech Connect

    Mazur, P.; Schreuders, P.D.; Cole, K.W.; Hall, J.W. ); Mahowald, A.P. )

    1992-12-18

    The inability to cryobiologically preserve the fruit fly Drosophila melanogaster has required that fly stocks be maintained by frequent transfer of adults. This method is costly in terms of time and can lead to loss of stocks. Traditional slow freezing methods do not succeed because the embryos are highly sensitive to chilling. With the procedures described here, 68 percent of precisely staged 15-hour Oregon R (wild-type) embryos hatch after vitrification at -205[degree]C, and 40 percent of the resulting larvae develop into normal adult flies. These embryos are among the most complex organisms successfully preserved by cryobiology.

  17. The Drosophila midkine/pleiotrophin homologues Miple1 and Miple2 affect adult lifespan but are dispensable for alk signaling during embryonic gut formation.

    PubMed

    Hugosson, Fredrik; Sjögren, Camilla; Birve, Anna; Hedlund, Ludmilla; Eriksson, Therese; Palmer, Ruth H

    2014-01-01

    Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.

  18. Response of the common cutworm Spodoptera litura to lead stress: changes in sex ratio, Pb accumulations, midgut cell ultrastructure.

    PubMed

    Shu, Yinghua; Zhou, Jialiang; Lu, Kai; Li, Keqing; Zhou, Qiang

    2015-11-01

    When cutworm Spodoptera litura larvae were fed on the diets with different lead (Pb) concentrations for one or five generations, changes in growth and food utilization were recorded; Pb accumulations were detected by Atomic Absorption Spectrophotometer; changes in midgut cell ultrastructure were observed by Transmission Electron Microscopy (TEM). The effects of Pb stress on S. litura growth and food utilization differed significantly between insects of the 1st and 5th generation. The male-female rate of 200mgkg(-1) Pb treatment from the 1st generation and 50mgkg(-1) Pb treatment from the 5th generation was significantly higher than control. No significant difference of Pb accumulations was found in larvae, pupae and adults between the 1st and 5th generation. No significant difference of Pb accumulations in corresponding tissues of larvae was found between male and female. Compared to fat body, hemolymph, head, foregut and hindgut, the highest Pb accumulation was found in migut of larvae exposed to 200mgkg(-1) Pb. TEM showed that expanded intercellular spaces were observed in Pb-treated midgut cells. The nuclei were strongly destroyed by Pb stress, evidenced by chromatin condensation and destroyed nuclear envelope. Mitochondria became swollen with some broken cristae after exposure to Pb. Therefore, neither gender nor progeny difference was present in Pb accumulations of S. litura, although effects of Pb stress on S. litura growth and food utilization differed from different generations and genders. Pb accumulations in midgut caused pathological changes in cells ultrastructure, possibly reflected the growth and food utilization of S. litura.

  19. Characterization of the Cis-Regulatory Region of the Drosophila Homeotic Gene Sex Combs Reduced

    PubMed Central

    Gindhart-Jr., J. G.; King, A. N.; Kaufman, T. C.

    1995-01-01

    The Drosophila homeotic gene Sex combs reduced (Scr) controls the segmental identity of the labial and prothoracic segments in the embryo and adult. It encodes a sequence-specific transcription factor that controls, in concert with other gene products, differentiative pathways of tissues in which Scr is expressed. During embryogenesis, Scr accumulation is observed in a discrete spatiotemporal pattern that includes the labial and prothoracic ectoderm, the subesophageal ganglion of the ventral nerve cord and the visceral mesoderm of the anterior and posterior midgut. Previous analyses have demonstrated that breakpoint mutations located in a 75-kb interval, including the Scr transcription unit and 50 kb of upstream DNA, cause Scr misexpression during development, presumably because these mutations remove Scr cis-regulatory sequences from the proximity of the Scr promoter. To gain a better understanding of the regulatory interactions necessary for the control of Scr transcription during embryogenesis, we have begun a molecular analysis of the Scr regulatory interval. DNA fragments from this 75-kb region were subcloned into P-element vectors containing either an Scr-lacZ or hsp70-lacZ fusion gene, and patterns of reporter gene expression were assayed in transgenic embryos. Several fragments appear to contain Scr regulatory sequences, as they direct reporter gene expression in patterns similar to those normally observed for Scr, whereas other DNA fragments direct Scr reporter gene expression in developmentally interesting but non-Scr-like patterns during embryogenesis. Scr expression in some tissues appears to be controlled by multiple regulatory elements that are separated, in some cases, by more than 20 kb of intervening DNA. Interestingly, regulatory sequences that direct reporter gene expression in an Scr-like pattern in the anterior and posterior midgut are imbedded in the regulatory region of the segmentation gene fushi tarazu (ftz), which is normally located

  20. Optogenetics in Drosophila Neuroscience.

    PubMed

    Riemensperger, Thomas; Kittel, Robert J; Fiala, André

    2016-01-01

    Optogenetic techniques enable one to target specific neurons with light-sensitive proteins, e.g., ion channels, ion pumps, or enzymes, and to manipulate their physiological state through illumination. Such artificial interference with selected elements of complex neuronal circuits can help to determine causal relationships between neuronal activity and the effect on the functioning of neuronal circuits controlling animal behavior. The advantages of optogenetics can best be exploited in genetically tractable animals whose nervous systems are, on the one hand, small enough in terms of cell numbers and to a certain degree stereotypically organized, such that distinct and identifiable neurons can be targeted reproducibly. On the other hand, the neuronal circuitry and the behavioral repertoire should be complex enough to enable one to address interesting questions. The fruit fly Drosophila melanogaster is a favorable model organism in this regard. However, the application of optogenetic tools to depolarize or hyperpolarize neurons through light-induced ionic currents has been difficult in adult flies. Only recently, several variants of Channelrhodopsin-2 (ChR2) have been introduced that provide sufficient light sensitivity, expression, and stability to depolarize central brain neurons efficiently in adult Drosophila. Here, we focus on the version currently providing highest photostimulation efficiency, ChR2-XXL. We exemplify the use of this optogenetic tool by applying it to a widely used aversive olfactory learning paradigm. Optogenetic activation of a population of dopamine-releasing neurons mimics the reinforcing properties of a punitive electric shock typically used as an unconditioned stimulus. In temporal coincidence with an odor stimulus this artificially induced neuronal activity causes learning of the odor signal, thereby creating a light-induced memory.

  1. Leigh Syndrome in Drosophila melanogaster

    PubMed Central

    Da-Rè, Caterina; von Stockum, Sophia; Biscontin, Alberto; Millino, Caterina; Cisotto, Paola; Zordan, Mauro A.; Zeviani, Massimo; Bernardi, Paolo; De Pittà, Cristiano; Costa, Rodolfo

    2014-01-01

    Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LSSurf1 patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R+ cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS. PMID:25164807

  2. Brugia malayi microfilariae transport alphaviruses across the mosquito midgut.

    PubMed

    Vaughan, Jefferson A; Turell, Michael J

    2017-01-01

    Concurrent ingestion of microfilariae (MF) and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Within hours of being ingested, MF penetrate the mosquito midgut and introduce virus into mosquito hemocoel, creating a disseminated viral infection much sooner than normal. How virus is actually introduced is not known. In this report, we present experimental evidence that suggests that certain alphaviruses may adhere or otherwise associate with sheathed Brugia malayi MF in the blood of a dually-infected host and that the virus is carried into the mosquito hemocoel by the MF during their penetration of the mosquito midgut. The mechanism of MF enhancement may be more complex than simple leakage of viremic blood into the hemocoel during MF penetration. The affinity of arboviruses to adhere to or otherwise associate with MF may depend on the specific combination of the virus and MF involved in a dual host infection. This in turn may determine the relative importance that MF enhancement has within an arbovirus transmission system.

  3. Brugia malayi microfilariae transport alphaviruses across the mosquito midgut

    PubMed Central

    Turell, Michael J.

    2017-01-01

    Concurrent ingestion of microfilariae (MF) and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Within hours of being ingested, MF penetrate the mosquito midgut and introduce virus into mosquito hemocoel, creating a disseminated viral infection much sooner than normal. How virus is actually introduced is not known. In this report, we present experimental evidence that suggests that certain alphaviruses may adhere or otherwise associate with sheathed Brugia malayi MF in the blood of a dually-infected host and that the virus is carried into the mosquito hemocoel by the MF during their penetration of the mosquito midgut. The mechanism of MF enhancement may be more complex than simple leakage of viremic blood into the hemocoel during MF penetration. The affinity of arboviruses to adhere to or otherwise associate with MF may depend on the specific combination of the virus and MF involved in a dual host infection. This in turn may determine the relative importance that MF enhancement has within an arbovirus transmission system. PMID:28222120

  4. Morpho-functional characterization and esterase patterns of the midgut of Tribolium castaneum Herbst, 1797 (Coleoptera: Tenebrionidae) parasitized by Gregarina cuneata (Apicomplexa: Eugregarinidae).

    PubMed

    Gigliolli, Adriana A Sinópolis; Lapenta, Ana Silva; Ruvolo-Takasusuki, Maria Claudia Colla; Abrahão, Josielle; Conte, Hélio

    2015-09-01

    Tribolium castaneum (Coleoptera: Tenebrionidae) is a common pest of stored grains and byproducts and is normally infected by Gregarina cuneata (Apicomplexa: Eugregarinidae). The life cycle of this parasite includes the sporozoite, trophozoite, gamont, gametocyte, and oocyst stages, which occur between the epithelium and lumen of the host's midgut. This study aims to describe the morphofunctional alterations in the midgut and determine the esterase patterns in T. castaneum when parasitized by gregarines. To achieve this purpose, midguts of adult insects were isolated, processed, and analysed using light and electron microscopy. We determined total protein content, amylase activity, and the expression and related activities of the esterases by using polyacrylamide gel electrophoresis (PAGE). The midgut of T. castaneum is formed by digestive, regenerative, and endocrine cells. The effects of parasitism on the digestive cells are severe, because the gregarines remain attached to these cells to absorb all the nutrients they need throughout their development. In these cells, the most common alterations observed include expansion and fragmentation of the rough endoplasmic reticulum, development of the smooth endoplasmic reticulum, changes in mitochondrial cristae, cytoplasmic vacuolization, formation of myelin structures, spherites, large intercellular spaces, autophagic vesicles, expansion of the basal labyrinth, and cytoplasmic protrusions. Deposits of glycogen granules were also observed. Amylase activity was reduced in parasitized insects. Regenerative cells were found in disorganized crypts and did not differentiate into new cells, thus, compromising the restoration of the damaged epithelium. Though few morphological alterations were observed in the endocrine cells, results suggest that the synthesis and/or release of hormones might be impaired. Nine esterases (EST-1 to 9) were identified in the midgut of T. castaneum and were expressed in varying levels in response

  5. Bowman-Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster.

    PubMed

    Li, H-M; Sun, L; Mittapalli, O; Muir, W M; Xie, J; Wu, J; Schemerhorn, B J; Jannasch, A; Chen, J Y; Zhang, F; Adamec, J; Murdock, L L; Pittendrigh, B R

    2010-06-01

    Bowman-Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy-associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster. In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI-fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.

  6. Characterization of an Obligate Intracellular Bacterium in the Midgut Epithelium of the Bulrush Bug Chilacis typhae (Heteroptera, Lygaeidae, Artheneinae)▿

    PubMed Central

    Kuechler, Stefan Martin; Dettner, Konrad; Kehl, Siegfried

    2011-01-01

    Many members of the suborder Heteroptera have symbiotic bacteria, which are usually found extracellularly in specific sacs or tubular outgrowths of the midgut or intracellularly in mycetomes. In this study, we describe the second molecular characterization of a symbiotic bacterium in a monophagous, seed-sucking stink bug of the family Lygaeidae (sensu stricto). Chilacis typhae possesses at the end of the first section of the midgut a structure which is composed of circularly arranged, strongly enlarged midgut epithelial cells. It is filled with an intracellular endosymbiont. This “mycetocytic belt” might represent an evolutionarily intermediate stage of the usual symbiotic structures found in stink bugs. Phylogenetic analysis based on the 16S rRNA and the groEL genes showed that the bacterium belongs to the Gammaproteobacteria, and it revealed a phylogenetic relationship with a secondary bacterial endosymbiont of Cimex lectularius and free-living plant pathogens such as Pectobacterium and Dickeya. The distribution and ultrastructure of the rod-shaped Chilacis endosymbiont were studied in adults and nymph stages using fluorescence in situ hybridization (FISH) and electron microscopy. The detection of symbionts at the anterior poles of developing eggs indicates that endosymbionts are transmitted vertically. A new genus and species name, “Candidatus Rohrkolberia cinguli,” is proposed for this newly characterized clade of symbiotic bacteria. PMID:21378044

  7. Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in drosophila.

    PubMed

    Strilbytska, Olha M; Semaniuk, Uliana V; Storey, Kenneth B; Edgar, Bruce A; Lushchak, Oleh V

    2017-01-01

    The TOR (target of rapamycin) signaling pathway and the transcriptional factor Myc play important roles in growth control. Myc acts, in part, as a downstream target of TOR to regulate the activity and functioning of stem cells. Here we explore the role of TOR-Myc axis in stem and progenitor cells in the regulation of lifespan, stress resistance and metabolism in Drosophila. We found that both overexpression of rheb and myc-rheb in midgut stem and progenitor cells decreased the lifespan and starvation resistance of flies. TOR activation caused higher survival under malnutrition conditions. Furthermore, we demonstrate gut-specific activation of JAK/STAT and insulin signaling pathways to control gut integrity. Both genetic manipulations had an impact on carbohydrate metabolism and transcriptional levels of metabolic genes. Our findings indicate that activation of the TOR-Myc axis in midgut stem and progenitor cells influences a variety of traits in Drosophila.

  8. Ultrastructural changes of the midgut epithelium in Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada: Eutardigrada) during oogenesis.

    PubMed

    Rost-Roszkowska, Magdalena M; Poprawa, Izabela; Wójtowicz, Maria; Kaczmarek, Lukasz

    2011-04-01

    The midgut epithelium of Isohypsibius granulifer granulifer (Eutardigrada) is composed of columnar digestive cells. At its anterior end, a group of cells with cytoplasm which differs from the cytoplasm of digestive cells is present. Probably, those cells respond to crescent-like cells (midgut regenerative cells) described for some tardigrade species. Their mitotic divisions have not been observed. We analyzed the ultrastructure of midgut digestive cells in relation to five different stages of oogenesis (previtellogenesis, beginning of the vitellogenesis, vitellogenesis--early choriogenesis, vitellogenesis--middle choriogenesis, late choriogenesis). In the midgut epithelium cells, the gradual accumulation of glycogen granules, lipid droplets and structures of varying electron density occurs. During vitellogenesis and choriogenesis, in the cytoplasm of midgut cells we observed the increasing number of organelles which are responsible for the intensive synthesis of lipids, proteins and saccharides such as cisterns of endoplasmic reticulum and Golgi complexes. At the end of oogenesis, autophagy also intensifies in midgut epithelial cells, which is probably caused by the great amount of reserve material. Midgut epithelium of analyzed species takes part in the yolk precursor synthesis.

  9. Anopheles stephensi Heme Peroxidase HPX15 Suppresses Midgut Immunity to Support Plasmodium Development

    PubMed Central

    Kajla, Mithilesh; Kakani, Parik; Choudhury, Tania Pal; Kumar, Vikas; Gupta, Kuldeep; Dhawan, Rini; Gupta, Lalita; Kumar, Sanjeev

    2017-01-01

    The heme peroxidase HPX15 is an evolutionary conserved anopheline lineage-specific gene. Previously, we found that this gene is present in the genome of 19 worldwide distributed different species of Anopheles mosquito and its orthologs are absent in other mosquitoes, insects, or human. In addition, 65–99% amino acid identity among these 19 orthologs permitted us to hypothesize that the functional aspects of this gene might be also conserved in different anophelines. In this study, we found that Anopheles stephensi AsHPX15 gene is mainly expressed in the midgut and highly induced after uninfected or Plasmodium berghei-infected blood feeding. RNA interference-mediated silencing of midgut AsHPX15 gene drastically reduced the number of developing P. berghei oocysts. An antiplasmodial gene nitric oxide synthase was induced 13-fold in silenced midguts when compared to the unsilenced controls. Interestingly, the induction of antiplasmodial immunity in AsHPX15-silenced midguts is in absolute agreement with Anopheles gambiae. In A. gambiae, AgHPX15 catalyzes the formation of a dityrosine network at luminal side of the midgut that suppresses the activation of mosquito immunity against the bolus bacteria. Thus, a low-immunity zone created by this mechanism indirectly supports Plasmodium development inside the midgut lumen. These indistinguishable functional behaviors and conserved homology indicates that HPX15 might be a potent target to manipulate the antiplasmodial immunity of the anopheline midgut, and it will open new frontiers in the field of malaria control. PMID:28352267

  10. Temporal and spatial expression of caudal-type homeobox proteins in the midgut of human embryos

    PubMed Central

    Tang, Xiao-Bing; Zhang, Jin; Wang, Wei-Lin; Yuan, Zheng-Wei; Bai, Yu-Zuo

    2015-01-01

    Background: This study aimed to determine the spatiotemporal expression of caudal-type homeobox genes (CDX1, CDX2 and CDX4) during development of the midgut in human embryos and to explore the possible roles of CDX genes during the morphogenesis of human midgut. Human embryos (n=28) were sectioned serially and sagittally and CDX1, CDX2 and CDX4 proteins were detected on the midline from the 5th to 9th weeks of gestation by immunohistochemical staining. Results: CDX1, CDX2 and CDX4 proteins were weakly expressed in epithelium and mesenchyme of the midgut in the 6th and 7th weeks of gestation and reached estimated optimal level on the 8th and 9th weeks of gestation. In the 9th week of gestation, immunoreactivities specific to CDX1, CDX2 and CDX4 were restricted in epithelium of the midgut. Conclusions: CDX1, CDX2 and CDX4 proteins began to express in human midgut in the 6th week of gestation. From the 6th to 9th week of gastation, the expression of CDX1, CDX2 and CDX4 proteins gradually increase and exhibited overlapping expression patterns, suggesting that CDX genes may be involved in early development of the epithelium of human midgut. Cross-regulatory interactions may exist among CDX genes with respect to human midgut development. PMID:26884902

  11. Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.

    PubMed

    Elpidina, E N; Vinokurov, K S; Gromenko, V A; Rudenskaya, Y A; Dunaevsky, Y E; Zhuzhikov, D P

    2001-12-01

    Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.

  12. Sugar digestion in mosquitoes: identification and characterization of three midgut alpha-glucosidases of the neo-tropical malaria vector Anopheles aquasalis (Diptera: Culicidae).

    PubMed

    Souza-Neto, Jayme A; Machado, Fábio P; Lima, José B; Valle, Denise; Ribolla, Paulo E M

    2007-08-01

    Dietary carbohydrates provide an important source of energy for flight, and contribute to longevity and fecundity of mosquitoes. The most common sugar mosquitoes ingest is sucrose, and digestion of this substance is carried out mainly by alpha-glucosidases. In the current work, we tested the efficiency of sucrose on Anopheles aquasalis female diet. The best longevity (days) was reached when sugar was available in the diet, whereas most only blood fed females were dead 6 days after emergence. Three alpha-glucosidase isoforms were detected in the adult female midgut, named alphaGlu1, alphaGlu2 and alphaGlu3. These are acidic alpha-glucosidases with optima pH around pH 5.5. alphaGlu1 and alphaGlu2 are present in both secreted and membrane-bound forms, whereas alpha-Glu3 only in anchored to membranes. The alpha-glucosidase activity is concentrated mainly in the posterior midgut (70%), both in non-fed or 10% sucrose fed females. The single form of these alpha-glucosidases seemed to be approximately 70 kDa polypeptides, although alphaGlu2 is presented in >or=600 kDa self-aggregates. Km values of alphaGlu1, alphaGlu2 and alphaGlu3 differed significantly from each other, supporting the statement that three alpha-glucosidases are produced in the female midgut. Together, all data suggest that sugar is an essential component of A. aquasalis female diet. In addition, alpha-glucosidases are synthesized in the same place where sucrose is digested and absorbed, the midgut.

  13. Antioxidant defenses in caterpillars: role of the ascorbate-recycling system in the midgut lumen.

    PubMed

    Barbehenn, R V; Bumgarner, S L; Roosen, E F; Martin, M M

    2001-04-01

    This study demonstrates that an ascorbate-recycling system in the midgut lumen can act as an effective antioxidant defense in caterpillars that feed on prooxidant-rich foods. In tannin-sensitive larvae of the forest tent caterpillar, Malacosoma disstria (Lasiocampidae), ingested tannic acid is oxidized in the midgut lumen, generating significant quantities of peroxides, including hydrogen peroxide, which readily diffuses across cell membranes and is a powerful cytotoxin. By contrast, in the tannin-tolerant larvae of the white-marked tussock moth, Orgyia leucostigma (Lymantriidae), tannic acid oxidation and the generation of peroxides are suppressed. The superior defense of O. leucostigma against oxidative stress imposed by the oxidation of ingested polyphenols can be explained by the presence of higher concentrations of ascorbate and glutathione in the midgut lumen. In O. leucostigma at least 50% of the ingested ascorbate present in the anterior midgut is still present in the posterior midgut, whereas in M. disstria, only 10% of the ascorbate is present in the posterior half of the midgut. We propose that the maintenance of higher levels of ascorbate in the midgut lumen of O. leucostigma than in M. disstria is explained by the secretion of glutathione into the midgut lumen by O. leucostigma, thereby forming a complete ascorbate-recycling system. The concentration of glutathione in the midgut lumen of O. leucostigma is 3.5-fold higher than in M. disstria and more than double the concentration in the diet. Our results emphasize the importance of a defensive strategy in herbivorous insects based on the maintenance of conditions in the gut lumen that reduce or eliminate the potential prooxidant behavior of ingested phenols.

  14. Post-zygotic isolation in cactophilic Drosophila: larval viability and adult life-history traits of D. mojavensis/D. arizonae hybrids

    PubMed Central

    BONO, J. M.; MARKOW, T. A.

    2015-01-01

    Drosophila mojavensis and Drosophila arizonae are cactophilic flies that have been used extensively in speciation studies. Incomplete premating isolation, evidence of reinforcement, and a lack of recent introgression between these species point to a potentially important role for post-zygotic isolating barriers in this system. Other than hybrid male sterility, however, post-zygotic isolation between D. mojavensis and D. arizonae has received little attention. In this study, we examined viability and life-history traits of D. mojavensis/D. arizonae F1 hybrids from sympatric crosses. Specifically, we reared hybrids and purebreds on the natural host cacti of each parental species and compared viability, development time, thorax length, and desiccation resistance between hybrids and purebreds. Interestingly, hybrid females from both crosses performed similarly or even better than purebred females. In contrast, hybrid sons of D. arizonae mothers, in addition to being sterile, had shorter average thorax length than males of both parental species, and hybrid males from both crosses had substantially lower desiccation resistance than D. mojavensis males. The probable cost to hybridization for D. mojavensis females resulting from reduced desiccation resistance of hybrid sons may have been an important selective factor in the history of reinforcement for crosses involving these females. PMID:19508411

  15. Calcium tartrate crystals in the midgut of the grape leafhopper.

    PubMed

    Böll, S; Schmitt, T; Burschka, C; Schreier, P; Schwappach, P; Herrmann, J V

    2005-12-01

    Calcium tartrate crystals were observed in the midgut of grape leafhoppers. This unique compound was found for the first time in insects. The size of the crystals varied strongly between and within individuals with a mean length of 153 +/- 87 microm and a mean width of 71 +/- 46 microm. In addition, the number of crystals per individual showed a broad variation and ranged from 1 to 150 crystals/individual. The occurrence of calcium tartrate crystals as well as the number of crystals per individual followed the same seasonal pattern as seasonal vine leaf concentrations of tartaric acid found in a previous study, indicating that calcium tartrate is formed to neutralize the tartaric acid in the gut system. It further implies that the grape leafhopper, rather than being a pure phloem sucker, employs a mixed feeding strategy to satisfy its demands for calcium uptake.

  16. Characterization of a midgut-specific chitin synthase gene (LmCHS2) responsible for biosynthesis of chitin of peritrophic matrix in Locusta migratoria.

    PubMed

    Liu, Xiaojian; Zhang, Huanhuan; Li, Sheng; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2012-12-01

    Chitin, an essential component of peritrophic matrix (PM), is produced by a series of biochemical reactions. Chitin synthase plays a crucial role in chitin polymerization in chitin biosynthetic pathway. In this study, we identified and characterized a full-length cDNA of chitin synthase 2 gene (LmCHS2) from Locusta migratoria. The cDNA contains an open reading frame of 4569 nucleotides that encode 1523 amino acid residues, and 76- and 373-nucleotides for 5'- and 3'-noncoding regions, respectively. Analysis of LmCHS2 transcript in different tissues of the locust by using real-time quantitative PCR indicated that LmCHS2 was exclusively expressed in midgut and gastric caeca (a part of the midgut). The highest expression was found in the anterior midgut with a decline of the transcript level from the anterior to posterior regions. During growth and development of locusts, there was only a slight expression in eggs, but the expression gradually increased from nymphs to adults. In situ hybridization further revealed that LmCHS2 transcript mainly presented in the apical regions of brush border forming columnar cells of gastric caeca. LmCHS2 dsRNA was injected to fifth-instar nymphs to further explore biological functions of LmCHS2. Significantly down-regulated transcript of LmCHS2 resulted in a cessation of feeding and a high mortality of the insect. However, no visible abnormal morphological change of locusts was observed until insects molted to adults. After dissection, we found that the average length of midguts from the LmCHS2 dsRNA-injected locusts was shorter than that of the control insects that were injected with dsGFP. Furthermore, microsection of midguts showed that the PM of the LmCHS2 dsRNA-injected nymphs was amorphous and thin as compared with the controls. Our results demonstrate that LmCHS2 is responsible for the biosynthesis of chitin associated with PM and plays an essential role in locust growth and development.

  17. Optogenetic pacing in Drosophila melanogaster

    PubMed Central

    Alex, Aneesh; Li, Airong; Tanzi, Rudolph E.; Zhou, Chao

    2015-01-01

    Electrical stimulation is currently the gold standard for cardiac pacing. However, it is invasive and nonspecific for cardiac tissues. We recently developed a noninvasive cardiac pacing technique using optogenetic tools, which are widely used in neuroscience. Optogenetic pacing of the heart provides high spatial and temporal precisions, is specific for cardiac tissues, avoids artifacts associated with electrical stimulation, and therefore promises to be a powerful tool in basic cardiac research. We demonstrated optogenetic control of heart rhythm in a well-established model organism, Drosophila melanogaster. We developed transgenic flies expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), specifically in their hearts and demonstrated successful optogenetic pacing of ChR2-expressing Drosophila at different developmental stages, including the larva, pupa, and adult stages. A high-speed and ultrahigh-resolution optical coherence microscopy imaging system that is capable of providing images at a rate of 130 frames/s with axial and transverse resolutions of 1.5 and 3.9 μm, respectively, was used to noninvasively monitor Drosophila cardiac function and its response to pacing stimulation. The development of a noninvasive integrated optical pacing and imaging system provides a novel platform for performing research studies in developmental cardiology. PMID:26601299

  18. Isolation and characterization of bacteria from midgut of the rice water weevil (Coleoptera: Curculionidae).

    PubMed

    Lu, Fang; Kang, Xiaoying; Jiang, Cong; Lou, Binggan; Jiang, Mingxing; Way, Michael O

    2013-10-01

    Gut bacteria are known to play important and often essential roles in the biology of insects. Theoretically, they can be genetically manipulated, then reintroduced into insects to negatively modify specific biological features. The weevil superfamily Curculionoidea is one of the most species-rich and successful animal groups on earth, but currently the overall knowledge of the bacterial communities in weevils and their associations with hosts is still limited. In this study, we isolated and characterized the bacteria in the midgut of an invasive weevil, Lissorhoptrus oryzophilus Kuschel, by culturing methods. Female adults of this weevil were collected from four different geographic regions of the United States and mainland China. Sequencing of the bacterial 16S rRNA amplicons demonstrated that the major culturable gut bacteria of rice water weevil are γ-proteobacteria and Bacilli. The gut bacterial composition differs among regions, with many of the bacteria isolated from only a single region while several were detected from more than one region. Overall, the diversity of gut bacteria in rice water weevil is relatively low. The possible origins of certain bacteria are discussed in relation to the weevil, rice plant, and bacteria.

  19. Toward new Drosophila paradigms.

    PubMed

    Andrioli, Luiz Paulo

    2012-08-01

    The fruit fly Drosophila melanogaster is a great model system in developmental biology studies and related disciplines. In a historical perspective, I focus on the formation of the Drosophila segmental body plan using a comparative approach. I highlight the evolutionary trend of increasing complexity of the molecular segmentation network in arthropods that resulted in an incredible degree of complexity at the gap gene level in derived Diptera. There is growing evidence that Drosophila is a highly derived insect, and we are still far from fully understanding the underlying evolutionary mechanisms that led to its complexity. In addition, recent data have altered how we view the transcriptional regulatory mechanisms that control segmentation in Drosophila. However, these observations are not all bad news for the field. Instead, they stimulate further study of segmentation in Drosophila and in other species as well. To me, these seemingly new Drosophila paradigms are very challenging ones.

  20. Axon and dendrite pruning in Drosophila.

    PubMed

    Yu, Fengwei; Schuldiner, Oren

    2014-08-01

    Pruning, a process by which neurons selectively remove exuberant or unnecessary processes without causing cell death, is crucial for the establishment of mature neural circuits during animal development. Yet relatively little is known about molecular and cellular mechanisms that govern neuronal pruning. Holometabolous insects, such as Drosophila, undergo complete metamorphosis and their larval nervous systems are replaced with adult-specific ones, thus providing attractive models for studying neuronal pruning. Drosophila mushroom body and dendritic arborization neurons have been utilized as two appealing systems to elucidate the underlying mechanisms of axon and dendrite pruning, respectively. In this review we highlight recent developments and discuss some similarities and differences in the mechanisms that regulate these two distinct modes of neuronal pruning in Drosophila.

  1. Developmental Toxicity Assays Using the Drosophila Model

    PubMed Central

    Rand, Matthew D.; Montgomery, Sara L.; Prince, Lisa; Vorojeikina, Daria

    2014-01-01

    The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. The utility of Drosophila for toxicology studies has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants and drugs. The second assay engages straightforward exposures to developing larvae and easy to score outcomes of adult development. With the extensive collections of flies that are publicly available and the ease with which to create transgenic flies, these two assays have a unique power for identifying and characterizing molecular mechanisms and cellular pathways specific to the mode of action of a number of toxicants and drugs. PMID:24789363

  2. Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

    PubMed Central

    Khattab, Ayman; Barroso, Marta; Miettinen, Tiera; Meri, Seppo

    2015-01-01

    Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood. PMID:25679788

  3. Comparison of midgut bacterial diversity in tropical caterpillars (Lepidoptera: Saturniidae) fed on different diets.

    PubMed

    Pinto-Tomás, Adrián A; Sittenfeld, Ana; Uribe-Lorío, Lorena; Chavarría, Felipe; Mora, Marielos; Janzen, Daniel H; Goodman, Robert M; Simon, Holly M

    2011-10-01

    As primary consumers of foliage, caterpillars play essential roles in shaping the trophic structure of tropical forests. The caterpillar midgut is specialized in plant tissue processing; its pH is exceptionally alkaline and contains high concentrations of toxic compounds derived from the ingested plant material (secondary compounds or allelochemicals) and from the insect itself. The midgut, therefore, represents an extreme environment for microbial life. Isolates from different bacterial taxa have been recovered from caterpillar midguts, but little is known about the impact of these microorganisms on caterpillar biology. Our long-term goals are to identify midgut symbionts and to investigate their functions. As a first step, different diet formulations were evaluated for rearing two species of tropical saturniid caterpillars. Using the polymerase chain reaction (PCR) with primers hybridizing broadly to sequences from the bacterial domain, 16S rRNA gene libraries were constructed with midgut DNA extracted from caterpillars reared on different diets. Amplified rDNA restriction analysis indicated that bacterial sequences recovered from the midguts of caterpillars fed on foliage were more diverse than those from caterpillars fed on artificial diet. Sequences related to Methylobacterium sp., Bradyrhizobium sp., and Propionibacterium sp. were detected in all caterpillar libraries regardless of diet, but were not detected in a library constructed from the diet itself. Furthermore, libraries constructed with DNA recovered from surface-sterilized eggs indicated potential for vertical transmission of midgut symbionts. Taken together, these results suggest that microorganisms associated with the tropical caterpillar midgut may engage in symbiotic interactions with these ecologically important insects.

  4. Identification of the Midgut Microbiota of An. stephensi and An. maculipennis for Their Application as a Paratransgenic Tool against Malaria

    PubMed Central

    Dinparast Djadid, Navid; Jazayeri, Hoda; Raz, Abbasali; Favia, Guido; Ricci, Ignacio; Zakeri, Sedigheh

    2011-01-01

    The midgut microbiota associated with Anopheles stephensi and Anopheles maculipennis (Diptera: Culicidae) was investigated for development of a paratransgenesis-based approach to control malaria transmission in Eastern Mediterranean Region (EMR). Here, we present the results of a polymerase chain reaction (PCR) and biochemical-based approaches to identify the female adult and larvae mosquitoe microbiota of these two major malaria vectors, originated from South Eastern and North of Iran. Plating the mosquito midgut contents from lab-reared and field-collected Anopheles spp. was used for microbiota isolation. The Gram-negative and Gram-positive bacterial colonies were identified by Gram staining and specific mediums. Selected colonies were identified by differential biochemical tests and 16S rRNA gene sequence analysis. A number of 10 An. stephensi and 32 An. maculipennis adult mosquitoes and 15 An. stephensi and 7 An. maculipennis larvae were analyzed and 13 sequences of 16S rRNA gene bacterial species were retrieved, that were categorized in 3 classes and 8 families. The majority of the identified bacteria were belonged to the γ-proteobacteria class, including Pseudomonas sp. and Aeromonas sp. and the others were some closely related to those found in other vector mosquitoes, including Pantoea, Acinetobacter, Brevundimonas, Bacillus, Sphingomonas, Lysinibacillus and Rahnella. The 16S rRNA sequences in the current study aligned with the reference strains available in GenBank were used for construction of the phylogenetic tree that revealed the relatedness among the bacteria identified. The presented data strongly encourage further investigations, to verify the potential role of the detected bacteria for the malaria control in Iran and neighboring countries. PMID:22163022

  5. Identification of the midgut microbiota of An. stephensi and An. maculipennis for their application as a paratransgenic tool against malaria.

    PubMed

    Dinparast Djadid, Navid; Jazayeri, Hoda; Raz, Abbasali; Favia, Guido; Ricci, Ignacio; Zakeri, Sedigheh

    2011-01-01

    The midgut microbiota associated with Anopheles stephensi and Anopheles maculipennis (Diptera: Culicidae) was investigated for development of a paratransgenesis-based approach to control malaria transmission in Eastern Mediterranean Region (EMR). Here, we present the results of a polymerase chain reaction (PCR) and biochemical-based approaches to identify the female adult and larvae mosquitoe microbiota of these two major malaria vectors, originated from South Eastern and North of Iran. Plating the mosquito midgut contents from lab-reared and field-collected Anopheles spp. was used for microbiota isolation. The gram-negative and gram-positive bacterial colonies were identified by Gram staining and specific mediums. Selected colonies were identified by differential biochemical tests and 16S rRNA gene sequence analysis. A number of 10 An. stephensi and 32 An. maculipennis adult mosquitoes and 15 An. stephensi and 7 An. maculipennis larvae were analyzed and 13 sequences of 16S rRNA gene bacterial species were retrieved, that were categorized in 3 classes and 8 families. The majority of the identified bacteria were belonged to the γ-proteobacteria class, including Pseudomonas sp. and Aeromonas sp. and the others were some closely related to those found in other vector mosquitoes, including Pantoea, Acinetobacter, Brevundimonas, Bacillus, Sphingomonas, Lysinibacillus and Rahnella. The 16S rRNA sequences in the current study aligned with the reference strains available in GenBank were used for construction of the phylogenetic tree that revealed the relatedness among the bacteria identified. The presented data strongly encourage further investigations, to verify the potential role of the detected bacteria for the malaria control in Iran and neighboring countries.

  6. The midgut epithelium of aquatic arthropods: a critical target organ in environmental toxicology.

    PubMed Central

    Beaty, Barry J; Mackie, Ryan S; Mattingly, Kimberly S; Carlson, Jonathan O; Rayms-Keller, Alfredo

    2002-01-01

    The midgut epithelium of aquatic arthropods is emerging as an important and toxicologically relevant organ system for monitoring environmental pollution. The peritrophic matrix of aquatic arthropods, which is secreted by the midgut epithelium cells, is perturbed by copper or cadmium. Molecular biological studies have identified and characterized two midgut genes induced by heavy metals in the midgut epithelium. Many other metal-responsive genes (MRGs) await characterization. One of the MRGs codes for an intestinal mucin, which is critical for protecting the midgut from toxins and pathogens. Another codes for a tubulin gene, which is critical for structure and function of the midgut epithelial cells. Perturbation of expression of either gene could condition aquatic arthropod survivorship. Induction of these MRGs is a more sensitive and rapid indicator of heavy-metal pollution than biological assays. Characterization of genes induced by pollutants could provide mechanistic understanding of fundamental cellular responses to pollutants and insight into determinants of aquatic arthropod population genetic structure and survivorship in nature. PMID:12634118

  7. Juvenile hormone regulation of Drosophila aging

    PubMed Central

    2013-01-01

    Background Juvenile hormone (JH) has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone’s source. In contrast, little is known about how juvenile hormone affects adult Drosophila melanogaster. Previous work suggests that insulin signaling may modulate Drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control Drosophila life span. Here we adapt a genetic approach to knock out the corpora allata in adult Drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. With this system we test potential explanations for how juvenile hormone modulates aging. Results A tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. Corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. Treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. Knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. Dietary restriction further extended the lifespan of knockout females. In an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about 100 genes changed in response to loss of juvenile hormone independent of reproductive state. Conclusions Reduced juvenile hormone alone is sufficient to extend the lifespan of Drosophila melanogaster. Reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. Our data do not support a mechanism for juvenile hormone control

  8. Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

    PubMed Central

    Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian

    2015-01-01

    ABSTRACT Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the

  9. Midgut Barrier Imparts Selective Resistance to Filarial Worm Infection in Culex pipiens pipiens

    PubMed Central

    Michalski, Michelle L.; Erickson, Sara M.; Bartholomay, Lyric C.; Christensen, Bruce M.

    2010-01-01

    Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF) caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies of these worms

  10. Infection Dynamics and Immune Response in a Newly Described Drosophila-Trypanosomatid Association

    PubMed Central

    Votýpka, Jan; Dostálová, Anna; Yurchenko, Vyacheslav; Bird, Nathan H.; Lukeš, Julius; Lemaitre, Bruno

    2015-01-01

    ABSTRACT Trypanosomatid parasites are significant causes of human disease and are ubiquitous in insects. Despite the importance of Drosophila melanogaster as a model of infection and immunity and a long awareness that trypanosomatid infection is common in the genus, no trypanosomatid parasites naturally infecting Drosophila have been characterized. Here, we establish a new model of trypanosomatid infection in Drosophila—Jaenimonas drosophilae, gen. et sp. nov. As far as we are aware, this is the first Drosophila-parasitic trypanosomatid to be cultured and characterized. Through experimental infections, we find that Drosophila falleni, the natural host, is highly susceptible to infection, leading to a substantial decrease in host fecundity. J. drosophilae has a broad host range, readily infecting a number of Drosophila species, including D. melanogaster, with oral infection of D. melanogaster larvae resulting in the induction of numerous immune genes. When injected into adult hemolymph, J. drosophilae kills D. melanogaster, although interestingly, neither the Imd nor the Toll pathway is induced and Imd mutants do not show increased susceptibility to infection. In contrast, mutants deficient in drosocrystallin, a major component of the peritrophic matrix, are more severely infected during oral infection, suggesting that the peritrophic matrix plays an important role in mediating trypanosomatid infection in Drosophila. This work demonstrates that the J. drosophilae-Drosophila system can be a powerful model to uncover the effects of trypanosomatids in their insect hosts. PMID:26374124

  11. Rapid Establishment of a Regular Distribution of Adult Tropical Drosophila Parasitoids in a Multi-Patch Environment by Patch Defence Behaviour

    PubMed Central

    de Jong, Peter W.; Hemerik, Lia; Gort, Gerrit; van Alphen, Jacques J. M.

    2011-01-01

    Females of the larval parasitoid of Drosophila, Asobara citri, from sub-Saharan Africa, defend patches with hosts by fighting and chasing conspecific females upon encounter. Females of the closely related, palearctic species Asobara tabida do not defend patches and often search simultaneously in the same patch. The effect of patch defence by A. citri females on their distribution in a multi-patch environment was investigated, and their distributions were compared with those of A. tabida. For both species 20 females were released from two release-points in replicate experiments. Females of A. citri quickly reached a regular distribution across 16 patches, with a small variance/mean ratio per patch. Conversely, A. tabida females initially showed a clumped distribution, and after gradual dispersion, a more Poisson-like distribution across patches resulted (variance/mean ratio was closer to 1 and higher than for A. citri). The dispersion of A. tabida was most probably an effect of exploitation: these parasitoids increasingly made shorter visits to already exploited patches. We briefly discuss hypotheses on the adaptive significance of patch defence behaviour or its absence in the light of differences in the natural history of both parasitoid species, notably the spatial distribution of their hosts. PMID:21765889

  12. Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut.

    PubMed

    Kelkenberg, Marco; Odman-Naresh, Jothini; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

    2015-01-01

    In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB

  13. Morphological alterations induced by boric acid and fipronil in the midgut of worker honeybee (Apis mellifera L.) larvae : Morphological alterations in the midgut of A. mellifera.

    PubMed

    da Silva Cruz, Aline; da Silva-Zacarin, Elaine C M; Bueno, Odair C; Malaspina, Osmar

    2010-04-01

    Morphological alterations, by means of histological and ultrastructural analysis, have been used to determine the effects of boric acid and fipronil on midgut tissues of honeybee worker, Apis mellifera L. larvae. In order to observe possible morphological alterations in the midgut, two groups of bioassays were performed. In the first one, the larvae were chronically treated with different concentrations of boric acid added to the food (1.0, 2.5 and 7.5 mg/g). In the second group, the larvae were fed with diets containing different concentrations of fipronil (0.1 and 1 microg/g) and compared with control groups without these chemical compounds. In the first bioassay, the larvae were collected on day 3 and in the second bioassay on day 4, when the mortality rate obtained in the toxicological bioassay was not very high. The larval midguts were removed and processed for morphological analyses using a light and transmission electron microscopy. We observed cytoplasmic vacuolizations, with the absence of autophagic vacuoles, and chromatinic compacting in most of the cells in the groups treated with pesticides. The morphological alterations were far greater in the larvae treated with boric acid than in the larvae treated with fipronil. Our data suggest that the midgut cell death observed was in response to boric acid and fipronil action. This study significantly improves the understanding of the toxicological effect of these insecticides from the ecotoxicological perspective.

  14. Regulation of chitin synthesis in the larval midgut of Manduca sexta.

    PubMed

    Zimoch, L; Hogenkamp, D G; Kramer, K J; Muthukrishnan, S; Merzendorfer, H

    2005-06-01

    In insects, chitin is not only synthesized by ectodermal cells that form chitinous cuticles, but also by endodermal cells of the midgut that secrete a chitinous peritrophic matrix. Using anti-chitin synthase (CHS) antibodies, we previously demonstrated that in the midgut of Manduca sexta, CHS is expressed by two cell types, tracheal cells forming a basal tracheal network and columnar cells forming the apical brush border [Zimoch and Merzendorfer, 2002, Cell Tissue Res. 308, 287-297]. Now, we show that two different genes, MsCHS1 and MsCHS2, encode CHSs of midgut tracheae and columnar cells, respectively. To investigate MsCHS2 expression and activity in the course of the larval development, we monitored chitin synthesis, enzyme levels as well as mRNA amounts. All of the tested parameters were significantly reduced during molting and in the wandering stage when compared to the values obtained from intermolt feeding larvae. By contrast, MsCHS1 appeared to be inversely regulated because its mRNA was detectable only during the molt at the time when tracheal growth occurs at the basal site of the midgut. To further examine midgut chitin synthesis, we measured enzyme activity in crude midgut extracts and different membrane fractions. When we analysed trypsin-mediated proteolytic activation, a phenomenon previously reported for insect and fungal systems, we recognized that midgut chitin synthesis was only activated in crude extracts, but not in the 12,000 g membrane fraction. However, proteolytic activation by trypsin in the 12,000 g membrane fraction could be reconstituted by re-adding a soluble fraction, indicating that limited proteolysis affects an unknown soluble factor, a process that in turn activates chitin synthesis.

  15. Biotic and abiotic factors impacting development, behavior, phenology, and reproductive biology of Drosophila suzukii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spotted wing drosophila, Drosophila suzukii, quickly emerged as a devastating invasive pest of small and stone fruits in the Americas and Europe. To better understand the population dynamics of D. suzukii, we reviewed recent work on juvenile development, adult reproduction, and seasonal variation in...

  16. Dimethylnitrosamine demethylase activity in Drosophila melanogaster

    SciTech Connect

    Waters, L.C.; Nix, C.E.; Epler, J.L.

    1982-06-15

    A dimethylnitrosamine (DMN) demethylase with levels of activity comparable to that in uninduced rat liver was demonstrated in both larval and adult forms of the Hikone-R strain of Drosophila. A microsomal enzyme, it has many properties of a cytochrome P-450-containing mixed-function oxidase. Kinetic analysis indicates only a single enzyme with an apparent K/sub m/ of 10.5 mM DMN.

  17. Effects of Periplocoside P from Periploca sepium on the Midgut Transmembrane Potential of Mythimna separata Larvae

    PubMed Central

    Wang, YingYing; Qi, Zhijun; Qi, Meng; Hu, Zhaonong; Wu, Wenjun

    2016-01-01

    Periplocoside P (PSP) isolated from the root bark of Periploca sepium contains a pregnane glycoside skeleton and possesses high insecticidal properties. Preliminary studies indicated that PSP disrupts epithelial functions in the midgut of lepidopteran larvae. In the present study, we examined the effects of PSP on the apical and basolateral membrane voltages, Va and Vbl, respectively, of cells from (1) midguts isolated from the larvae of the oriental armyworm Mythimna separata that were in vitro incubated with toxins and (2) midguts isolated from M. separata larvae force-fed with PSP. We compared the effects of PSP with the effects of the Bacillus thuringiensis toxin Cry1Ab and inactive periplocoside E (PSE) on the midgut epithelial cells. The results showed that Va rapidly decreased in the presence of PSP in a time- and dose-dependent manner, similar to the effects of Cry1Ab. By contrast, PSE did not affect the Va and Vbl. Additionally, PSP did not influence the Vbl. Given these results, we speculate that PSP may modulate transport mechanisms at the apical membrane of the midgut epithelial cells by inhibiting the V-type H+ ATPase. PMID:27833169

  18. Mod(mdg4) participates in hormonally regulated midgut programmed cell death during metamorphosis.

    PubMed

    Cai, Mei-Juan; Liu, Wen; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2012-12-01

    The insect midgut undergoes programmed cell death (PCD) during metamorphosis, but the molecular basis for this phenomenon has not been demonstrated. We report a mod(mdg4) protein [designated as mod(mdg4)1A] that is involved in hormonally regulated insect midgut PCD, from the lepidopteran Helicoverpa armigera. Mod(mdg4)1A is localized in the larval midgut and is highly expressed during metamorphosis. Knockdown of mod(mdg4)1a by feeding dsRNA to the larvae suppressed midgut PCD and delayed metamorphosis. The mechanism is that mod(mdg4)1a knockdown decreased the transcript levels of genes involved in PCD and metamorphosis, but increased the transcript level of inhibitor of apoptosis survivin. The transcript level of mod(mdg4)1a is independently upregulated by 20-hydroxyecdysone (20E) or juvenile hormone (JH) analog methoprene. Overlapped 20E and methoprene counteractively regulate the transcript level of mod(mdg4)1a. 20E upregulates the mod(mdg4)1a transcript level not through its nuclear receptor EcRB1. Methoprene upregulates the mod(mdg4)1a transcript level through the juvenile hormone candidate receptor Met. These findings indicate that mod(mdg4)1a participates in midgut PCD and metamorphosis by regulating the transcript levels of a network of genes via different pathways under 20E and JH regulation.

  19. Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

    PubMed

    Lomate, Purushottam R; Jadhav, Bhakti R; Giri, Ashok P; Hivrale, Vandana K

    2013-01-01

    Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

  20. Investigation of the midgut structure and ultrastructure in Cimex lectularius and Cimex pipistrelli (Hemiptera: Cimicidae).

    PubMed

    Rost-Roszkowska, M M; Vilimova, J; Włodarczyk, A; Sonakowska, L; Kamińska, K; Kaszuba, F; Marchewka, A; Sadílek, D

    2017-02-01

    Cimicidae are temporary ectoparasites, which means that they cannot obtain food continuously. Both Cimex species examined here, Cimex lectularius (Linnaeus 1758) and Cimex pipistrelli (Jenyns 1839), can feed on a non-natal host, C. lectularius from humans on bats, C. pipistrelli on humans, but never naturally. The midgut of C. lectularius and C. pipistrelli is composed of three distinct regions-the anterior midgut (AMG), which has a sack-like shape, the long tube-shaped middle midgut (MMG), and the posterior midgut (PMG). The different ultrastructures of the AMG, MMG, and PMG in both of the species examined suggest that these regions must fulfill different functions in the digestive system. Ultrastructural analysis showed that the AMG fulfills the role of storing food and synthesizing and secreting enzymes, while the MMG is the main organ for the synthesis of enzymes, secretion, and the storage of the reserve material. Additionally, both regions, the AMG and MMG, are involved in water absorption in the digestive system of both Cimex species. The PMG is the part of the midgut in which spherites accumulate. The results of our studies confirm the suggestion of former authors that the structure of the digestive tract of insects is not attributed solely to diet but to the basic adaptation of an ancestor.

  1. Drosophila Blastorderm Analysis Software

    SciTech Connect

    2006-10-25

    PointCloudMake analyzes 3D fluorescent images of whole Drosophila embryo and produces a table-style "PointCloud" file which contains the coordinates and volumes of all the nuclei, cells, their associated relative gene expression levels along with morphological features of the embryo. See: Luengo Hendrix et at 2006 3D Morphology and Gene Expression in the Drosophila Blastoderm at Cellular Resolution manuscript submitted LBNL # LBNL-60178 Knowles DW, Keranen SVE, Biggin M. Sudar S (2002) Mapping organism expression levels at cellular resolution in developing Drosophila. In: Conchello JA, Cogswell CJ, Wilson T, editors. Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX. pp. 57-64

  2. Olfactory Learning in Individually Assayed Drosophila Larvae

    PubMed Central

    Scherer, Sabine; Stocker, Reinhard F.; Gerber, Bertram

    2003-01-01

    Insect and mammalian olfactory systems are strikingly similar. Therefore, Drosophila can be used as a simple model for olfaction and olfactory learning. The brain of adult Drosophila, however, is still complex. We therefore chose to work on the larva with its yet simpler but adult-like olfactory system and provide evidence for olfactory learning in individually assayed Drosophila larvae. We developed a differential conditioning paradigm in which odorants are paired with positive (“+” fructose) or negative (“-” quinine or sodium chloride) gustatory reinforcers. Test performance of individuals from two treatment conditions is compared—one received odorant A with the positive reinforcer and odorant B with a negative reinforcer (A+/B-); animals from the other treatment condition were trained reciprocally (A-/B+). During test, differences in choice between A and B of individuals having undergone either A+/B- or A-/B+ training therefore indicate associative learning. We provide such evidence for both combinations of reinforcers; this was replicable across repetitions, laboratories, and experimenters. We further show that breaks improve performance, in accord with basic principles of associative learning. The present individual assay will facilitate electrophysiological studies, which necessarily use individuals. As such approaches are established for the larval neuromuscular synapse, but not in adults, an individual larval learning paradigm will serve to link behavioral levels of analysis to synaptic physiology. PMID:12773586

  3. The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.

    PubMed Central

    Spradling, A C; Stern, D; Beaton, A; Rhem, E J; Laverty, T; Mozden, N; Misra, S; Rubin, G M

    1999-01-01

    A fundamental goal of genetics and functional genomics is to identify and mutate every gene in model organisms such as Drosophila melanogaster. The Berkeley Drosophila Genome Project (BDGP) gene disruption project generates single P-element insertion strains that each mutate unique genomic open reading frames. Such strains strongly facilitate further genetic and molecular studies of the disrupted loci, but it has remained unclear if P elements can be used to mutate all Drosophila genes. We now report that the primary collection has grown to contain 1045 strains that disrupt more than 25% of the estimated 3600 Drosophila genes that are essential for adult viability. Of these P insertions, 67% have been verified by genetic tests to cause the associated recessive mutant phenotypes, and the validity of most of the remaining lines is predicted on statistical grounds. Sequences flanking >920 insertions have been determined to exactly position them in the genome and to identify 376 potentially affected transcripts from collections of EST sequences. Strains in the BDGP collection are available from the Bloomington Stock Center and have already assisted the research community in characterizing >250 Drosophila genes. The likely identity of 131 additional genes in the collection is reported here. Our results show that Drosophila genes have a wide range of sensitivity to inactivation by P elements, and provide a rationale for greatly expanding the BDGP primary collection based entirely on insertion site sequencing. We predict that this approach can bring >85% of all Drosophila open reading frames under experimental control. PMID:10471706

  4. Metallothionein genes in Drosophila melanogaster constitute a dual system.

    PubMed Central

    Mokdad, R; Debec, A; Wegnez, M

    1987-01-01

    We have selected a metallothionein (MT) cDNA clone from a cadmium-resistant Drosophila melanogaster cell line. This clone includes an open reading frame coding for a 43-amino acid protein whose characteristics are a high cysteine content (12 cysteines, 28% of all residues) and a lack of aromatic amino acids. This protein differs markedly from the Drosophila MT (Mtn gene) previously reported [Lastowski-Perry, D., Otto, E. & Maroni, G. (1985) J. Biol. Chem. 260, 1527-1530). The MT system of Drosophila thus consists of at least two distantly related genes, in sharp contrast with vertebrate MT systems, in which the different members of MT gene families display high similarity. The gene corresponding to our MT cDNA (Mto) is inducible in Drosophila cell lines and in both larval and adult flies. Images PMID:3106973

  5. The Utilization of Sugars and Other Substances by Drosophila,

    DTIC Science & Technology

    1948-03-01

    Anastrepha ludens (Baker, et al., 1944), and a number of others. The reviews of Trager, 1941.and 1947, 4nd Uvarov, 1928, furnish extensive re...investigated for several species. The results in hand for the adult and larval bee, the adult blowfly, and for the adult fruit flies Anastrepha and...follows: mannose is used by Calliphora, Anastrepha and Drosophila, but not by the bee. Indeed von Frisch (1934:) .and Staudenmayer (1936)),Ihave

  6. Meiosis in male Drosophila

    PubMed Central

    McKee, Bruce D.; Yan, Rihui; Tsai, Jui-He

    2012-01-01

    Meiosis entails sorting and separating both homologous and sister chromatids. The mechanisms for connecting sister chromatids and homologs during meiosis are highly conserved and include specialized forms of the cohesin complex and a tightly regulated homolog synapsis/recombination pathway designed to yield regular crossovers between homologous chromatids. Drosophila male meiosis is of special interest because it dispenses with large segments of the standard meiotic script, particularly recombination, synapsis and the associated structures. Instead, Drosophila relies on a unique protein complex composed of at least two novel proteins, SNM and MNM, to provide stable connections between homologs during meiosis I. Sister chromatid cohesion in Drosophila is mediated by cohesins, ring-shaped complexes that entrap sister chromatids. However, unlike other eukaryotes Drosophila does not rely on the highly conserved Rec8 cohesin in meiosis, but instead utilizes two novel cohesion proteins, ORD and SOLO, which interact with the SMC1/3 cohesin components in providing meiotic cohesion. PMID:23087836

  7. Drosophila comet assay: insights, uses, and future perspectives

    PubMed Central

    Gaivão, Isabel; Sierra, L. María

    2014-01-01

    The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type. PMID:25221574

  8. Ferritin Is Required in Multiple Tissues during Drosophila melanogaster Development

    PubMed Central

    Blowes, Liisa M.; Missirlis, Fanis; Riesgo-Escovar, Juan R.

    2015-01-01

    In Drosophila melanogaster, iron is stored in the cellular endomembrane system inside a protein cage formed by 24 ferritin subunits of two types (Fer1HCH and Fer2LCH) in a 1:1 stoichiometry. In larvae, ferritin accumulates in the midgut, hemolymph, garland, pericardial cells and in the nervous system. Here we present analyses of embryonic phenotypes for mutations in Fer1HCH, Fer2LCH and in both genes simultaneously. Mutations in either gene or deletion of both genes results in a similar set of cuticular embryonic phenotypes, ranging from non-deposition of cuticle to defects associated with germ band retraction, dorsal closure and head involution. A fraction of ferritin mutants have embryonic nervous systems with ventral nerve cord disruptions, misguided axonal projections and brain malformations. Ferritin mutants die with ectopic apoptotic events. Furthermore, we show that ferritin maternal contribution, which varies reflecting the mother’s iron stores, is used in early development. We also evaluated phenotypes arising from the blockage of COPII transport from the endoplasmic reticulum to the Golgi apparatus, feeding the secretory pathway, plus analysis of ectopically expressed and fluorescently marked Fer1HCH and Fer2LCH. Overall, our results are consistent with insect ferritin combining three functions: iron storage, intercellular iron transport, and protection from iron-induced oxidative stress. These functions are required in multiple tissues during Drosophila embryonic development. PMID:26192321

  9. Ferritin Is Required in Multiple Tissues during Drosophila melanogaster Development.

    PubMed

    González-Morales, Nicanor; Mendoza-Ortíz, Miguel Ángel; Blowes, Liisa M; Missirlis, Fanis; Riesgo-Escovar, Juan R

    2015-01-01

    In Drosophila melanogaster, iron is stored in the cellular endomembrane system inside a protein cage formed by 24 ferritin subunits of two types (Fer1HCH and Fer2LCH) in a 1:1 stoichiometry. In larvae, ferritin accumulates in the midgut, hemolymph, garland, pericardial cells and in the nervous system. Here we present analyses of embryonic phenotypes for mutations in Fer1HCH, Fer2LCH and in both genes simultaneously. Mutations in either gene or deletion of both genes results in a similar set of cuticular embryonic phenotypes, ranging from non-deposition of cuticle to defects associated with germ band retraction, dorsal closure and head involution. A fraction of ferritin mutants have embryonic nervous systems with ventral nerve cord disruptions, misguided axonal projections and brain malformations. Ferritin mutants die with ectopic apoptotic events. Furthermore, we show that ferritin maternal contribution, which varies reflecting the mother's iron stores, is used in early development. We also evaluated phenotypes arising from the blockage of COPII transport from the endoplasmic reticulum to the Golgi apparatus, feeding the secretory pathway, plus analysis of ectopically expressed and fluorescently marked Fer1HCH and Fer2LCH. Overall, our results are consistent with insect ferritin combining three functions: iron storage, intercellular iron transport, and protection from iron-induced oxidative stress. These functions are required in multiple tissues during Drosophila embryonic development.

  10. Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae).

    PubMed

    Fialho, Maria C Q; Moreira, Nathalia R; Zanuncio, José C; Ribeiro, Alberto F; Terra, Walter R; Serrão, José E

    2012-06-01

    Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 μM); CAL2 (17 kDa, pHo 5.5, km 11 μM Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM l-Leucine-p-nitroanilide), α-glucosidase (90 kDa, pHo 5.0, km 5mM with p-nitrophenyl α-d-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans.

  11. Two essential peritrophic matrix proteins mediate matrix barrier functions in the insect midgut.

    PubMed

    Agrawal, Sinu; Kelkenberg, Marco; Begum, Khurshida; Steinfeld, Lea; Williams, Clay E; Kramer, Karl J; Beeman, Richard W; Park, Yoonseong; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

    2014-06-01

    The peritrophic matrix (PM) in the midgut of insects consists primarily of chitin and proteins and is thought to support digestion and provide protection from abrasive food particles and enteric pathogens. We examined the physiological roles of 11 putative peritrophic matrix protein (PMP) genes of the red flour beetle, Tribolium castaneum (TcPMPs). TcPMP genes are differentially expressed along the length of the midgut epithelium of feeding larvae. RNAi of individual PMP genes revealed no abnormal developmental phenotypes for 9 of the 11 TcPMPs. However, RNAi for two PMP genes, TcPMP3 and TcPMP5-B, resulted in depletion of the fat body, growth arrest, molting defects and mortality. In situ permeability assays after oral administration of different-sized FITC-dextran beads demonstrated that the exclusion size of the larval peritrophic matrix (PM) decreases progressively from >2 MDa to <4 kDa from the anterior to the most posterior regions of the midgut. In the median midguts of control larvae, 2 MDa dextrans were completely retained within the PM lumen, whereas after RNAi for TcPMP3 and TcPMP5-B, these dextrans penetrated the epithelium of the median midgut, indicating loss of structural integrity and barrier function of the larval PM. In contrast, RNAi for TcPMP5-B, but not RNAi for TcPMP3, resulted in breakdown of impermeability to 4 and 40 kDa dextrans in the PM of the posterior midgut. These results suggest that specific PMPs are involved in the regulation of PM permeability, and that a gradient of barrier function is essential for survival and fat body maintenance.

  12. A comparison of Frost expression among species and life stages of Drosophila.

    PubMed

    Bing, X; Zhang, J; Sinclair, Brent J

    2012-02-01

    Frost (Fst) is a gene associated with cold exposure in Drosophila melanogaster. We used real-time PCR to assess whether cold exposure induces expression of Fst in 10 different life stages of D. melanogaster, and adults of seven other Drosophila species. We exposed groups of individuals to 0 °C (2 h), followed by 1 h recovery (22 °C). Frost was significantly upregulated in response to cold in eggs, third instar larvae, and 2- and 5-day-old male and female adults in D. melanogaster. Life stages in which cold did not upregulate Fst had high constitutive expression. Frost is located on the opposite strand of an intron of Diuretic hormone (DH), but cold exposure did not upregulate DH. Frost orthologues were identified in six other species within the Melanogaster group (Drosophila sechellia, Drosophila simulans, Drosophila yakuba, Drosophila erecta, Drosophila ananassae and Drosophila mauritiana). Frost orthologues were upregulated in response to cold exposure in both sexes in adults of all of these species. The predicted structure of a putative Frost consensus protein shows highly conserved tandem repeats of motifs involved in cell signalling (PEST and TRAF2), suggesting that Fst might encode an adaptor protein involved in acute stress or apoptosis signalling in vivo.

  13. In focus: spotted wing drosophila, Drosophila suzukii, across perspectives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An effective response to the invasion of spotted wing Drosophila (SWD), Drosophila suzukii, requires proper taxonomic identification at the initial phase, understanding its basic biology and phenology, developing management tools, transferring information and technology quickly to user groups, and e...

  14. Determination of Blastoderm Cells in Drosophila melanogaster

    PubMed Central

    Chan, L.-N.; Gehring, W.

    1971-01-01

    A method for culturing blastoderm cells of Drosophila in vivo has been developed that allows these cells to differentiate into larval or adult structures. By intermixture of genetically marked cells from bisected and whole embryos, it was shown that blastoderm cells are restricted in their potential for forming adult epidermal structures. Cells isolated from anterior-half embryos are determined for forming head and thoracic structures, whereas cells from posterior-half embryos are determined for forming thoracic and abdominal structures. The specificity of determination and the localization of determinative factors is discussed. Images PMID:5002429

  15. Functional morphology of the midgut of a sandfly as compared to other hematophagous nematocera.

    PubMed

    Rudin, W; Hecker, H

    1982-01-01

    The midgut epithelium of female Lutzomyia longipalpis was investigated by means of electron microscopic morphometry before and during blood digestion. Ultrastructure and cytological changes of the stomach cells upon blood feeding were generally similar to the ones described for Phlebotomus longipes (Gemetchu, 1974) and for mosquitoes (Hecker, 1977). In addition, the quantitative composition of the cells resembled the one of mosquitoes in many respects. Despite some morphological differences in the functional gut cytology, it can be admitted that, in general, digestive processes may run similarly in the midguts of sandflies and mosquitoes.

  16. Detection of heparin in the salivary gland and midgut of Aedes togoi.

    PubMed

    Ha, Young-Ran; Oh, So-Ra; Seo, Eun-Seok; Kim, Bo-Heum; Lee, Dong-Kyu; Lee, Sang-Joon

    2014-04-01

    Mosquitoes secrete saliva that contains biological substances, including anticoagulants that counteract a host's hemostatic response and prevent blood clotting during blood feeding. This study aimed to detect heparin, an anticoagulant in Aedes togoi using an immunohistochemical detection method, in the salivary canal, salivary gland, and midgut of male and female mosquitoes. Comparisons showed that female mosquitoes contained higher concentrations of heparin than male mosquitoes. On average, the level of heparin was higher in blood-fed female mosquitoes than in non-blood-fed female mosquitoes. Heparin concentrations were higher in the midgut than in the salivary gland. This indicates presence of heparin in tissues of A. togoi.

  17. The Dopaminergic System in the Aging Brain of Drosophila

    PubMed Central

    White, Katherine E.; Humphrey, Dickon M.; Hirth, Frank

    2010-01-01

    Drosophila models of Parkinson's disease are characterized by two principal phenotypes: the specific loss of dopaminergic (DA) neurons in the aging brain and defects in motor behavior. However, an age-related analysis of these baseline parameters in wildtype Drosophila is lacking. Here we analyzed the DA system and motor behavior in aging Drosophila. DA neurons in the adult brain can be grouped into bilateral symmetric clusters, each comprising a stereotypical number of cells. Analysis of TH > mCD8::GFP and cell type-specific MARCM clones revealed that DA neurons show cluster-specific, stereotypical projection patterns with terminal arborization in target regions that represent distinct functional areas of the adult brain. Target areas include the mushroom bodies, involved in memory formation and motivation, and the central complex, involved in the control of motor behavior, indicating that similar to the mammalian brain, DA neurons in the fly brain are involved in the regulation of specific behaviors. Behavioral analysis revealed that Drosophila show an age-related decline in startle-induced locomotion and negative geotaxis. Motion tracking however, revealed that walking activity, and exploration behavior, but not centrophobism increase at late stages of life. Analysis of TH > Dcr2, mCD8::GFP revealed a specific effect of Dcr2 expression on walking activity but not on exploratory or centrophobic behavior, indicating that the siRNA pathway may modulate distinct DA behaviors in Drosophila. Moreover, DA neurons were maintained between early- and late life, as quantified by TH > mCD8::GFP and anti-TH labeling, indicating that adult onset, age-related degeneration of DA neurons does not occur in the aging brain of Drosophila. Taken together, our data establish baseline parameters in Drosophila for the study of Parkinson's disease as well as other disorders affecting DA neurons and movement control. PMID:21165178

  18. Silencing of Anopheles stephensi Heme Peroxidase HPX15 Activates Diverse Immune Pathways to Regulate the Growth of Midgut Bacteria

    PubMed Central

    Kajla, Mithilesh; Choudhury, Tania P.; Kakani, Parik; Gupta, Kuldeep; Dhawan, Rini; Gupta, Lalita; Kumar, Sanjeev

    2016-01-01

    Anopheles mosquito midgut harbors a diverse group of endogenous bacteria that grow extensively after the blood feeding and help in food digestion and nutrition in many ways. Although, the growth of endogenous bacteria is regulated by various factors, however, the robust antibacterial immune reactions are generally suppressed in this body compartment by a heme peroxidase HPX15 crosslinked mucins barrier. This barrier is formed on the luminal side of the midgut and blocks the direct interactions and recognition of bacteria or their elicitors by the immune reactive midgut epithelium. We hypothesized that in the absence of HPX15, an increased load of exogenous bacteria will enormously induce the mosquito midgut immunity and this situation in turn, can easily regulate mosquito-pathogen interactions. In this study, we found that the blood feeding induced AsHPX15 gene in Anopheles stephensi midgut and promoted the growth of endogenous as well as exogenous fed bacteria. In addition, the mosquito midgut also efficiently regulated the number of these bacteria through the induction of classical Toll and Imd immune pathways. In case of AsHPX15 silenced midguts, the growth of midgut bacteria was largely reduced through the induction of nitric oxide synthase (NOS) gene, a downstream effector molecule of the JAK/STAT pathway. Interestingly, no significant induction of the classical immune pathways was observed in these midguts. Importantly, the NOS is a well known negative regulator of Plasmodium development, thus, we proposed that the induction of diverged immune pathways in the absence of HPX15 mediated midgut barrier might be one of the strategies to manipulate the vectorial capacity of Anopheles mosquito. PMID:27630620

  19. New insights into Drosophila vision.

    PubMed

    Dolph, Patrick

    2008-01-10

    Studies of the Drosophila visual system have provided valuable insights into the function and regulation of phototransduction signaling pathways. Much of this work has stemmed from or relied upon the genetic tools offered by the Drosophila system. In this issue of Neuron, Wang and colleagues and Acharya and colleagues have further exploited the Drosophila genetic system to characterize two new phototransduction players.

  20. Ion and solute transport by Prestin in Drosophila and Anopheles.

    PubMed

    Hirata, Taku; Czapar, Anna; Brin, Lauren; Haritonova, Alyona; Bondeson, Daniel P; Linser, Paul; Cabrero, Pablo; Thompson, James; Dow, Julian A T; Romero, Michael F

    2012-04-01

    The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO(3)(-) transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (Prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl(-)/nHCO(3)(-), Cl(-)/SO(4)(2-) and Cl(-)/oxalate(2-)) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.

  1. Neem oil (Azadirachta indica A. Juss) affects the ultrastructure of the midgut muscle of Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae).

    PubMed

    Scudeler, Elton Luiz; Garcia, Ana Silvia Gimenes; Pinheiro, Patricia Fernanda Felipe; Santos, Daniela Carvalho Dos

    2017-01-01

    Cytomorphological changes, by means of ultrastructural analyses, have been used to determine the effects of the biopesticide neem oil on the muscle fibers of the midgut of the predator Ceraeochrysa claveri. Insects, throughout the larval period, were fed eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% or 2%. In the adult stage, the midgut was collected from female insects at two stages of adulthood (newly emerged and at the start of oviposition) and processed for ultrastructural analyses. In the newly emerged insects obtained from neem oil treatments, muscle fibers showed a reduction of myofilaments as well as swollen mitochondria and an accumulation of membranous structures. Muscular fibers responded to those cellular injuries with the initiation of detoxification mechanisms, in which acid phosphatase activity was observed in large vesicles located at the periphery of the muscle fiber. At the start of oviposition in the neem oil treated insects, muscle fibers exhibited signs of degeneration, containing vacant areas in which contractile myofilaments were reduced or completely absent, and an accumulation of myelin structures, a dilatation of cisternae of sarcoplasmic reticulum, and mitochondrial swelling and cristolysis were observed. Enzymatic activity for acid phosphatase was present in large vesicles, indicating that mechanisms of lytic activity during the cell injury were utilized but insufficient for recovery from all the cellular damage. The results indicate that the visceral muscle layer is also the target of action of neem oil, and the cytotoxic effects observed may compromise the function of that organ.

  2. Identification of a polymorphic mucin-like gene expressed in the midgut of the mosquito, Aedes aegypti, using an integrated bulked segregant and differential display analysis.

    PubMed Central

    Morlais, I; Severson, D W

    2001-01-01

    The identification of putative differentially expressed genes within genome regions containing QTL determining susceptibility of the mosquito, Aedes aegypti, to the malarial parasite, Plasmodium gallinaceum, was investigated using an integrated, targeted approach based on bulked segregant and differential display analysis. A mosquito F2 population was obtained from pairwise matings between the parasite-susceptible RED strain and the resistant MOYO-R substrain. DNA from female carcasses was used to genotype individuals at RFLP markers of known chromosomal position around the major QTL (pgs 1). Midguts, dissected 48 hr after an infected blood meal, were used to prepare two RNA bulks, each representing one of the parental genotypes at the QTL interval. The RNA bulks were compared by differential display PCR. A mucin-like protein gene (AeIMUC1) was isolated and characterized. The gene maps within the pgs 1 QTL interval and is expressed in the adult female midgut. AeIMUC1 RNA abundance decreased with time after blood meal ingestion. No differential expression was observed between the two mosquito strains but three different alleles with inter- and intrastrain allelic polymorphisms including indels and SNPs were characterized. The AeIMUC1 gene chromosome location and allelic polymorphisms raise the possibility that the protein might be involved in parasite-mosquito interactions. PMID:11454761

  3. Maintenance of a Drosophila melanogaster Population Cage.

    PubMed

    Caravaca, Juan Manuel; Lei, Elissa P

    2016-03-15

    Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

  4. Maintenance of a Drosophila melanogaster Population Cage

    PubMed Central

    Caravaca, Juan Manuel; Lei, Elissa P.

    2016-01-01

    Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle. PMID:27023790

  5. Predatory cannibalism in Drosophila melanogaster larvae.

    PubMed

    Vijendravarma, Roshan K; Narasimha, Sunitha; Kawecki, Tadeusz J

    2013-01-01

    Hunting live prey is risky and thought to require specialized adaptations. Therefore, observations of predatory cannibalism in otherwise non-carnivorous animals raise questions about its function, adaptive significance and evolutionary potential. Here we document predatory cannibalism on larger conspecifics in Drosophila melanogaster larvae and address its evolutionary significance. We found that under crowded laboratory conditions younger larvae regularly attack and consume 'wandering-stage' conspecifics, forming aggregations mediated by chemical cues from the attacked victim. Nutrition gained this way can be significant: an exclusively cannibalistic diet was sufficient for normal development from eggs to fertile adults. Cannibalistic diet also induced plasticity of larval mouth parts. Finally, during 118 generations of experimental evolution, replicated populations maintained under larval malnutrition evolved enhanced propensity towards cannibalism. These results suggest that, at least under laboratory conditions, predation on conspecifics in Drosophila is a functional, adaptive behaviour, which can rapidly evolve in response to nutritional conditions.

  6. The bacterial communities of Drosophila suzukii collected from undamaged cherries.

    PubMed

    Chandler, James Angus; James, Pamela M; Jospin, Guillaume; Lang, Jenna M

    2014-01-01

    Drosophila suzukii is an introduced pest insect that feeds on undamaged, attached fruit. This diet is distinct from the fallen, discomposing fruits utilized by most other species of Drosophila. Since the bacterial microbiota of Drosophila, and of many other animals, is affected by diet, we hypothesized that the bacteria associated with D. suzukii are distinct from that of other Drosophila. Using 16S rDNA PCR and Illumina sequencing, we characterized the bacterial communities of larval and adult D. suzukii collected from undamaged, attached cherries in California, USA. We find that the bacterial communities associated with these samples of D. suzukii contain a high frequency of Tatumella. Gluconobacter and Acetobacter, two taxa with known associations with Drosophila, were also found, although at lower frequency than Tatumella in four of the five samples examined. Sampling D. suzukii from different locations and/or while feeding on different fruits is needed to determine the generality of the results determined by these samples. Nevertheless this is, to our knowledge, the first study characterizing the bacterial communities of this ecologically unique and economically important species of Drosophila.

  7. Synergistic mitotoxicity of chloromethanes and fullerene C60 nanoaggregates in Daphnia magna midgut epithelial cells.

    PubMed

    Seke, Mariana; Markelic, Milica; Morina, Arian; Jovic, Danica; Korac, Aleksandra; Milicic, Dragana; Djordjevic, Aleksandar

    2016-12-03

    Adsorption of non-polar compounds by suspended fullerene nanoaggregates (nC60) may enhance their toxicity and affect the fate, transformation, and transport of non-polar compounds in the environment. The potential of stable fullerene nanoaggregates as contaminant carriers in aqueous systems and the influence of chloromethanes (trichloromethane and dichloromethane) were studied on the midgut epithelial cells of Daphnia magna by light and electron microscopy. The size and shape of fullerene nanoaggregates were observed and measured using dynamic light scattering, transmission electron microscopy, and low vacuum scanning electron microscopy. The nC60 in suspension appeared as a bulk of aggregates of irregular shape with a surface consisting of small clumps 20-30 nm in diameter. The presence of nC60 aggregates was confirmed in midgut lumen and epithelial cells of D. magna. After in vivo acute exposure to chloromethane, light and electron microscopy revealed an extensive cytoplasmic vacuolization with disruption and loss of specific structures of D. magna midgut epithelium (mitochondria, endoplasmic reticulum, microvilli, peritrophic membrane) and increased appearance of necrotic cells. The degree of observed changes depended on the type of treatment: trichloromethane (TCM) induced the most notable changes, whereas fullerene nanoaggregates alone had no negative effects. Transmission electron microscopy also indicated increased lysosomal degradation and severe peroxidative damages of enterocyte mitochondria following combined exposure to chloromethane and fullerene nanoaggregates. In conclusion, the adsorption of chloromethane by fullerene nanoaggregates enhances their toxicity and induces peroxidative mitochondrial damage in midgut enterocytes.

  8. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    PubMed Central

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-01-01

    Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania. PMID:24037187

  9. Determination of pH in regions of the midguts of acaridid mites.

    PubMed

    Erban, Tomas; Hubert, Jan

    2010-01-01

    The pH of the guts of mites strongly affects their digestive processes. This study was carried out to determine the pH in the guts of 12 species of stored product and house dust mites. Eighteen pH indicators were chosen and offered to the mites in the feeding biotest. Based on the color changes of the indicators, the gut contents of acaridid mites were determined to be within a pH range of 4 to neutral. The gut contents showed a gradient in pH from the anterior to the posterior part. The anterior midgut (ventriculus and caeca) of most species had a pH ranging from 4.5 to 5, or slightly more alkaline for most of the species, while the middle midgut (intercolon/colon) had a pH of 5 to 6. Finally, the pH of the posterior midgut (postcolon) was between 5.5 and 7. Except for Dermatophagoides spp., no remarkable differences in the pH of the gut were observed among the tested species. Dermatophagoides spp. had a more acidic anterior midgut (a pH of 4 to 5) and colon (a pH of 5) with postcolon (a pH of below 6). The results characterizing in vivo conditions in the mite gut offer useful information to study the activity of mite digestive enzymes including their inhibitors and gut microflora.

  10. Midgut transcriptome profiling of Anoplophora glabripennis, a lignocellulose degrading Cerambycid beetle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Wood-feeding insects often work in collaboration with microbial symbionts to degrade lignin biopolymers and release glucose and other fermentable sugars from recalcitrant plant cell wall carbohydrates, including cellulose and hemicellulose. Here, we present the midgut transcriptome of la...

  11. Localization of two post-proline cleaving peptidases in the midgut of Tenebrio molitor larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in the midgut of Tenebrio molitor larvae with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activit...

  12. Midgut epithelium in molting silkworm: A fine balance among cell growth, differentiation, and survival.

    PubMed

    Franzetti, Eleonora; Casartelli, Morena; D'Antona, Paola; Montali, Aurora; Romanelli, Davide; Cappellozza, Silvia; Caccia, Silvia; Grimaldi, Annalisa; de Eguileor, Magda; Tettamanti, Gianluca

    2016-07-01

    The midgut of insects has attracted great attention as a system for studying intestinal stem cells (ISCs) as well as cell death-related processes, such as apoptosis and autophagy. Among insects, Lepidoptera represent a good model to analyze these cells and processes. In particular, larva-larva molting is an interesting developmental phase since the larva must deal with nutrient starvation and its organs are subjected to rearrangements due to proliferation and differentiation events. Several studies have analyzed ISCs in vitro and characterized key factors involved in their division and differentiation during molt. However, in vivo studies performed during larva-larva transition on these cells, and on the whole midgut epithelium, are fragmentary. In the present study, we analyzed the larval midgut epithelium of the silkworm, Bombyx mori, during larva-larva molting, focusing our attention on ISCs. Moreover, we investigated the metabolic changes that occur in the epithelium and evaluated the intervention of autophagy. Our data on ISCs proliferation and differentiation, autophagy activation, and metabolic and functional activities of the midgut cells shed light on the complexity of this organ during the molting phase.

  13. Variation in Vector Competence for Dengue Viruses Does Not Depend on Mosquito Midgut Binding Affinity

    PubMed Central

    Cox, Jonathan; Brown, Heidi E.; Rico-Hesse, Rebeca

    2011-01-01

    Background Dengue virus genotypes of Southeast Asian origin have been associated with higher virulence and transmission compared to other genotypes of serotype 2 (DEN-2). We tested the hypothesis that genetic differences in dengue viruses may result in differential binding to the midgut of the primary vector, Aedes aegypti, resulting in increased transmission or vectorial capacity. Methodology/Principal Finding Two strains of each of the four DEN-2 genotypes (Southeast Asian, American, Indian, and West African) were tested to determine their binding affinity for mosquito midguts from two distinct populations (Tapachula, Chiapas, Mexico and McAllen, Texas, USA). Our previous studies demonstrated that Southeast Asian viruses disseminated up to 65-fold more rapidly in Ae. aegypti from Texas and were therefore more likely to be transmitted to humans. Results shown here demonstrate that viruses from all four genotypes bind to midguts at the same rate, in a titer-dependent manner. In addition, we show population differences when comparing binding affinity for DEN-2 between the Tapachula and McAllen mosquito colonies. Conclusions If midgut binding potential is the same for all DEN-2 viruses, then viral replication differences in these tissues and throughout the mosquito can thus probably explain the significant differences in dissemination and vector competence. These conclusions differ from the established paradigms to explain mosquito barriers to infection, dissemination, and transmission. PMID:21610852

  14. Apoptosis and necrosis during the circadian cycle in the centipede midgut.

    PubMed

    Rost-Roszkowska, M M; Chajec, Ł; Vilimova, J; Tajovský, K

    2016-07-01

    Three types of cells have been distinguished in the midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata: digestive, secretory, and regenerative cells. According to the results of our previous studies, we decided to analyze the relationship between apoptosis and necrosis in their midgut epithelium and circadian rhythms. Ultrastructural analysis showed that these processes proceed in a continuous manner that is independent of the circadian rhythm in L. forficatus, while in S. cingulata necrosis is activated at midnight. Additionally, the description of apoptosis and necrosis showed no differences between males and females of both species analyzed. At the beginning of apoptosis, the cell cytoplasm becomes electron-dense, apparently in response to shrinkage of the cell. Organelles such as the mitochondria, cisterns of endoplasmic reticulum transform and degenerate. Nuclei gradually assume lobular shapes before the apoptotic cell is discharged into the midgut lumen. During necrosis, however, the cytoplasm of the cell becomes electron-lucent, and the number of organelles decreases. While the digestive cells of about 10 % of L. forficatus contain rickettsia-like pathogens, the corresponding cells in S. cingulata are free of rickettsia. As a result, we can state that apoptosis in L. forficatus is presumably responsible for protecting the organism against infections, while in S. cingulata apoptosis is not associated with the elimination of pathogens. Necrosis is attributed to mechanical damage, and the activation of this process coincides with proliferation of the midgut regenerative cells at midnight in S. cingulata.

  15. Regulation of Stem Cell Proliferation and Cell Fate Specification by Wingless/Wnt Signaling Gradients Enriched at Adult Intestinal Compartment Boundaries

    PubMed Central

    Tian, Ai; Benchabane, Hassina; Wang, Zhenghan; Ahmed, Yashi

    2016-01-01

    Intestinal stem cell (ISC) self-renewal and proliferation are directed by Wnt/β-catenin signaling in mammals, whereas aberrant Wnt pathway activation in ISCs triggers the development of human colorectal carcinoma. Herein, we have utilized the Drosophila midgut, a powerful model for ISC regulation, to elucidate the mechanisms by which Wingless (Wg)/Wnt regulates intestinal homeostasis and development. We provide evidence that the Wg signaling pathway, activation of which peaks at each of the major compartment boundaries of the adult intestine, has essential functions. Wg pathway activation in the intestinal epithelium is required not only to specify cell fate near compartment boundaries during development, but also to control ISC proliferation within compartments during homeostasis. Further, in contrast with the previous focus on Wg pathway activation within ISCs, we demonstrate that the primary mechanism by which Wg signaling regulates ISC proliferation during homeostasis is non-autonomous. Activation of the Wg pathway in absorptive enterocytes is required to suppress JAK-STAT signaling in neighboring ISCs, and thereby their proliferation. We conclude that Wg signaling gradients have essential roles during homeostasis and development of the adult intestine, non-autonomously controlling stem cell proliferation inside compartments, and autonomously specifying cell fate near compartment boundaries. PMID:26845150

  16. Queuine metabolism and cadmium toxicity in Drosophila

    SciTech Connect

    Farkas, W.R.; Siard, T. ); Jacobson, K.B. )

    1991-03-11

    Queuine is a derivative of guanine found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His and Tyr. The transcripts of these tRNAs contain a guanine in this position. This guanine is enzymatically excised and replaced by queuine. The ratio of queuine-containing or (q+) tRNA to its precursor or (q{minus}) tRNA changes throughout the Drosophila life cycle. in the egg 10% of the tRNA is (q+). During the three larval stages this ratio drops to zero. In the one day old adult it is about 10%. It has previously been shown that when flies are selected for the ability to grow in the presence of cadmium, the tolerant flies had 100% (q+) tRNA at the first day after pupation instead of 10%. However, it was not known whether the elevated level of (q+) tRNA was a coincidence or if the elevated levels of (q+) tRNA was protective. The authors explored this problem using germfree Drosophila. The first thing was to determine if Drosophila can synthesize queuine. Sterilized eggs were seeded onto sterile chemically defined medium. The flies were grown to the adult stage. This study showed that Drosophila like mammals cannot synthesize queuine. A second result of this research was the demonstration that the authors could alter the ratio of (q+) to (q{minus}) tRNA by adding exogenous queuine to the medium e.g. at 0.008 mM queuine the (q+) tRNA was 95% instead of {lt} 5% in the last instar stage. Finally, the authors investigated whether or not queuine gave protection against cadmium. The results were that when the flies were grown in the presence of 0.2 mM cadmium queuine at 0.008 mM gave a statistically significant increase in the number of survivors.

  17. Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

    PubMed Central

    2010-01-01

    Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora. PMID:20663211

  18. Midgut Transcriptome of the Cockroach Periplaneta americana and Its Microbiota: Digestion, Detoxification and Oxidative Stress Response

    PubMed Central

    Zhang, Jianhua; Zhang, Yixi; Li, Jingjing; Liu, Meiling; Liu, Zewen

    2016-01-01

    The cockroach, Periplaneta americana, is an obnoxious and notorious pest of the world, with a strong ability to adapt to a variety of complex environments. However, the molecular mechanism of this adaptability is mostly unknown. In this study, the genes and microbiota composition associated with the adaptation mechanism were studied by analyzing the transcriptome and 16S rDNA pyrosequencing of the P. americana midgut, respectively. Midgut transcriptome analysis identified 82,905 unigenes, among which 64 genes putatively involved in digestion (11 genes), detoxification (37 genes) and oxidative stress response (16 genes) were found. Evaluation of gene expression following treatment with cycloxaprid further revealed that the selected genes (CYP6J1, CYP4C1, CYP6K1, Delta GST, alpha-amylase, beta-glucosidase and aminopeptidase) were upregulated at least 2.0-fold at the transcriptional level, and four genes were upregulated more than 10.0-fold. An interesting finding was that three digestive enzymes positively responded to cycloxaprid application. Tissue expression profiles further showed that most of the selected genes were midgut-biased, with the exception of CYP6K1. The midgut microbiota composition was obtained via 16S rDNA pyrosequencing and was found to be mainly dominated by organisms from the Firmicutes phylum, among which Clostridiales, Lactobacillales and Burkholderiales were the main orders which might assist the host in the food digestion or detoxification of noxious compounds. The preponderant species, Clostridium cellulovorans, was previously reported to degrade lignocellulose efficiently in insects. The abundance of genes involved in digestion, detoxification and response to oxidative stress, and the diversity of microbiota in the midgut might provide P. americana high capacity to adapt to complex environments. PMID:27153200

  19. Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells

    PubMed Central

    Ramphul, Urvashi N.; Garver, Lindsey S.; Molina-Cruz, Alvaro; Canepa, Gaspar E.; Barillas-Mury, Carolina

    2015-01-01

    The malaria parasite, Plasmodium, must survive and develop in the mosquito vector to be successfully transmitted to a new host. The Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. Two alternative approaches were used to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses, and show that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 disrupts JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h postfeeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system. PMID:25552553

  20. Midgut Transcriptome of the Cockroach Periplaneta americana and Its Microbiota: Digestion, Detoxification and Oxidative Stress Response.

    PubMed

    Zhang, Jianhua; Zhang, Yixi; Li, Jingjing; Liu, Meiling; Liu, Zewen

    2016-01-01

    The cockroach, Periplaneta americana, is an obnoxious and notorious pest of the world, with a strong ability to adapt to a variety of complex environments. However, the molecular mechanism of this adaptability is mostly unknown. In this study, the genes and microbiota composition associated with the adaptation mechanism were studied by analyzing the transcriptome and 16S rDNA pyrosequencing of the P. americana midgut, respectively. Midgut transcriptome analysis identified 82,905 unigenes, among which 64 genes putatively involved in digestion (11 genes), detoxification (37 genes) and oxidative stress response (16 genes) were found. Evaluation of gene expression following treatment with cycloxaprid further revealed that the selected genes (CYP6J1, CYP4C1, CYP6K1, Delta GST, alpha-amylase, beta-glucosidase and aminopeptidase) were upregulated at least 2.0-fold at the transcriptional level, and four genes were upregulated more than 10.0-fold. An interesting finding was that three digestive enzymes positively responded to cycloxaprid application. Tissue expression profiles further showed that most of the selected genes were midgut-biased, with the exception of CYP6K1. The midgut microbiota composition was obtained via 16S rDNA pyrosequencing and was found to be mainly dominated by organisms from the Firmicutes phylum, among which Clostridiales, Lactobacillales and Burkholderiales were the main orders which might assist the host in the food digestion or detoxification of noxious compounds. The preponderant species, Clostridium cellulovorans, was previously reported to degrade lignocellulose efficiently in insects. The abundance of genes involved in digestion, detoxification and response to oxidative stress, and the diversity of microbiota in the midgut might provide P. americana high capacity to adapt to complex environments.

  1. Composition of the Spruce Budworm (Choristoneura fumiferana) Midgut Microbiota as Affected by Rearing Conditions.

    PubMed

    Landry, Mathieu; Comeau, André M; Derome, Nicolas; Cusson, Michel; Levesque, Roger C

    2015-01-01

    The eastern spruce budworm (Choristoneura fumiferana) is one of the most destructive forest insect pests in Canada. Little is known about its intestinal microbiota, which could play a role in digestion, immune protection, communication and/or development. The present study was designed to provide a first characterization of the effects of rearing conditions on the taxonomic diversity and structure of the C. fumiferana midgut microbiota, using a culture-independent approach. Three diets and insect sources were examined: larvae from a laboratory colony reared on a synthetic diet and field-collected larvae reared on balsam fir or black spruce foliage. Bacterial DNA from the larval midguts was extracted to amplify and sequence the V6-V8 region of the 16S rRNA gene, using the Roche 454 GS-FLX technology. Our results showed a dominance of Proteobacteria, mainly Pseudomonas spp., in the spruce budworm midgut, irrespective of treatment group. Taxonomic diversity of the midgut microbiota was greater for larvae reared on synthetic diet than for those collected and reared on host plants, a difference that is likely accounted for by several factors. A greater proportion of bacteria from the phylum Bacteroidetes in insects fed artificial diet constituted the main difference between this group and those reared on foliage; within the phylum Proteobacteria, the presence of the genus Bradyrhizobium was also unique to insects reared on artificial diet. Strikingly, a Bray-Curtis analysis showed important differences in microbial diversity among the treatment groups, pointing to the importance of diet and environment in defining the spruce budworm midgut microbiota.

  2. Composition of the Spruce Budworm (Choristoneura fumiferana) Midgut Microbiota as Affected by Rearing Conditions

    PubMed Central

    Landry, Mathieu; Comeau, André M.; Derome, Nicolas; Cusson, Michel; Levesque, Roger C.

    2015-01-01

    The eastern spruce budworm (Choristoneura fumiferana) is one of the most destructive forest insect pests in Canada. Little is known about its intestinal microbiota, which could play a role in digestion, immune protection, communication and/or development. The present study was designed to provide a first characterization of the effects of rearing conditions on the taxonomic diversity and structure of the C. fumiferana midgut microbiota, using a culture-independent approach. Three diets and insect sources were examined: larvae from a laboratory colony reared on a synthetic diet and field-collected larvae reared on balsam fir or black spruce foliage. Bacterial DNA from the larval midguts was extracted to amplify and sequence the V6-V8 region of the 16S rRNA gene, using the Roche 454 GS-FLX technology. Our results showed a dominance of Proteobacteria, mainly Pseudomonas spp., in the spruce budworm midgut, irrespective of treatment group. Taxonomic diversity of the midgut microbiota was greater for larvae reared on synthetic diet than for those collected and reared on host plants, a difference that is likely accounted for by several factors. A greater proportion of bacteria from the phylum Bacteroidetes in insects fed artificial diet constituted the main difference between this group and those reared on foliage; within the phylum Proteobacteria, the presence of the genus Bradyrhizobium was also unique to insects reared on artificial diet. Strikingly, a Bray-Curtis analysis showed important differences in microbial diversity among the treatment groups, pointing to the importance of diet and environment in defining the spruce budworm midgut microbiota. PMID:26636571

  3. Transporters involved in glucose and water absorption in the Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) anterior midgut.

    PubMed

    Bifano, Thaís D; Alegria, Thiago G P; Terra, Walter R

    2010-09-01

    Little is known about insect intestinal sugar absorption, in spite of the recent findings, and even less has been published regarding water absorption. The aim of this study was to shed light on putative transporters of water and glucose in the insect midgut. Glucose and water absorptions by the anterior ventriculus of Dysdercus peruvianus midgut were determined by feeding the insects with a glucose and a non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventricular contents. Glucose absorption decreases glucose/dye ratios and water absorption increases dye concentrations. Water and glucose transports are activated (water 50%, glucose 33%) by 50 mM K(2)SO(4) and are inhibited (water 46%, glucose 82%) by 0.2 mM phloretin, the inhibitor of the facilitative hexose transporter (GLUT) or are inhibited (water 45%, glucose 35%) by 0.1 mM phlorizin, the inhibitor of the Na(+)-glucose cotransporter (SGLT). The results also showed that the putative SGLT transports about two times more water relative to glucose than the putative GLUT. These results mean that D. peruvianus uses a GLUT-like transporter and an SGLT-like transporter (with K(+) instead of Na(+)) to absorb dietary glucose and water. A cDNA library from D. peruvianus midgut was screened and we found one sequence homologous to GLUT1, named DpGLUT, and another to a sodium/solute symporter, named DpSGLT. Semi-quantitative RT-PCR studies revealed that DpGLUT and DpSGLTs mRNA were expressed in the anterior midgut, where glucose and water are absorbed, but not in fat body, salivary gland and Malpighian tubules. This is the first report showing the involvement of putative GLUT and SGLT in both water and glucose midgut absorption in insects.

  4. Retinoids regulate a developmental checkpoint for tissue regeneration in Drosophila

    PubMed Central

    Halme, Adrian; Cheng, Michelle; Hariharan, Iswar K.

    2010-01-01

    Summary Drosophila melanogaster larvae have a remarkable capacity for regenerative growth: Damage to their imaginal discs, the larval precursors of adult structures, elicits a robust proliferative response from the surviving tissue [1–4]. However, as in other organisms, developmental progression and differentiation can restrict regenerative capacity of Drosophila tissues. Experiments in Drosophila and other holometabolous insects have demonstrated that either damage to imaginal tissues [5, 6] or transplantation of a damaged imaginal disc [7, 8] delays the onset of metamorphosis, a time when the imaginal discs undergo morphogenesis and differentiation into their adult structures. Therefore, in Drosophila there appears to be a mechanism that senses tissue damage and extends the larval phase to coordinate tissue regeneration with the overall developmental program of the organism. However, how such a pathway functions remains unknown. Here we demonstrate that a developmental checkpoint extends larval growth after imaginal disc damage by inhibiting the transcription of the gene encoding PTTH, a neuropeptide that promotes the release of the steroid hormone ecdysone. Using a genetic screen, we identify a previously unsuspected role for retinoid biosynthesis in regulating PTTH expression and delaying development in response to tissue damage. Retinoid signaling plays an important, but poorly defined role in several vertebrate regeneration models [9–11]. Our findings demonstrate that retinoid biosynthesis in Drosophila is important for the maintenance of a permissive condition for regenerative growth. PMID:20189388

  5. Hearing regulates Drosophila aggression.

    PubMed

    Versteven, Marijke; Vanden Broeck, Lies; Geurten, Bart; Zwarts, Liesbeth; Decraecker, Lisse; Beelen, Melissa; Göpfert, Martin C; Heinrich, Ralf; Callaerts, Patrick

    2017-02-21

    Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly's auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level.

  6. Binding of insecticidal lectin Colocasia esculenta tuber agglutinin (CEA) to midgut receptors of Bemisia tabaci and Lipaphis erysimi provides clues to its insecticidal potential.

    PubMed

    Roy, Amit; Gupta, Sumanti; Hess, Daniel; Das, Kali Pada; Das, Sampa

    2014-07-01

    The insecticidal potential of Galanthus nivalis agglutinin-related lectins against hemipterans has been experimentally proven. However, the basis behind the toxicity of these lectins against hemipterans remains elusive. The present study elucidates the molecular basis behind insecticidal efficacy of Colocasia esculenta tuber agglutinin (CEA) against Bemisia tabaci and Lipaphis erysimi. Confocal microscopic analyses highlighted the binding of 25 kDa stable homodimeric lectin to insect midgut. Ligand blots followed by LC MS/MS analyses identified binding partners of CEA as vacuolar ATP synthase and sarcoplasmic endoplasmic reticulum type Ca(2+) ATPase from B. tabaci, and ATP synthase, heat shock protein 70 and clathrin heavy chain assembly protein from L. erysimi. Internalization of CEA into hemolymph was confirmed by Western blotting. Glycoprotein nature of the receptors was identified through glycospecific staining. Deglycosylation assay indicated the interaction of CEA with its receptors to be probably glycan mediated. Surface plasmon resonance analysis revealed the interaction kinetics between ATP synthase of B. tabaci with CEA. Pathway prediction study based on Drosophila homologs suggested the interaction of CEA with insect receptors that probably led to disruption of cellular processes causing growth retardation and loss of fecundity of target insects. Thus, the present findings strengthen our current understanding of the entomotoxic potentiality of CEA, which will facilitate its future biotechnological applications.

  7. Chemical sensing in Drosophila.

    PubMed

    Benton, Richard

    2008-08-01

    Chemical sensing begins when peripheral receptor proteins recognise specific environmental stimuli and translate them into spatial and temporal patterns of sensory neuron activity. The chemosensory system of the fruit fly, Drosophila melanogaster, has become a dominant model to understand this process, through its accessibility to a powerful combination of molecular, genetic and electrophysiological analysis. Recent results have revealed many surprises in the biology of peripheral chemosensation in Drosophila, including novel structural and signalling properties of the insect odorant receptors (ORs), combinatorial mechanisms of chemical recognition by the gustatory receptors (GRs), and the implication of Transient Receptor Potential (TRP) ion channels as a novel class of chemosensory receptors.

  8. Studying aging in Drosophila.

    PubMed

    He, Ying; Jasper, Heinrich

    2014-06-15

    Drosophila melanogaster represents one of the most important genetically accessible model organisms for aging research. Studies in flies have identified single gene mutations that influence lifespan and have characterized endocrine signaling interactions that control homeostasis systemically. Recent studies have focused on the effects of aging on specific tissues and physiological processes, providing a comprehensive picture of age-related tissue dysfunction and the loss of systemic homeostasis. Here we review methodological aspects of this work and highlight technical considerations when using Drosophila to study aging and age-related diseases.

  9. Development-based compartmentalization of the Drosophila central brain.

    PubMed

    Pereanu, Wayne; Kumar, Abilasha; Jennett, Arnim; Reichert, Heinrich; Hartenstein, Volker

    2010-08-01

    The neuropile of the Drosophila brain is subdivided into anatomically discrete compartments. Compartments are rich in terminal neurite branching and synapses; they are the neuropile domains in which signal processing takes place. Compartment boundaries are defined by more or less dense layers of glial cells as well as long neurite fascicles. These fascicles are formed during the larval period, when the approximately 100 neuronal lineages that constitute the Drosophila central brain differentiate. Each lineage forms an axon tract with a characteristic trajectory in the neuropile; groups of spatially related tracts congregate into the brain fascicles that can be followed from the larva throughout metamorphosis into the adult stage. Here we provide a map of the adult brain compartments and the relevant fascicles defining compartmental boundaries. We have identified the neuronal lineages contributing to each fascicle, which allowed us to compare compartments of the larval and adult brain directly. Most adult compartments can be recognized already in the early larval brain, where they form a "protomap" of the later adult compartments. Our analysis highlights the morphogenetic changes shaping the Drosophila brain; the data will be important for studies that link early-acting genetic mechanisms to the adult neuronal structures and circuits controlled by these mechanisms.

  10. Gp93, the Drosophila GRP94 ortholog, is required for gut epithelial homeostasis and nutrient assimilation-coupled growth control

    PubMed Central

    Maynard, Jason C.; Pham, Trang; Zheng, Tianli; Jockheck-Clark, Angela; Rankin, Helen B.; Newgard, Christopher B.; Spana, Eric P.; Nicchitta, Christopher V.

    2010-01-01

    GRP94, the endoplasmic reticulum Hsp90, is a metazoan-restricted chaperone essential for early development in mammals, yet dispensable for mammalian cell viability. This dichotomy suggests that GRP94 is required for the functional expression of secretory and/or membrane proteins that enable the integration of cells into tissues. To explore this hypothesis, we have identified the Drosophila ortholog of GRP94, Gp93, and report that Gp93 is an essential gene in Drosophila. Loss of zygotic Gp93 expression is late larval lethal and causes prominent defects in the larval midgut, the sole endoderm-derived larval tissue. Gp93 mutant larvae display pronounced defects in the midgut epithelium, with aberrant copper cell structure, markedly reduced gut acidification, atypical septate junction structure, depressed gut motility, and deficits in intestinal nutrient uptake. The metabolic consequences of the loss of Gp93-expression are profound; Gp93 mutant larvae exhibit a starvation-like metabolic phenotype, including suppression of insulin signaling and extensive mobilization of amino acids and triglycerides. The defects in copper cell structure/function accompanying loss of Gp93 expression resemble those reported for mutations in labial, an endodermal homeotic gene required for copper cell specification, and α-spectrin, thus suggesting an essential role for Gp93 in the functional expression of secretory/integral membrane protein-encoding lab protein target genes and/or integral membrane protein(s) that interact with the spectrin cytoskeleton to confer epithelial membrane specialization. PMID:20044986

  11. Regional repression of a Drosophila POU box gene in the endoderm involves inductive interactions between germ layers.

    PubMed

    Affolter, M; Walldorf, U; Kloter, U; Schier, A F; Gehring, W J

    1993-04-01

    An induction process occurring between the mesodermal and the endodermal germ layers has recently been described in the regulation of the Drosophila homeotic gene labial (lab). We report here that proper spatial regulation of the Drosophila POU box gene pdm-1 products also involves interaction between these two germ layers. pdm-1 transcripts are initially present in both the anterior and the posterior endodermal midgut primordia. Upon fusion of the two primordia, transcripts disappear from two regions in the endoderm, a central domain and an anterior domain. The anterior repression domain of pdm-1 is independent of the expression of known homeotic genes and genes encoding secreted signalling molecules in the visceral mesoderm, both for its positioning and its repression. Repression in the central domain requires both the homeotic gene Ultrabithorax (Ubx) and the decapentaplegic (dpp) gene, which encodes a secreted protein. Both of these genes are also required for lab induction. However, the analysis of pdm-1 expression in various mutant backgrounds indicates that the regulation of lab and pdm-1 across germ layers is controlled by different genetic cascades. Our study indicates that dpp is not the signal that dictates central pdm-1 repression across germ layers and suggests that in the same midgut region, different signalling pathways result in the differential activation or repression of potential transcription factors.

  12. Towards a Drosophila model of Hutchinson-Gilford progeria syndrome.

    PubMed

    Beard, Gemma S; Bridger, Joanna M; Kill, Ian R; Tree, David R P

    2008-12-01

    The laminopathy Hutchinson-Gilford progeria syndrome (HGPS) is caused by the mutant lamin A protein progerin and leads to premature aging of affected children. Despite numerous cell biological and biochemical insights into the basis for the cellular abnormalities seen in HGPS, the mechanism linking progerin to the organismal phenotype is not fully understood. To begin to address the mechanism behind HGPS using Drosophila melanogaster, we have ectopically expressed progerin and lamin A. We found that ectopic progerin and lamin A phenocopy several effects of laminopathies in developing and adult Drosophila, but that progerin causes a stronger phenotype than wild-type lamin A.

  13. Heritable Endosymbionts of Drosophila

    PubMed Central

    Mateos, Mariana; Castrezana, Sergio J.; Nankivell, Becky J.; Estes, Anne M.; Markow, Therese A.; Moran, Nancy A.

    2006-01-01

    Although heritable microorganisms are increasingly recognized as widespread in insects, no systematic screens for such symbionts have been conducted in Drosophila species (the primary insect genetic models for studies of evolution, development, and innate immunity). Previous efforts screened relatively few Drosophila lineages, mainly for Wolbachia. We conducted an extensive survey of potentially heritable endosymbionts from any bacterial lineage via PCR screens of mature ovaries in 181 recently collected fly strains representing 35 species from 11 species groups. Due to our fly sampling methods, however, we are likely to have missed fly strains infected with sex ratio-distorting endosymbionts. Only Wolbachia and Spiroplasma, both widespread in insects, were confirmed as symbionts. These findings indicate that in contrast to some other insect groups, other heritable symbionts are uncommon in Drosophila species, possibly reflecting a robust innate immune response that eliminates many bacteria. A more extensive survey targeted these two symbiont types through diagnostic PCR in 1225 strains representing 225 species from 32 species groups. Of these, 19 species were infected by Wolbachia while only 3 species had Spiroplasma. Several new strains of Wolbachia and Spiroplasma were discovered, including ones divergent from any reported to date. The phylogenetic distribution of Wolbachia and Spiroplasma in Drosophila is discussed. PMID:16783009

  14. Visual learning in individually assayed Drosophila larvae.

    PubMed

    Gerber, B; Scherer, S; Neuser, K; Michels, B; Hendel, T; Stocker, R F; Heisenberg, M

    2004-01-01

    An understanding of associative learning is facilitated if it can be analyzed in a simple animal like the fruit fly Drosophila. Here, we introduce the first visual associative learning paradigm for larval Drosophila; this is remarkable as larvae have an order of magnitude fewer neurons than adult flies. Larvae were subjected to either of two reciprocal training regimes: Light+/Dark- or Light-/Dark+. Subsequently, all larvae were individually tested for their preference between Light versus Dark. The difference between training regimes was therefore exclusively which visual situation was associated with which reinforcer; differences observed during the test thus reflected exclusively associative learning. For positive reinforcement (+) we used fructose (FRU), and for negative reinforcement (-) either quinine or sodium chloride (QUI, NaCl). Under these conditions, associative learning could be reproducibly observed in both wild-type strains tested. We then compared the effectiveness of training using differential conditioning, with both positive and negative reinforcement, to that using only positive or only negative reinforcement. We found that FRU only, but neither QUI nor NaCl, was in itself effective as a reinforcer. This is the first demonstration of appetitive learning in larval Drosophila. It is now possible to investigate the behavioral and neuronal organization of appetitive visual learning in this simple and genetically easy-to-manipulate experimental system.

  15. SPARC–Dependent Cardiomyopathy in Drosophila

    PubMed Central

    Motamedchaboki, Khatereh; Bodmer, Rolf

    2016-01-01

    Background— The Drosophila heart is an important model for studying the genetics underpinning mammalian cardiac function. The system comprises contractile cardiomyocytes, adjacent to which are pairs of highly endocytic pericardial nephrocytes that modulate cardiac function by uncharacterized mechanisms. Identifying these mechanisms and the molecules involved is important because they may be relevant to human cardiac physiology. Methods and Results— This work aimed to identify circulating cardiomodulatory factors of potential relevance to humans using the Drosophila nephrocyte–cardiomyocyte system. A Kruppel-like factor 15 (dKlf15) loss-of-function strategy was used to ablate nephrocytes and then heart function and the hemolymph proteome were analyzed. Ablation of nephrocytes led to a severe cardiomyopathy characterized by a lengthening of diastolic interval. Rendering adult nephrocytes dysfunctional by disrupting their endocytic function or temporally conditional knockdown of dKlf15 led to a similar cardiomyopathy. Proteomics revealed that nephrocytes regulate the circulating levels of many secreted proteins, the most notable of which was the evolutionarily conserved matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC), a protein involved in mammalian cardiac function. Finally, reducing SPARC gene dosage ameliorated the cardiomyopathy that developed in the absence of nephrocytes. Conclusions— The data implicate SPARC in the noncell autonomous control of cardiac function in Drosophila and suggest that modulation of SPARC gene expression may ameliorate cardiac dysfunction in humans. PMID:26839388

  16. [Estimation of the biological age in males of the taiga tick (Ixodes persulcatus: Ixodinae) by fat reserves in the midgut].

    PubMed

    Grigor'eva, L A

    2012-01-01

    Some criteria for the estimation of the biological and calendar age by the fat storage in midgut cells of Ixodes persulcatus males were established on the basis of examination of ticks from the laboratory culture.

  17. Regenerative Inflammation: Lessons from Drosophila Intestinal Epithelium in Health and Disease

    PubMed Central

    Panayidou, Stavria; Apidianakis, Yiorgos

    2013-01-01

    Intestinal inflammation is widely recognized as a pivotal player in health and disease. Defined cytologically as the infiltration of leukocytes in the lamina propria layer of the intestine, it can damage the epithelium and, on a chronic basis, induce inflammatory bowel disease and potentially cancer. The current view thus dictates that blood cell infiltration is the instigator of intestinal inflammation and tumor-promoting inflammation. This is based partially on work in humans and mice showing that intestinal damage during microbially mediated inflammation activates phagocytic cells and lymphocytes that secrete inflammatory signals promoting tissue damage and tumorigenesis. Nevertheless, extensive parallel work in the Drosophila midgut shows that intestinal epithelium damage induces inflammatory signals and growth factors acting mainly in a paracrine manner to induce intestinal stem cell proliferation and tumor formation when genetically predisposed. This is accomplished without any apparent need to involve Drosophila hemocytes. Therefore, recent work on Drosophila host defense to infection by expanding its main focus on systemic immunity signaling pathways to include the study of organ homeostasis in health and disease shapes a new notion that epithelially emanating cytokines and growth factors can directly act on the intestinal stem cell niche to promote “regenerative inflammation” and potentially cancer. PMID:25437036

  18. Intestinal peptides as circulating hormones: release of tachykinin-related peptide from the locust and cockroach midgut.

    PubMed

    Winther, A M; Nässel, D R

    2001-04-01

    Tachykinin-related peptides (TRPs) in the locust Locusta migratoria and the cockroach Leucophaea maderae have stimulatory effects on some muscles that are not innervated by TRP-containing neurons. Thus, these tissues may be affected by circulating TRPs. Here, we have investigated whether the midgut is the source of circulating TRPs. TRP-immunoreactive material in the locust midgut is found only in the endocrine cells of the gut epithelium. In both species of insect, the endocrine cells contain several isoforms of TRPs, as determined by immunocytochemistry and a combination of chromatography (HPLC) and enzyme immunoassay (ELISA). The release of TRPs was investigated by ELISA using isolated midguts of the locust and cockroach. Elevated levels of K(+) in the bathing saline induced the release of TRP from the midgut of both species. To examine the release of TRPs into the circulation in vivo, we measured haemolymph levels of TRPs in fed and starved locusts. The concentration of TRP-immunoreactive material in fed locusts was estimated to be 0.15 nmol l(-1), and this increased approximately fourfold in insects starved for 24 h. In accordance with this observation, the content of TRP-immunoreactive material in the midgut was lower in starved locusts than in fed locusts. Although part of the increased blood concentration of TRPs may be due to reduced blood volume, our data suggest that TRPs are released as hormones from the midgut of the locust and cockroach and that this release may be linked to nutritional status.

  19. Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae).

    PubMed

    Suneja, Amita; Gulia, Monika; Gakhar, S K

    2003-02-01

    Rabbits were immunized three times with extracts of Anopheles stephensi midgut. Immunized rabbits showed a high titer of antibodies when characterized by ELISA. We investigated the effect of anti-mosquito midgut antibodies on mosquito fecundity, longevity, mortality, engorgement, and the development of the malaria parasite in mosquitoes. Fecundity was reduced significantly (38%) and similarly hatchability by about 43.5%. There was no statistically significant effect on mortality, longevity, and engorgement. When the mosquito blood meal contained anti-midgut antibodies, fewer oocysts of Plasmodium vivax developed in the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. We also found that the midgut antibodies inhibit the development and/or translocation of the sporozoites. Antisera raised against midgut of A. stephensi recognized eight polypeptides (110, 92, 70, 45, 38, 29, 15, 13 kDa) by Western blotting. Cross-reactive antigens/epitopes present in other tissues of A. stephensi were also examined both by Western blotting and in vivo ELISA. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission blocking vaccine and/or anti-mosquito vaccine.

  20. RNA editing in Drosophila melanogaster: new targets and functionalconsequences

    SciTech Connect

    Stapleton, Mark; Carlson, Joseph W.; Celniker, Susan E.

    2006-09-05

    Adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine in primary mRNA transcripts. These re-coding events affect coding potential, splice-sites, and stability of mature mRNAs. ADAR is an essential gene and studies in mouse, C. elegans, and Drosophila suggest its primary function is to modify adult behavior by altering signaling components in the nervous system. By comparing the sequence of isogenic cDNAs to genomic DNA, we have identified and experimentally verified 27 new targets of Drosophila ADAR. Our analyses lead us to identify new classes of genes whose transcripts are targets of ADAR including components of the actin cytoskeleton, and genes involved in ion homeostasis and signal transduction. Our results indicate that editing in Drosophila increases the diversity of the proteome, and does so in a manner that has direct functional consequences on protein function.

  1. RNA editing in Drosophila melanogaster: New targets and functional consequences

    PubMed Central

    Carlson, Joseph W.; Celniker, Susan E.

    2006-01-01

    Adenosine deaminases that act on RNA [adenosine deaminase, RNA specific (ADAR)] catalyze the site-specific conversion of adenosine to inosine in primary mRNA transcripts. These re-coding events affect coding potential, splice sites, and stability of mature mRNAs. ADAR is an essential gene, and studies in mouse, Caenorhabditis elegans, and Drosophila suggest that its primary function is to modify adult behavior by altering signaling components in the nervous system. By comparing the sequence of isogenic cDNAs to genomic DNA, we have identified and experimentally verified 27 new targets of Drosophila ADAR. Our analyses led us to identify new classes of genes whose transcripts are targets of ADAR, including components of the actin cytoskeleton and genes involved in ion homeostasis and signal transduction. Our results indicate that editing in Drosophila increases the diversity of the proteome, and does so in a manner that has direct functional consequences on protein function. PMID:17018572

  2. Aging studies in Drosophila melanogaster.

    PubMed

    Sun, Yaning; Yolitz, Jason; Wang, Cecilia; Spangler, Edward; Zhan, Ming; Zou, Sige

    2013-01-01

    Drosophila is a genetically tractable system ideal for investigating the mechanisms of aging and developing interventions for promoting healthy aging. Here we describe methods commonly used in Drosophila aging research. These include basic approaches for preparation of diets and measurements of lifespan, food intake, and reproductive output. We also describe some commonly used assays to measure changes in physiological and behavioral functions of Drosophila in aging, such as stress resistance and locomotor activity.

  3. Aging Studies in Drosophila melanogaster

    PubMed Central

    Sun, Yaning; Yolitz, Jason; Wang, Cecilia; Spangler, Edward; Zhan, Ming; Zou, Sige

    2015-01-01

    Summary Drosophila is a genetically tractable system ideal for investigating the mechanisms of aging and developing interventions for promoting healthy aging. Here we describe methods commonly used in Drosophila aging research. These include basic approaches for preparation of diets and measurements of lifespan, food intake and reproductive output. We also describe some commonly used assays to measure changes in physiological and behavioral functions of Drosophila in aging, such as stress resistance and locomotor activity. PMID:23929099

  4. Aggregation of Bacillus thuringiensis Cry1A Toxins upon Binding to Target Insect Larval Midgut Vesicles

    PubMed Central

    Aronson, Arthur I.; Geng, Chaoxian; Wu, Lan

    1999-01-01

    During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of Manduca sexta larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the Heliothis virescens midgut than with those from the M. sexta midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity

  5. Histochemical analysis of the goblet cell matrix in the larval midgut of Manduca sexta

    SciTech Connect

    Schultz, T.W.; Lozano, G.; Cajina-Quezada, M.

    1981-01-01

    Experimental analyses were made to histochemically determine the composition of the goblet cell matrix material in the larval midgut of the tobacco hornworm, Manduca sexta. Techniques employed following fixation in Carnoy fluid were the periodic acid-Schiff reaction and the alcian blue stain at pH 1.0 and pH 2.5 and following methylation and subsequent saponification. The cumulative evidence suggests that the plug material is an acid mucosubstance.

  6. Shifts in the Midgut/Pyloric Microbiota Composition within a Honey Bee Apiary throughout a Season.

    PubMed

    Ludvigsen, Jane; Rangberg, Anbjørg; Avershina, Ekaterina; Sekelja, Monika; Kreibich, Claus; Amdam, Gro; Rudi, Knut

    2015-01-01

    Honey bees (Apis mellifera) are prominent crop pollinators and are, thus, important for effective food production. The honey bee gut microbiota is mainly host specific, with only a few species being shared with other insects. It currently remains unclear how environmental/dietary conditions affect the microbiota within a honey bee population over time. Therefore, the aim of the present study was to characterize the composition of the midgut/pyloric microbiota of a honey bee apiary throughout a season. The rationale for investigating the midgut/pyloric microbiota is its dynamic nature. Monthly sampling of a demographic homogenous population of bees was performed between May and October, with concordant recording of the honey bee diet. Mixed Sanger-and Illumina 16S rRNA gene sequencing in combination with a quantitative PCR analysis were used to determine the bacterial composition. A marked increase in α-diversity was detected between May and June. Furthermore, we found that four distinct phylotypes belonging to the Proteobacteria dominated the microbiota, and these displayed major shifts throughout the season. Gilliamella apicola dominated the composition early on, and Snodgrassella alvi began to dominate when the other bacteria declined to an absolute low in October. In vitro co-culturing revealed that G. apicola suppressed S. alvi. No shift was detected in the composition of the microbiota under stable environment/dietary conditions between November and February. Therefore, environmental/dietary changes may trigger the shifts observed in the honey bee midgut/pyloric microbiota throughout a season.

  7. Responses of midgut amylases of Helicoverpa armigera to feeding on various host plants.

    PubMed

    Kotkar, Hemlata M; Sarate, Priya J; Tamhane, Vaijayanti A; Gupta, Vidya S; Giri, Ashok P

    2009-08-01

    Midgut digestive amylases and proteinases of Helicoverpa armigera, a polyphagous and devastating insect pest of economic importance have been studied. We also identified the potential of a sorghum amylase inhibitor against H. armigera midgut amylase. Amylase activities were detected in all the larval instars, pupae, moths and eggs; early instars had lower amylase levels which steadily increased up to the sixth larval instar. Qualitative and quantitative differences in midgut amylases of H. armigera upon feeding on natural and artificial diets were evident. Natural diets were categorized as one or more members of legumes, vegetables, flowers and cereals belonging to different plant families. Amylase activity and isoform patterns varied depending on host plant and/or artificial diet. Artificial diet-fed H. armigera larvae had comparatively high amylase activity and several unique amylase isoforms. Correlation of amylase and proteinase activities of H. armigera with the protein and carbohydrate content of various diets suggested that H. armigera regulates the levels of these digestive enzymes in response to macromolecular composition of the diet. These adjustments in the digestive enzymes of H. armigera may be to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. H. armigera, a generalist feeder experiences a great degree of nutritional heterogeneity in its diet. An investigation of the differences in enzyme levels in response to macronutrient balance and imbalance highlight their importance in insect nutrition.

  8. Multiple Modes of Action of the Squamocin in the Midgut Cells of Aedes aegypti Larvae

    PubMed Central

    de Paula, Sérgio Oliveira; Martins, Gustavo Ferreira; Zanuncio, José Cola

    2016-01-01

    Annonaceous acetogenins are botanical compounds with good potential for use as insecticides. In the vector, Aedes aegypti (L.) (Diptera: Culicidae), squamocin (acetogenin) has been reported to be a larvicide and cytotoxic, but the modes of action of this molecule are still poorly understood. This study evaluated the changes in the cell morphology, and in the expression of genes, for autophagy (Atg1 and Atg8), for membrane ion transporter V-ATPase, and for water channel aquaporin-4 (Aqp4) in the midgut of A. aegypti larvae exposed to squamocin from Annona mucosa Jacq. (Annonaceae). Squamocin showed cytotoxic action with changes in the midgut epithelium and digestive cells of A. aegypti larvae, increase in the expression for autophagy gene Atg1 and Atg8, decrease in the expression of V-ATPase, decrease in the expression of Aqp4 gene in LC20 and inhibition of Apq4 genes in the midgut of this vector in LC50. These multiple modes of action for squamocin are described for the first time in insects, and they are important because different sites of action of squamocin from A. mucosa may reduce the possibility of resistance of A. aegypti to this molecule. PMID:27532504

  9. Toxoneuron nigriceps parasitization delays midgut replacement in fifth-instar Heliothis virescens larvae.

    PubMed

    Tettamanti, Gianluca; Grimaldi, Annalisa; Pennacchio, Francesco; de Eguileor, Magda

    2008-05-01

    We have analyzed the effects of Toxoneuron nigriceps parasitization on the midgut development of its host Heliothis virescens. In parasitized H. virescens larvae, the midgut epithelium undergoes a complete replacement, which is qualitatively not different to that observed in synchronous unparasitized larvae, with similar temporal profiles of cell death and metabolic activity. However, the whole gut replacement process is significantly delayed in parasitized larvae, with complete differentiation of the new gut epithelium being observed 4 days later than in unparasitized controls. The administration of juvenile hormone before commitment and of 20-hydroxyecdysone (20E) after commitment delays and fosters, respectively, the replacement process of the midgut epithelium; moreover, the injection of 20E into developmentally arrested and 20E-deficient host last-instar larvae parasitized by T. nigriceps immediately triggers regular gut development. These hormone-based experiments suggest that endocrine alterations in the larval host, induced by T. nigriceps parasitism, are responsible for the temporal alterations in the gut replacement process. The role of this parasitoid-induced developmental change in the host regulation process is discussed.

  10. Cytotoxic effects of thiamethoxam in the midgut and malpighian tubules of Africanized Apis mellifera (Hymenoptera: Apidae).

    PubMed

    Catae, Aline Fernanda; Roat, Thaisa Cristina; De Oliveira, Regiane Alves; Nocelli, Roberta Cornélio Ferreira; Malaspina, Osmar

    2014-04-01

    Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non-target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera. Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC₅₀ (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide.

  11. Observations on house fly larvae midgut ultrastructure after Brevibacillus laterosporus ingestion.

    PubMed

    Ruiu, Luca; Satta, Alberto; Floris, Ignazio

    2012-11-01

    The pathological and histopathological course caused by Brevibacillus laterosporus on house fly larvae has been investigated conducting observations on insect behavior and midgut ultrastructure. After dissection and fixation, gut tissues were analyzed under transmission electron microscopy (TEM) in order to compare in vivo-treated and non-treated (control) fly specimens. Treated larvae showed extensively reduced feeding and growth rate, then became sluggish and died within 72 h. A progressive midgut epithelium deterioration was observed in treated larvae, compared to the control. Ultrastructural changes consisted of microvilli disruption, cytoplasm vacuolization and general disorganization, endoplasmic reticulum deformation, mitochondria alteration. Deterioration became progressively more dramatic until the infected cells released their content into the gut lumen. Disruption was associated also with midgut muscular sheath and connective tissue. These ultrastructural changes are similar to those widely described for other entomopathogenic bacteria, such as Bacillus thuringiensis, against different insect species. The rapid disruption of cellular fine structure supports a hypothesis based on an interaction of toxins with the epithelial cell membranes reminiscent of the specific B. thuringiensis δ-endotoxins mechanism of action on other insect targets.

  12. Shifts in the Midgut/Pyloric Microbiota Composition within a Honey Bee Apiary throughout a Season

    PubMed Central

    Ludvigsen, Jane; Rangberg, Anbjørg; Avershina, Ekaterina; Sekelja, Monika; Kreibich, Claus; Amdam, Gro; Rudi, Knut

    2015-01-01

    Honey bees (Apis mellifera) are prominent crop pollinators and are, thus, important for effective food production. The honey bee gut microbiota is mainly host specific, with only a few species being shared with other insects. It currently remains unclear how environmental/dietary conditions affect the microbiota within a honey bee population over time. Therefore, the aim of the present study was to characterize the composition of the midgut/pyloric microbiota of a honey bee apiary throughout a season. The rationale for investigating the midgut/pyloric microbiota is its dynamic nature. Monthly sampling of a demographic homogenous population of bees was performed between May and October, with concordant recording of the honey bee diet. Mixed Sanger-and Illumina 16S rRNA gene sequencing in combination with a quantitative PCR analysis were used to determine the bacterial composition. A marked increase in α-diversity was detected between May and June. Furthermore, we found that four distinct phylotypes belonging to the Proteobacteria dominated the microbiota, and these displayed major shifts throughout the season. Gilliamella apicola dominated the composition early on, and Snodgrassella alvi began to dominate when the other bacteria declined to an absolute low in October. In vitro co-culturing revealed that G. apicola suppressed S. alvi. No shift was detected in the composition of the microbiota under stable environment/dietary conditions between November and February. Therefore, environmental/dietary changes may trigger the shifts observed in the honey bee midgut/pyloric microbiota throughout a season. PMID:26330094

  13. Bioimage Informatics in the context of Drosophila research.

    PubMed

    Jug, Florian; Pietzsch, Tobias; Preibisch, Stephan; Tomancak, Pavel

    2014-06-15

    Modern biological research relies heavily on microscopic imaging. The advanced genetic toolkit of Drosophila makes it possible to label molecular and cellular components with unprecedented level of specificity necessitating the application of the most sophisticated imaging technologies. Imaging in Drosophila spans all scales from single molecules to the entire populations of adult organisms, from electron microscopy to live imaging of developmental processes. As the imaging approaches become more complex and ambitious, there is an increasing need for quantitative, computer-mediated image processing and analysis to make sense of the imagery. Bioimage Informatics is an emerging research field that covers all aspects of biological image analysis from data handling, through processing, to quantitative measurements, analysis and data presentation. Some of the most advanced, large scale projects, combining cutting edge imaging with complex bioimage informatics pipelines, are realized in the Drosophila research community. In this review, we discuss the current research in biological image analysis specifically relevant to the type of systems level image datasets that are uniquely available for the Drosophila model system. We focus on how state-of-the-art computer vision algorithms are impacting the ability of Drosophila researchers to analyze biological systems in space and time. We pay particular attention to how these algorithmic advances from computer science are made usable to practicing biologists through open source platforms and how biologists can themselves participate in their further development.

  14. Hsp27 gene in Drosophila ananassae subgroup was split by a recently acquired intron.

    PubMed

    Zhang, Li; Kang, Han; Jin, Shan; Zeng, Qing Tao; Yang, Yong

    2016-06-01

    In Drosophila, heat shock protein 27 (Hsp27) is a critical single-copy intron-free nuclear gene involved in the defense response against fungi and bacteria, and is a regulator of adult lifespan. In the present study, 33 homologous Hsp27 nucleotide sequences from different Drosophila species were amplified by PCR and reverse transcription PCR, and the phylogenetic relationships were analysed using neighbour-joining, maximum-likelihood and Bayesian methods. The phylogenetic topologies from analysis with different algorithms were similar, suggesting that the Hsp27 gene was split by a recently acquired intron during the evolution of the Drosophila ananassae subgroup.

  15. The effect of Emblica officinalis diet on lifespan, sexual behavior, and fitness characters in Drosophila melanogaster.

    PubMed

    Pathak, Pankaj; Prasad, B R Guru; Murthy, N Anjaneya; Hegde, S N

    2011-04-01

    Drosophila is an excellent organism to test Ayurvedic medicines. The objective of our study was to explore the potential of Emblica officinalis drug on longevity, sexual behavior, and reproductive fitness of Drosophila melanogaster using adult feeding method. Increase in the lifespan, fecundity, fertility, ovarioles number, and developmental time was observed in both parents and F1 generation, but not in the F2 generation in experimental culture (control + E. officinalis). According to the Duncan's multiple range test and ANOVA, there is a significant difference between two cultures. It was also noticed that E. officinalis influence some fitness characters in Drosophila along with sexual behavior.

  16. Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster

    PubMed Central

    Remnant, Emily J.; Williams, Adam; Lumb, Chris; Yang, Ying Ting; Chan, Janice; Duchêne, Sebastian; Daborn, Phillip J.; Batterham, Philip; Perry, Trent

    2016-01-01

    Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster. Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets. PMID:27172217

  17. Conserved genetic pathways controlling the development of the diffuse endocrine system in vertebrates and Drosophila.

    PubMed

    Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina L

    2010-05-01

    The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type.

  18. Reduced Gut Acidity Induces an Obese-Like Phenotype in Drosophila melanogaster and in Mice

    PubMed Central

    Yen, Jui-Hung; Kuo, Ping-Chang; Yeh, Sheng-Rong; Lin, Hung-Yu; Fu, Tsai-Feng; Wu, Ming-Shiang; Wang, Horng-Dar; Wang, Pei-Yu

    2015-01-01

    In order to identify genes involved in stress and metabolic regulation, we carried out a Drosophila P-element-mediated mutagenesis screen for starvation resistance. We isolated a mutant, m2, that showed a 23% increase in survival time under starvation conditions. The P-element insertion was mapped to the region upstream of the vha16-1 gene, which encodes the c subunit of the vacuolar-type H+-ATPase. We found that vha16-1 is highly expressed in the fly midgut, and that m2 mutant flies are hypomorphic for vha16-1 and also exhibit reduced midgut acidity. This deficit is likely to induce altered metabolism and contribute to accelerated aging, since vha16-1 mutant flies are short-lived and display increases in body weight and lipid accumulation. Similar phenotypes were also induced by pharmacological treatment, through feeding normal flies and mice with a carbonic anhydrase inhibitor (acetazolamide) or proton pump inhibitor (PPI, lansoprazole) to suppress gut acid production. Our study may thus provide a useful model for investigating chronic acid suppression in patients. PMID:26436771

  19. Tre1, a G Protein-Coupled Receptor, Directs Transepithelial Migration of Drosophila Germ Cells

    PubMed Central

    Bainton, Roland J; Heberlein, Ulrike

    2003-01-01

    In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target. PMID:14691551

  20. Conserved Genetic Pathways Controlling the Development of the Diffuse Endocrine System in Vertebrates and Drosophila

    PubMed Central

    Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina

    2014-01-01

    The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. PMID:20005229

  1. The Drosophila Auditory System

    PubMed Central

    Boekhoff-Falk, Grace; Eberl, Daniel F.

    2013-01-01

    Development of a functional auditory system in Drosophila requires specification and differentiation of the chordotonal sensilla of Johnston’s organ (JO) in the antenna, correct axonal targeting to the antennal mechanosensory and motor center (AMMC) in the brain, and synaptic connections to neurons in the downstream circuit. Chordotonal development in JO is functionally complicated by structural, molecular and functional diversity that is not yet fully understood, and construction of the auditory neural circuitry is only beginning to unfold. Here we describe our current understanding of developmental and molecular mechanisms that generate the exquisite functions of the Drosophila auditory system, emphasizing recent progress and highlighting important new questions arising from research on this remarkable sensory system. PMID:24719289

  2. Drosophila by the dozen

    SciTech Connect

    Celniker, Susan E.; Hoskins, Roger A.

    2007-07-13

    This year's conference on Drosophila research illustratedwell the current focus of Drosophila genomics on the comprehensiveidentification of functional elements in the genome sequence, includingmRNA transcripts arising from multiple alternative start sites and splicesites, a multiplicity of noncoding transcripts and small RNAs,identification of binding sites for transcription factors, sequenceconservation in related species and sequence variation within species.Resources and technologies for genetics and functional genomics aresteadily being improved, including the building of collections oftransposon insertion mutants and hairpin constructs for RNA interference(RNAi). The conference also highlighted progress in the use of genomicinformation by many laboratories to study diverse aspects of biology andmodels of human disease. Here we will review a few highlights of especialinterest to readers of Genome Biology.

  3. The bHLH transcription factor Hand is regulated by Alk in the Drosophila embryonic gut

    SciTech Connect

    Varshney, Gaurav K.; Palmer, Ruth H. . E-mail: Ruth.Palmer@ucmp.umu.se

    2006-12-29

    During embryonic development the midgut visceral muscle is formed by fusion of cells within the visceral mesoderm, a process initiated by the specification of a specialised cell type, the founder cell, within this tissue. Activation of the receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) in the developing visceral muscle of Drosophila melanogaster initiates a signal transduction pathway required for muscle fusion. In this paper, we have investigated downstream components which are regulated by this novel signalling pathway. Here we show that Alk-mediated signal transduction drives the expression of the bHLH transcription factor Hand in vivo. Loss of Alk function results in a complete lack of Hand expression in this tissue, whereas Alk gain of function results in an expansion of Hand expression. Finally, we have investigated the process of muscle fusion in the gut of Hand mutant animals and can find no obvious defects in this process, suggesting that Hand is not critical for visceral muscle fusion per se.

  4. Sexual circuitry in Drosophila.

    PubMed

    Auer, Thomas O; Benton, Richard

    2016-06-01

    The sexual behavior of Drosophila melanogaster is an outstanding paradigm to understand the molecular and neuronal basis of sophisticated animal actions. We discuss recent advances in our knowledge of the genetic hardwiring of the underlying neuronal circuitry, and how pertinent sensory cues are differentially detected and integrated in the male and female brain. We also consider how experience influences these circuits over short timescales, and the evolution of these pathways over longer timescales to endow species-specific sexual displays and responses.

  5. A Lepidopteran-Specific Gene Family Encoding Valine-Rich Midgut Proteins

    PubMed Central

    Odman-Naresh, Jothini; Duevel, Margret; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

    2013-01-01

    Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM), an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps), which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran digestive tract facing

  6. The fine structure of the midgut epithelium in a centipede, Scolopendra cingulata (Chilopoda, Scolopendridae), with the special emphasis on epithelial regeneration.

    PubMed

    Chajec, Lukasz; Sonakowska, Lidia; Rost-Roszkowska, Magdalena M

    2014-01-01

    Scolopendra cingulata has a tube-shaped digestive system that is divided into three distinct regions: fore-, mid- and hindgut. The midgut is lined with a pseudostratified columnar epithelium which is composed of digestive, secretory and regenerative cells. Hemocytes also appear between the digestive cells of the midgut epithelium. The ultrastructure of three types of epithelial cells and hemocytes of the midgut has been described with the special emphasis on the role of regenerative cells in the protection of midgut epithelium. The process of midgut epithelium regeneration proceeds due to the ability of regenerative cells to proliferate and differentiate according to a circadian rhythm. The regenerative cells serve as unipotent stem cells that divide in an asymmetric manner. Additionally, two types of hemocytes have been distinguished among midgut epithelial cells. They enter the midgut epithelium from the body cavity. Because of the fact that numerous microorganisms occur in the cytoplasm of midgut epithelial cells, we discuss the role of hemocytes in elimination of pathogens from the midgut epithelium. The studies were conducted with the use of transmission electron microscope and immunofluorescent methods.

  7. The heat shock response restricts virus infection in Drosophila

    PubMed Central

    Merkling, Sarah H.; Overheul, Gijs J.; van Mierlo, Joël T.; Arends, Daan; Gilissen, Christian; van Rij, Ronald P.

    2015-01-01

    Innate immunity is the first line of defence against pathogens and is essential for survival of the infected host. The fruit fly Drosophila melanogaster is an emerging model to study viral pathogenesis, yet antiviral defence responses remain poorly understood. Here, we describe the heat shock response, a cellular mechanism that prevents proteotoxicity, as a component of the antiviral immune response in Drosophila. Transcriptome analyses of Drosophila S2 cells and adult flies revealed strong induction of the heat shock response upon RNA virus infection. Dynamic induction patterns of heat shock pathway components were characterized in vitro and in vivo following infection with different classes of viruses. The heat shock transcription factor (Hsf), as well as active viral replication, were necessary for the induction of the response. Hsf-deficient adult flies were hypersensitive to virus infection, indicating a role of the heat shock response in antiviral defence. In accordance, transgenic activation of the heat shock response prolonged survival time after infection and enabled long-term control of virus replication to undetectable levels. Together, our results establish the heat shock response as an important constituent of innate antiviral immunity in Drosophila. PMID:26234525

  8. Fine structure of the midgut and Malpighian papillae in Campodea (Monocampa) quilisi Silvestri, 1932 (Hexapoda, Diplura) with special reference to the metal composition and physiological significance of midgut intracellular electron-dense granules.

    PubMed

    Pigino, G; Migliorini, M; Paccagnini, E; Bernini, F; Leonzio, C

    2005-06-01

    The fine structure of the midgut and the Malpighian papillae in Campodea (Monocampa) quilisi Silvestri, 1932 (Hexapoda, Diplura) specimens was described. We observed the presence of electron-dense granules (EDGs) in the midgut epithelial cells, similar in genesis, structure and aspect to the type A spherocrystals described in the midgut epithelium of Collembola and Diplopoda. Energy-dispersive X-ray microanalysis was used to detect the chemical composition of the granules and to relate it to the concentrations of some potential toxic heavy metals (Pb, Cu, Zn) in soil and litter. Chemical composition of the granules seems strongly influenced by the presence and bioavailability of heavy metals in the external environment. Specimens from a contaminated abandoned mining and smelting area (Colline Metallifere, southern Tuscany) were able to accumulate Fe, Mn, Zn, Pb and Cu in their midgut EDGs. In addition, we observed that C. (M.) quilisi was able to excrete the metal-containing granules into the external medium by the moulting of the intestinal epithelium. This confirms that the process of ionic retention of midgut cells is particularly significant in animals lacking Malpighian tubules.

  9. The Drosophila melanogaster host model

    PubMed Central

    Igboin, Christina O.; Griffen, Ann L.; Leys, Eugene J.

    2012-01-01

    The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed. PMID:22368770

  10. Antigenotoxicity studies in Drosophila melanogaster.

    PubMed

    Graf, U; Abraham, S K; Guzmán-Rincón, J; Würgler, F E

    1998-06-18

    The fruit fly Drosophila melangaster with its well developed array of genotoxicity test systems has been used in a number of studies on antigenotoxicity of various compounds and mixtures. In recent years, the newly developed Somatic Mutation and Recombination Tests (SMART) have mainly been employed. These one-generation tests make use of the wing or eye imaginal disc cells in larvae and have proven to be very efficient and sensitive. They are based on the principle that the loss of heterozygosity of suitable recessive markers can lead to the formation of mutant clones of cells that are then expressed as spots on the wings or eyes of the adult flies. We have employed the wing spot test with the two markers multiple wing hairs (mwh,3-0.3) and flare (flr,3-38.8). Three-day-old larvae, trans-heterozygous for these markers, are treated chronically or acutely by oral administration with the test compound(s) or complex mixtures. For antigenotoxicity studies, chronic co-treatments can be used, as well as separate pre-treatments with an antigenotoxic agent followed by a chronic treatment with a genotoxin. After eclosion, the wings of the adult flies are scored for the presence of single and twin spots. These spots can be due to different genotoxic events: either mitotic recombination or mutation (deletion, point mutation, specific types of translocation, etc.). The analysis of two different genotypes (one with structurally normal chromosomes, one with a multiply inverted balancer chromosome) allows for a quantitative determination of the recombinagenic activity of genotoxins. Results of two separate studies presented: (1) instant coffee has antirecombinagenic but not antimutagenic activity in the wing spot test; and (2) ascorbic acid and catechin are able to protect against in vivo nitrosation products of methyl urea in combination with sodium nitrite.

  11. Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix.

    PubMed

    Shi, Ji-Feng; Fu, Jia; Mu, Li-Li; Guo, Wen-Chao; Li, Guo-Qing

    2016-01-01

    Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata. In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAPs, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2, whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAPs responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval-larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.

  12. Hypergravity-induced altered behavior in Drosophila

    NASA Astrophysics Data System (ADS)

    Hosamani, Ravikumar; Wan, Judy; Marcu, Oana; Bhattacharya, Sharmila

    2012-07-01

    Microgravity and mechanical stress are important factors of the spaceflight environment, and affect astronaut health and behavior. Structural, functional, and behavioral mechanisms of all cells and organisms are adapted to Earth's gravitational force, 1G, while altered gravity can pose challenges to their adaptability to this new environment. On ground, hypergravity paradigms have been used to predict and complement studies on microgravity. Even small changes that take place at a molecular and genetic level during altered gravity may result in changes in phenotypic behavior. Drosophila provides a robust and simple, yet very reliable model system to understand the complexity of hypergravity-induced altered behavior, due to availability of a plethora of genetic tools. Locomotor behavior is a sensitive parameter that reflects the array of molecular adaptive mechanisms recruited during exposure to altered gravity. Thus, understanding the genetic basis of this behavior in a hypergravity environment could potentially extend our understanding of mechanisms of adaptation in microgravity. In our laboratory we are trying to dissect out the cellular and molecular mechanisms underlying hypergravity-induced oxidative stress, and its potential consequences on behavioral alterations by using Drosophila as a model system. In the present study, we employed pan-neuronal and mushroom body specific knock-down adult flies by using Gal4/UAS system to express inverted repeat transgenes (RNAi) to monitor and quantify the hypergravity-induced behavior in Drosophila. We established that acute hypergravity (3G for 60 min) causes a significant and robust decrease in the locomotor behavior in adult Drosophila, and that this change is dependent on genes related to Parkinson's disease, such as DJ-1α , DJ-1β , and parkin. In addition, we also showed that anatomically the control of this behavior is significantly processed in the mushroom body region of the fly brain. This work links a molecular

  13. Glial β-oxidation regulates Drosophila energy metabolism.

    PubMed

    Schulz, Joachim G; Laranjeira, Antonio; Van Huffel, Leen; Gärtner, Annette; Vilain, Sven; Bastianen, Jarl; Van Veldhoven, Paul P; Dotti, Carlos G

    2015-01-15

    The brain's impotence to utilize long-chain fatty acids as fuel, one of the dogmas in neuroscience, is surprising, since the nervous system is the tissue most energy consuming and most vulnerable to a lack of energy. Challenging this view, we here show in vivo that loss of the Drosophila carnitine palmitoyltransferase 2 (CPT2), an enzyme required for mitochondrial β-oxidation of long-chain fatty acids as substrates for energy production, results in the accumulation of triacylglyceride-filled lipid droplets in adult Drosophila brain but not in obesity. CPT2 rescue in glial cells alone is sufficient to restore triacylglyceride homeostasis, and we suggest that this is mediated by the release of ketone bodies from the rescued glial cells. These results demonstrate that the adult brain is able to catabolize fatty acids for cellular energy production.

  14. Glial β-Oxidation regulates Drosophila Energy Metabolism

    PubMed Central

    Schulz, Joachim G.; Laranjeira, Antonio; Van Huffel, Leen; Gärtner, Annette; Vilain, Sven; Bastianen, Jarl; Van Veldhoven, Paul P.; Dotti, Carlos G.

    2015-01-01

    The brain's impotence to utilize long-chain fatty acids as fuel, one of the dogmas in neuroscience, is surprising, since the nervous system is the tissue most energy consuming and most vulnerable to a lack of energy. Challenging this view, we here show in vivo that loss of the Drosophila carnitine palmitoyltransferase 2 (CPT2), an enzyme required for mitochondrial β-oxidation of long-chain fatty acids as substrates for energy production, results in the accumulation of triacylglyceride-filled lipid droplets in adult Drosophila brain but not in obesity. CPT2 rescue in glial cells alone is sufficient to restore triacylglyceride homeostasis, and we suggest that this is mediated by the release of ketone bodies from the rescued glial cells. These results demonstrate that the adult brain is able to catabolize fatty acids for cellular energy production. PMID:25588812

  15. The developmental transcriptome of Drosophila melanogaster

    SciTech Connect

    University of Connecticut; Graveley, Brenton R.; Brooks, Angela N.; Carlson, Joseph W.; Duff, Michael O.; Landolin, Jane M.; Yang, Li; Artieri, Carlo G.; van Baren, Marijke J.; Boley, Nathan; Booth, Benjamin W.; Brown, James B.; Cherbas, Lucy; Davis, Carrie A.; Dobin, Alex; Li, Renhua; Lin, Wei; Malone, John H.; Mattiuzzo, Nicolas R.; Miller, David; Sturgill, David; Tuch, Brian B.; Zaleski, Chris; Zhang, Dayu; Blanchette, Marco; Dudoit, Sandrine; Eads, Brian; Green, Richard E.; Hammonds, Ann; Jiang, Lichun; Kapranov, Phil; Langton, Laura; Perrimon, Norbert; Sandler, Jeremy E.; Wan, Kenneth H.; Willingham, Aarron; Zhang, Yu; Zou, Yi; Andrews, Justen; Bicke, Peter J.; Brenner, Steven E.; Brent, Michael R.; Cherbas, Peter; Gingeras, Thomas R.; Hoskins, Roger A.; Kaufman, Thomas C.; Oliver, Brian; Celniker, Susan E.

    2010-12-02

    . Whereas, 20% of Drosophila genes are annotated as encoding alternatively spliced premRNAs, splice-junction microarray experiments indicate that this number is at least 40% (ref. 7). Determining the diversity of mRNAs generated by alternative promoters, alternative splicing and RNA editing will substantially increase the inferred protein repertoire. Non-coding RNA genes (ncRNAs) including short interfering RNAs (siRNAs) and microRNAS (miRNAs) (reviewed in ref. 10), and longer ncRNAs such as bxd (ref. 11) and rox (ref. 12), have important roles in gene regulation, whereas others such as small nucleolar RNAs (snoRNAs)and small nuclear RNAs (snRNAs) are important components of macromolecular machines such as the ribosome and spliceosome. The transcription and processing of these ncRNAs must also be fully documented and mapped. As part of the modENCODE project to annotate the functional elements of the D. melanogaster and Caenorhabditis elegans genomes, we used RNA-Seq and tiling microarrays to sample the Drosophila transcriptome at unprecedented depth throughout development from early embryo to ageing male and female adults. We report on a high-resolution view of the discovery, structure and dynamic expression of the D. melanogaster transcriptome.

  16. Myc Function in Drosophila

    PubMed Central

    Gallant, Peter

    2013-01-01

    Drosophila contains a single MYC gene. Like its vertebrate homologs, it encodes a transcription factor that activates many targets, including prominently genes involved in ribosome biogenesis and translation. This activity makes Myc a central regulator of growth and/or proliferation of many cell types, such as imaginal disc cells, polyploid cells, stem cells, and blood cells. Importantly, not only does Myc act cell autonomously but it also affects the fate of adjacent cells and tissues. This potential of Myc is harnessed by many different signaling pathways, involving, among others, Wg, Dpp, Hpo, ecdysone, insulin, and mTOR. PMID:24086064

  17. Feeding regulation in Drosophila

    PubMed Central

    Pool, Allan-Hermann; Scott, Kristin

    2014-01-01

    Neuromodulators play a key role in adjusting animal behavior based on environmental cues and internal needs. Here, we review the regulation of Drosophila feeding behavior to illustrate how neuromodulators achieve behavioral plasticity. Recent studies have made rapid progress in determining molecular and cellular mechanisms that translate the metabolic needs of the fly into changes in neuroendocrine and neuromodulatory states. These neuromodulators in turn promote or inhibit discrete feeding behavioral subprograms. This review highlights the links between physiological needs, neuromodulatory states, and feeding decisions. PMID:24937262

  18. Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

    SciTech Connect

    Buarque, Diego S.; Spindola, Leticia M.N.; Martins, Rafael M.; Braz, Gloria R.C.; Tanaka, Aparecida S.

    2011-09-23

    Highlights: {yields} Tigutcystatin inhibits Trypanosoma cruzi cysteine proteases with high specificity. {yields} Tigutcystatin expression is up-regulated in response to T. cruzi infection. {yields} It is the first cysteine proteases inhibitor characterized from a triatomine insect. -- Abstract: The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K{sub i} = 3.29 nM) and human cathepsin L (K{sub i} = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.

  19. Cadmium resistance in Drosophila: a small cadmium binding substance

    SciTech Connect

    Jacobson, K.B.; Williams, M.W.; Richter, L.J.; Holt, S.E.; Hook, G.J.; Knoop, S.M.; Sloop, F.V.; Faust, J.B.

    1985-01-01

    A small cadmium-binding substance (CdBS) has been observed in adult Drosophila melanogaster that were raised for their entire growth cycle on a diet that contained 0.15 mM CdCl/sub 2/. Induction of CdBS was observed in strains that differed widely in their sensitivity of CdCl/sub 2/. This report describes the induction of CdBS and some of its characteristics. 17 refs., 4 figs., 1 tab.

  20. Phase 3 Trial of (177)Lu-Dotatate for Midgut Neuroendocrine Tumors.

    PubMed

    Strosberg, Jonathan; El-Haddad, Ghassan; Wolin, Edward; Hendifar, Andrew; Yao, James; Chasen, Beth; Mittra, Erik; Kunz, Pamela L; Kulke, Matthew H; Jacene, Heather; Bushnell, David; O'Dorisio, Thomas M; Baum, Richard P; Kulkarni, Harshad R; Caplin, Martyn; Lebtahi, Rachida; Hobday, Timothy; Delpassand, Ebrahim; Van Cutsem, Eric; Benson, Al; Srirajaskanthan, Rajaventhan; Pavel, Marianne; Mora, Jaime; Berlin, Jordan; Grande, Enrique; Reed, Nicholas; Seregni, Ettore; Öberg, Kjell; Lopera Sierra, Maribel; Santoro, Paola; Thevenet, Thomas; Erion, Jack L; Ruszniewski, Philippe; Kwekkeboom, Dik; Krenning, Eric

    2017-01-12

    Background Patients with advanced midgut neuroendocrine tumors who have had disease progression during first-line somatostatin analogue therapy have limited therapeutic options. This randomized, controlled trial evaluated the efficacy and safety of lutetium-177 ((177)Lu)-Dotatate in patients with advanced, progressive, somatostatin-receptor-positive midgut neuroendocrine tumors. Methods We randomly assigned 229 patients who had well-differentiated, metastatic midgut neuroendocrine tumors to receive either (177)Lu-Dotatate (116 patients) at a dose of 7.4 GBq every 8 weeks (four intravenous infusions, plus best supportive care including octreotide long-acting repeatable [LAR] administered intramuscularly at a dose of 30 mg) ((177)Lu-Dotatate group) or octreotide LAR alone (113 patients) administered intramuscularly at a dose of 60 mg every 4 weeks (control group). The primary end point was progression-free survival. Secondary end points included the objective response rate, overall survival, safety, and the side-effect profile. The final analysis of overall survival will be conducted in the future as specified in the protocol; a prespecified interim analysis of overall survival was conducted and is reported here. Results At the data-cutoff date for the primary analysis, the estimated rate of progression-free survival at month 20 was 65.2% (95% confidence interval [CI], 50.0 to 76.8) in the (177)Lu-Dotatate group and 10.8% (95% CI, 3.5 to 23.0) in the control group. The response rate was 18% in the (177)Lu-Dotatate group versus 3% in the control group (P<0.001). In the planned interim analysis of overall survival, 14 deaths occurred in the (177)Lu-Dotatate group and 26 in the control group (P=0.004). Grade 3 or 4 neutropenia, thrombocytopenia, and lymphopenia occurred in 1%, 2%, and 9%, respectively, of patients in the (177)Lu-Dotatate group as compared with no patients in the control group, with no evidence of renal toxic effects during the observed time frame

  1. Protein expression in the midgut of sugar-fed Aedes albopictus females

    PubMed Central

    2012-01-01

    Background Aedes albopictus is a vector for several fatal arboviruses in tropical and sub-tropical regions of the world. The midgut of the mosquito is the first barrier that pathogens must overcome to establish infection and represents one of the main immunologically active sites of the insect. Nevertheless, little is known about the proteins involved in the defense against pathogens, and even in the processing of food, and the detoxification of metabolites. The identification of proteins exclusively expressed in the midgut is the first step in understanding the complex physiology of this tissue and can provide insight into the mechanisms of pathogen-vector interaction. However, identification of the locally expressed proteins presents a challenge because the Ae. albopictus genome has not been sequenced. Methods In this study, two-dimensional electrophoresis (2DE) was combined with liquid chromatography in line with tandem mass spectrometry (LC-MS/MS) and data mining to identify the major proteins in the midgut of sugar-fed Ae. albopictus females. Results Fifty-six proteins were identified by sequence similarity to entries from the Ae. aegypti genome. In addition, two hypothetical proteins were experimentally confirmed. According to the gene ontology analysis, the identified proteins were classified into 16 clusters of biological processes. Use of the STRING database to investigate protein functional associations revealed five functional networks among the identified proteins, including a network for carbohydrate and amino acid metabolism, a group associated with ATP production and a network of proteins that interact during detoxification of toxic free radicals, among others. This analysis allowed the assignment of a potential role for proteins with unknown function based on their functional association with other characterized proteins. Conclusion Our findings represent the first proteome map of the Ae. albopictus midgut and denotes the first steps towards the

  2. Preduodenal portal vein in association with midgut malrotation and duodenal web-triple anomaly?

    PubMed

    Singal, Arbinder Kumar; Ramu, Chithra; Paul, Sarah; Matthai, John

    2009-02-01

    Preduodenal portal vein (PDPV) is a rare anomaly in which the portal vein passes anterior to the duodenum rather than posteriorly. Generally asymptomatic, PDPV may rarely cause duodenal obstruction or may coexist with other anomalies. We report a neonate who presented with duodenal obstruction and was found out to have 3 coexisting anomalies, each of which can lead to duodenal obstruction independently-PDPV, midgut malrotation, and duodenal web. A duodenoduodenostomy and a Ladd procedure were done, and the child recovered uneventfully. The mechanism of obstruction, interesting metabolic aberrations observed, outcome, and relevant literature are presented.

  3. Total management of short gut secondary to midgut volvulus without prolonged total parenteral alimentation.

    PubMed

    Tepas, J J; MacLean, W C; Kolbach, S; Shermeta, D W

    1978-12-01

    Absorption studies in rats have shown that intestinal adaptation after catastrophic injury can be stimulated by early enteral feeding. Using this concept, we have devised a technique of early initiation and advancement of oral feedings that begins with Cho-Free and Polycose and gradually adds sucrose and MCT in increasing proportions. The increasing complexity and caloric density of this diet provide sufficient nutrition to allow weaning from total parenteral alimentation within 2--3 wk. Our preliminary experience in babies with midgut volvulus, necrotizing enterocolitis, and gastroschisis has been successful and uncomplicated. These patients have demonstrated consistent weight gain and have been spared the complications associated with prolonged parenteral alimentation.

  4. Comparative evaluation of the genomes of three common Drosophila-associated bacteria

    PubMed Central

    Petkau, Kristina; Fast, David; Duggal, Aashna

    2016-01-01

    ABSTRACT Drosophila melanogaster is an excellent model to explore the molecular exchanges that occur between an animal intestine and associated microbes. Previous studies in Drosophila uncovered a sophisticated web of host responses to intestinal bacteria. The outcomes of these responses define critical events in the host, such as the establishment of immune responses, access to nutrients, and the rate of larval development. Despite our steady march towards illuminating the host machinery that responds to bacterial presence in the gut, there are significant gaps in our understanding of the microbial products that influence bacterial association with a fly host. We sequenced and characterized the genomes of three common Drosophila-associated microbes: Lactobacillus plantarum, Lactobacillus brevis and Acetobacter pasteurianus. For each species, we compared the genomes of Drosophila-associated strains to the genomes of strains isolated from alternative sources. We found that environmental Lactobacillus strains readily associated with adult Drosophila and were similar to fly isolates in terms of genome organization. In contrast, we identified a strain of A. pasteurianus that apparently fails to associate with adult Drosophila due to an inability to grow on fly nutrient food. Comparisons between association competent and incompetent A. pasteurianus strains identified a short list of candidate genes that may contribute to survival on fly medium. Many of the gene products unique to fly-associated strains have established roles in the stabilization of host-microbe interactions. These data add to a growing body of literature that examines the microbial perspective of host-microbe relationships. PMID:27493201

  5. iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

    PubMed

    Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie

    2017-01-30

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection.

  6. A midgut lysate of the Riptortus pedestris has antibacterial activity against LPS O-antigen-deficient Burkholderia mutants.

    PubMed

    Jang, Ho Am; Seo, Eun Sil; Seong, Min Young; Lee, Bok Luel

    2017-02-01

    Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O-antigen. The LPS O-antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild-type Burkholderia. To determine why LPS O-antigen-deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O-antigen-deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O-antigen-deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O-antigen-deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N-terminal sequence of the protein was determined to be a soybean Kunitz-type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O-antigen deficient (rough-type) Burkholderia.

  7. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus.

    PubMed

    Crava, Cristina M; Jakubowska, Agata K; Escriche, Baltasar; Herrero, Salvador; Bel, Yolanda

    2015-01-01

    Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity.

  8. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae

    PubMed Central

    Fernandez, Luisa E.; Aimanova, Karlygash G.; Gill, Sarjeet S.; Bravo, Alejandra; Soberón, Mario

    2005-01-01

    A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI–ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI–ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI–ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI–ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae. PMID:16255715

  9. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus

    PubMed Central

    Crava, Cristina M.; Jakubowska, Agata K.; Escriche, Baltasar; Herrero, Salvador; Bel, Yolanda

    2015-01-01

    Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity. PMID:25993013

  10. Myoblast fusion in Drosophila

    SciTech Connect

    Haralalka, Shruti; Abmayr, Susan M.

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  11. Deconstructing Memory in Drosophila

    PubMed Central

    Margulies, Carla; Tully, Tim; Dubnau, Josh

    2011-01-01

    Unlike most organ systems, which have evolved to maintain homeostasis, the brain has been selected to sense and adapt to environmental stimuli by constantly altering interactions in a gene network that functions within a larger neural network. This unique feature of the central nervous system provides a remarkable plasticity of behavior, but also makes experimental investigations challenging. Each experimental intervention ramifies through both gene and neural networks, resulting in unpredicted and sometimes confusing phenotypic adaptations. Experimental dissection of mechanisms underlying behavioral plasticity ultimately must accomplish an integration across many levels of biological organization, including genetic pathways acting within individual neurons, neural network interactions which feed back to gene function, and phenotypic observations at the behavioral level. This dissection will be more easily accomplished for model systems such as Drosophila, which, compared with mammals, have relatively simple and manipulable nervous systems and genomes. The evolutionary conservation of behavioral phenotype and the underlying gene function ensures that much of what we learn in such model systems will be relevant to human cognition. In this essay, we have not attempted to review the entire Drosophila memory field. Instead, we have tried to discuss particular findings that provide some level of intellectual synthesis across three levels of biological organization: behavior, neural circuitry and biochemical pathways. We have attempted to use this integrative approach to evaluate distinct mechanistic hypotheses, and to propose critical experiments that will advance this field. PMID:16139203

  12. Epigenetic regulation in Drosophila.

    PubMed

    Lyko, F; Beisel, C; Marhold, J; Paro, R

    2006-01-01

    Epigenetic regulation of gene transcription relies on molecular marks like DNA methylation or histone modifications. Here we review recent advances in our understanding of epigenetic regulation in the fruit fly Drosophila melanogaster. In the past, DNA methylation research has primarily utilized mammalian model systems. However, several recent landmark discoveries have been made in other organisms. For example, the interaction between DNA methylation and histone methylation was first described in the filamentous fungus Neurospora crassa. Another example is provided by the interaction between epigenetic modifications and the RNA interference (RNAi) machinery that was first reported in the fission yeast Schizosaccharomyces pombe. Another organism with great experimental power is the fruit fly Drosophila. Epigenetic regulation by chromatin has been extensively analyzed in the fly and several of the key components have been discovered in this organism. In this chapter, we will focus on three aspects that represent the complexity of epigenetic gene regulation. (1) We will discuss the available data about the DNA methylation system, (2) we will illuminate the interaction between DNA methylation and chromatin regulation, and (3) we will provide an overview over the Polycomb system of epigenetic chromatin modifiers that has proved to be an important paradigm for a chromatin system regulating epigenetic programming.

  13. [Studies of the biological age in adult taiga ticks Ixodes persulcatus (Ixodinae)].

    PubMed

    Grigor'eva, L A

    2013-01-01

    The history of studies of the biological age in ixodid ticks is discussed. A method of estimation of the biological age in adult ticks of the genus Ixodes by the degree of fat inclusions in midgut cells and in the fat body is developed. An "age scale" for the determination of the calendar age was assumed.

  14. Restriction of viral dissemination from the midgut determines incompetence of small brown planthopper as a vector of Southern rice black-streaked dwarf virus.

    PubMed

    Jia, Dongsheng; Chen, Hongyan; Mao, Qianzhuo; Liu, Qifei; Wei, Taiyun

    2012-08-01

    Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, is transmitted by the white-backed planthopper in a persistent-propagative manner. In this study, we found that another planthopper species, the small brown planthopper (SBPH), could acquire SRBSDV but not transmit it. To identify the transmission barrier for SRBSDV in SBPHs, sequential infection by SRBSDV in the organs of SBPHs was studied with immunofluorescence for viral antigens. SRBSDV initially entered the epithelial cells of the midgut, then viroplasms, the sites for viral replication, formed in the midgut of viruliferous SBPHs. Furthermore, SRBSDV spread within the midgut, but failed to disseminate from the midgut into the hemocoel or into the salivary glands. All these results indicated that the inability of SBPH to transmit SRBSDV could be due to the restriction of viral dissemination from the midgut of SBPH, which led to the failure of viral spread to the salivary glands for virus transmission.

  15. Glutathione S-transferase in the midgut tissue of gypsy moth (Lymantria dispar) caterpillars exposed to dietary cadmium.

    PubMed

    Vlahović, Milena; Ilijin, Larisa; Mrdaković, Marija; Todorović, Dajana; Matić, Dragana; Lazarević, Jelica; Mataruga, Vesna Perić

    2016-06-01

    Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 and 30μg Cd/g dry food was examined. Based on the enzyme reaction through conjugation with glutathione, overall activity remained unaltered after acute and chronic treatment. No-observed-effect-concentration (10μg Cd/g dry food) significantly increased activity only after 3-day recovery following cadmium administration. Almost all comparisons of the indices of phenotypic plasticity revealed statistically significant differences. Despite the facts that GST has important role in xenobiotic biotransformation, our results indicate that this enzyme in insect midgut does not represent the key factor in cadmium detoxification.

  16. The scoop on the fly brain: glial engulfment functions in Drosophila.

    PubMed

    Logan, Mary A; Freeman, Marc R

    2007-02-01

    Glial cells provide support and protection for neurons in the embryonic and adult brain, mediated in part through the phagocytic activity of glia. Glial cells engulf apoptotic cells and pruned neurites from the developing nervous system, and also clear degenerating neuronal debris from the adult brain after neural trauma. Studies indicate that Drosophila melanogaster is an ideal model system to elucidate the mechanisms of engulfment by glia. The recent studies reviewed here show that many features of glial engulfment are conserved across species and argue that work in Drosophila will provide valuable cellular and molecular insight into glial engulfment activity in mammals.

  17. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

    PubMed Central

    Goldberg, D A

    1980-01-01

    The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments. Images PMID:6777776

  18. Bacterial entomopathogens from the Drosophila paulistorum semispecies complex.

    PubMed

    Miller, S G; Campbell, B C; Becnel, J; Ehrman, L

    1995-03-01

    Bacteria which are infectious by inoculation in lepidoptera have been isolated and characterized from semispecies comprising the Drosophila paulistorum complex. These microorganisms are pathogenic toward lepidopteran hosts such as Heliothis virescens when introduced by injection of Drosophila tissue extracts and have been given the trivial name DpLE (D. paulistorum lepidopteran entomopathogen). The DpLE from two of the semispecies, Transitional and Andean, were determined to be related to Proteus vulgaris based upon nucleotide sequence comparisons of 16S rDNA genes. Infectivity and 16S rDNA-based PCR assays showed the bacterium to be localized in a number of drosophilid tissues except adult heads and thoraces. Based upon similar experiments, the DpLE in transinfected Heliothis larvae were found in all tissues assayed prior to the onset of mortality. Stocks of Drosophila which had spontaneously lost DpLE continued to produce sterile sons when crossed with incompatible semispecies' females, confirming that the bacilliform DpLE is not the causative agent of the Drosophila paulistorum intersemispecific hybrid male sterility. Acquisition of the sequences of the 16S rDNA molecules of DpLE from all six semispecies permitted the construction of a phylogenetic tree in which the groupings were found not to be congruent with the phylogenies of their insect hosts.

  19. Enhancing undergraduate teaching and research with a Drosophila virginizing system.

    PubMed

    Venema, Dennis R

    2006-01-01

    Laboratory exercises using Drosophila crosses are an effective pedagogical method to complement traditional lecture and textbook presentations of genetics. Undergraduate thesis research is another common setting for using Drosophila. A significant barrier to using Drosophila for undergraduate teaching or research is the time and skill required to accurately collect virgins for use in controlled crosses. Erroneously collecting males or nonvirgin females contaminates crosses with unintended genotypes and confounds the results. Collecting adequate numbers of virgins requires large amounts of time, even for those skilled in virgin collection. I have adapted an effective method for virgin collection that eliminates these concerns and is straightforward to use in undergraduate settings. Using a heat-shock-induced, conditional lethal transgene specifically in males, male larvae can be eliminated from a culture before adults eclose. Females thus eclose in the absence of males and remain virgin, eliminating the need to laboriously score and segregate freshly eclosed females. This method is reliable, easily adaptable to any desired phenotypic marker, and readily scaleable to provide sufficient virgins for large laboratory classes or undergraduate research projects. In addition, it allows instructors lacking Drosophila expertise to use this organism as a pedagogical tool.

  20. Enhancing Undergraduate Teaching and Research with a Drosophila Virginizing System

    PubMed Central

    2006-01-01

    Laboratory exercises using Drosophila crosses are an effective pedagogical method to complement traditional lecture and textbook presentations of genetics. Undergraduate thesis research is another common setting for using Drosophila. A significant barrier to using Drosophila for undergraduate teaching or research is the time and skill required to accurately collect virgins for use in controlled crosses. Erroneously collecting males or nonvirgin females contaminates crosses with unintended genotypes and confounds the results. Collecting adequate numbers of virgins requires large amounts of time, even for those skilled in virgin collection. I have adapted an effective method for virgin collection that eliminates these concerns and is straightforward to use in undergraduate settings. Using a heat-shock–induced, conditional lethal transgene specifically in males, male larvae can be eliminated from a culture before adults eclose. Females thus eclose in the absence of males and remain virgin, eliminating the need to laboriously score and segregate freshly eclosed females. This method is reliable, easily adaptable to any desired phenotypic marker, and readily scaleable to provide sufficient virgins for large laboratory classes or undergraduate research projects. In addition, it allows instructors lacking Drosophila expertise to use this organism as a pedagogical tool. PMID:17146043

  1. Identification of a Drosophila activin receptor.

    PubMed Central

    Childs, S R; Wrana, J L; Arora, K; Attisano, L; O'Connor, M B; Massagué, J

    1993-01-01

    Activins are cytokines of the transforming growth factor beta superfamily that control various events during vertebrate embryo development and cell differentiation in the adult, and act through transmembrane receptors that contain a cytoplasmic protein-serine/threonine kinase domain. We describe the identification, deduced primary structure, and expression pattern of Atr-II, a receptor serine/threonine kinase found in Drosophila. With the exception of the spacing of 10 cysteine residues, the extracellular domain of Atr-II is very dissimilar from those of vertebrate activin receptors, yet it binds activin with high affinity and specificity. The kinase domain sequence of Atr-II is 60% identical to those of activin receptors from vertebrates, suggesting similarities in their signaling mechanisms. Maternal Atr-II transcript and its product are abundant in the oocyte. During development, the highest levels of Atr-II transcript and protein are observed in the mesoderm and gut. The possible role of an activin signaling system in Drosophila development is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8415726

  2. Genetic analysis of glutamatergic function in Drosophila

    SciTech Connect

    Chase, B.A.; Kankel, D.R.

    1987-01-01

    Neurotransmitters are essential for communication between neurons and hence are vital in the overall integrative functioning of the nervous system. Previous work on acetylcholine metabolism in the fruit fly, Drosophila melanogaster, has also raised the possibility that transmitter metabolism may play a prominent role in either the achievement or maintenance of the normal structure of the central nervous system in this species. Unfortunately, acetylcholine is rather poorly characterized as a neurotransmitter in Drosophila; consequently, we have begun an analysis of the role of glutamate (probably the best characterized transmitter in this organism) in the formation and/or maintenance of nervous system structure. We present here the results of a series of preliminary analyses. To suggest where glutamatergic function may be localized, an examination of the spatial distribution of high affinity (/sup 3/H)-glutamate binding sites are presented. We present the results of an analysis of the spatial and temporal distribution of enzymatic activities thought to be important in the regulation of transmitter-glutamate pools (i.e., glutamate oxaloacetic transaminase, glutaminase, and glutamate dehydrogenase). To begin to examine whether mutations in any of these functions are capable of affecting glutamatergic activity, we present the results of an initial genetic analysis of one enzymatic function, glutamate oxaloacetic transaminase (GOT), chosen because of its differential distribution within the adult central nervous system and musculature.

  3. Mosquito ingestion of antibodies against mosquito midgut microbiota improves conversion of ookinetes to oocysts for Plasmodium falciparum, but not P. yoelii

    PubMed Central

    Vaughan, Jefferson A.; Pumpuni, Charles B.; Beier, John C.

    2011-01-01

    The mosquito midgut is a site of complex interactions between the mosquito, the malaria parasite and the resident bacterial flora. In laboratory experiments, we observed significant enhancement of Plasmodium falciparum oocyst production when Anopheles gambiae (Diptera: Culicidae) mosquitoes were membrane-fed on infected blood containing gametocytes from in vitro cultures mixed with sera from rabbits immunized with A. gambiae midguts. To identify specific mechanisms, we evaluated whether the immune sera was interfering with the usual limiting activity of gram-negative bacteria in An. gambiae midguts. Enhancement of P. falciparum infection rates occurred at some stage between the ookinete and oocyst stage and was associated with greater numbers of oocysts in mosquitoes fed on immune sera. The same immune sera did not affect the sporogonic development of P. yoelii, a rodent malaria parasite. Not only did antibodies in the immune sera recognize several types of midgut-derived gram-negative bacteria (Pseudomonas spp. and Cedecea spp.), but gentamicin provided in the sugar meal 3 days before an infectious P. falciparum blood meal mixed with immune sera eliminated the enhancing effect. These results suggest that gram-negative bacteria, which normally impair P. falciparum development between the ookinete and oocyst stage, were altered by specific anti-bacterial antibodies produced by immunizing rabbits with non-antibiotic-treated midgut lysates. Because of the differences in developmental kinetics between human and rodent malaria species, the anti-bacterial antibodies had no effect on P. yoelii because their ookinetes leave the midgut much earlier than P. falciparum and so are not influenced as strongly by resident midgut bacteria. While this study highlights the complex interactions occurring between the parasite, mosquito, and midgut microbiota, the ultimate goal is to determine the influence of midgut microbiota on Plasmodium development in anopheline midguts in malaria

  4. Bacteria in midguts of field-collected Anopheles albimanus block Plasmodium vivax sporogonic development.

    PubMed

    Gonzalez-Ceron, Lilia; Santillan, Frida; Rodriguez, Mario H; Mendez, Domingo; Hernandez-Avila, Juan E

    2003-05-01

    Bacterial infections were investigated in midguts of insectary and field-collected Anopheles albimanus Weidemann from southern Mexico. Serratia marcescens, Enterobacter cloacae and Enterobacter amnigenus 2, Enterobacter sp., and Serratia sp. were isolated in field samples obtained in 1998, but only Enterobacter sp. was recovered in field samples of 1997 and no bacteria were isolated from insectary specimens. These bacteria were offered along with Plasmodium vivax infected blood to aseptic insectary An. albimanus, and the number of infected mosquitoes as well as the oocyst densities assessed after 7d. Plasmodium vivax infections in mosquitoes co-infected with En. amnigenus 2, En. cloacae, and S. marcensces were 53, 17, and 210 times, respectively, lower than in control mosquitoes, and the mean oocyst density in mosquitoes co-infected with En. cloacae was 2.5 times lower than in controls. Mortality was 13 times higher in S. marcensces-infected mosquitoes compared with controls. The overall midgut bacterial infection in mosquito field populations may influence P. vivax transmission, and could contribute to explain the annual variations in malaria incidence observed in the area.

  5. Modulation of Malaria Infection in Anopheles gambiae Mosquitoes Exposed to Natural Midgut Bacteria

    PubMed Central

    Tchioffo, Majoline T.; Boissière, Anne; Churcher, Thomas S.; Abate, Luc; Gimonneau, Geoffrey; Nsango, Sandrine E.; Awono-Ambéné, Parfait H.; Christen, Richard; Berry, Antoine; Morlais, Isabelle

    2013-01-01

    The development of Plasmodium falciparum within the Anopheles gambiae mosquito relies on complex vector-parasite interactions, however the resident midgut microbiota also plays an important role in mediating parasite infection. In natural conditions, the mosquito microbial flora is diverse, composed of commensal and symbiotic bacteria. We report here the isolation of culturable midgut bacteria from mosquitoes collected in the field in Cameroon and their identification based on the 16S rRNA gene sequencing. We next measured the effect of selected natural bacterial isolates on Plasmodium falciparum infection prevalence and intensity over multiple infectious feedings and found that the bacteria significantly reduced the prevalence and intensity of infection. These results contrast with our previous study where the abundance of Enterobacteriaceae positively correlated with P. falciparum infection (Boissière et al. 2012). The oral infection of bacteria probably led to the disruption of the gut homeostasis and activated immune responses, and this pinpoints the importance of studying microbe-parasite interactions in natural conditions. Our results indicate that the effect of bacterial exposure on P. falciparum infection varies with factors from the parasite and the human host and calls for deeper dissection of these parameters for accurate interpretation of bacterial exposure results in laboratory settings. PMID:24324714

  6. Exosome Secretion by the Parasitic Protozoan Leishmania within the Sand Fly Midgut.

    PubMed

    Atayde, Vanessa Diniz; Aslan, Hamide; Townsend, Shannon; Hassani, Kasra; Kamhawi, Shaden; Olivier, Martin

    2015-11-03

    Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect's bite, possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in-vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite's infectious life cycle, and we propose to add these vesicles to the repertoire of virulence factors associated with vector-transmitted infections.

  7. West Nile Virus Infection Alters Midgut Gene Expression in Culex pipiens quinquefasciatus Say (Diptera: Culicidae)

    PubMed Central

    Smartt, Chelsea T.; Richards, Stephanie L.; Anderson, Sheri L.; Erickson, Jennifer S.

    2009-01-01

    Alterations in gene expression in the midgut of female Culex pipiens quinquefasciatus exposed to blood meals containing 6.8 logs plaque-forming units/mL of West Nile virus (WNV) were studied by fluorescent differential display. Twenty-six different cDNAs exhibited reproducible differences after feeding on infected blood. Of these, 21 cDNAs showed an increase in expression, and 5 showed a decrease in expression as a result of WNV presence in the blood meal. GenBank database searches showed that one clone with increased expression, CQ G12A2, shares 94% identity with a leucine-rich repeat-containing protein from Cx. p. quinquefasciatus and 32% identity to Toll-like receptors from Aedes aegypti. We present the first cDNA clone isolated from female Cx. p. quinquefasciatus midgut tissue whose expression changes on exposure to WNV. This cDNA represents a mosquito gene that is an excellent candidate for interacting with WNV in Cx. p. quinquefasciatus and may play a role in disease transmission. PMID:19635880

  8. Exosome secretion by the parasitic protozoan Leishmania within the sand fly midgut

    PubMed Central

    Atayde, Vanessa Diniz; Suau, Hamide Aslan; Townsend, Shannon; Hassani, Kasra; Kamhawi, Shaden; Olivier, Martin

    2015-01-01

    SUMMARY Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect’s bite possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite’s infectious life cycle and propose to add these vesicles to the repertoire of virulence factors associated to vector-transmitted infections. PMID:26565909

  9. Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut.

    PubMed

    Rupp, Ingrid; Sologub, Ludmilla; Williamson, Kim C; Scheuermayer, Matthias; Reininger, Luc; Doerig, Christian; Eksi, Saliha; Kombila, Davy U; Frank, Matthias; Pradel, Gabriele

    2011-04-01

    Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.

  10. The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects.

    PubMed

    Yu, C G; Mullins, M A; Warren, G W; Koziel, M G; Estruch, J J

    1997-02-01

    The Vip3A protein is a member of a newly discovered class of vegetative insecticidal proteins with activity against a broad spectrum of lepidopteran insects. Histopathological observations indicate that Vip3A ingestion by susceptible insects such as the black cutworm (Agrotis ipsilon) and fall armyworm (Spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm2 of diet and complete lysis of gut epithelium cells resulting in larval death at concentrations above 40 ng/cm2. The European corn borer (Ostrinia nubilalis), a nonsusceptible insect, does not develop any pathology upon ingesting Vip3A. While proteolytic processing of the Vip3A protein by midgut fluids obtained from susceptible and nonsusceptible insects is comparable, in vivo immunolocalization studies show that Vip3a binding is restricted to gut cells of susceptible insects. Therefore, the insect host range for Vip3A seems to be determined by its ability to bind gut cells. These results indicate that midgut epithelium cells of susceptible insects are the primary target for the Vip3A insecticidal protein and that their subsequent lysis is the primary mechanism of lethality. Disruption of gut cells appears to be the strategy adopted by the most effective insecticidal proteins.

  11. Cadmium-binding proteins in midgut gland of freshwater crayfish Procambarus clarkii

    SciTech Connect

    Del Ramo, J.; Pastor, A.; Torreblanca, A.; Medina, J.; Diza-Mayans, J.

    1989-02-01

    Metallothioneins, metal binding proteins, were originally isolated and characterized by Margoshes and Vallee. These proteins have a high affinity for various heavy metals, particularly cadmium and mercury and have extensively been studied in mammals. Metal binding proteins have been observed in a variety of marine invertebrates; however, there is very little information available on metal binding proteins in freshwater invertebrates, and particularly in freshwater crustaceans. Cadmium is an ubiquitous non essential element which possesses high toxicity to aquatic organisms. Cadmium binding proteins observed in invertebrates have similar characteristics to mammalian metallothioneins. In 1978, the American red crayfish appeared in Albufera Lake and the surrounding rice fields (Valencia, Spain). Albufera Lake and the surrounding rice fields waters are subjected to very heavy loads of sewage and toxic industrial residues (including heavy metals) from the many urban and wastewaters in this area. In previous reports the authors studied the toxicity and accumulation of cadmium on Procambarus clarkii of Albufera Lake. This crayfish shows a high resistance to cadmium and a great accumulation rate of this metal in several tissues, including midgut gland. Since Procambarus clarkii shows a high resistance to cadmium, the presence of cadmium binding proteins (Cd-BP) in midgut gland of these crayfish would be expected. This report describes results on the characterization of Cd-BPs obtained from cadmium exposed crayfish Procambarus clarkii, demonstrating their presence in this freshwater crayfish.

  12. Signaling by Drosophila capa neuropeptides.

    PubMed

    Davies, Shireen-A; Cabrero, Pablo; Povsic, Manca; Johnston, Natalie R; Terhzaz, Selim; Dow, Julian A T

    2013-07-01

    The capa peptide family, originally identified in the tobacco hawk moth, Manduca sexta, is now known to be present in many insect families, with increasing publications on capa neuropeptides each year. The physiological actions of capa peptides vary depending on the insect species but capa peptides have key myomodulatory and osmoregulatory functions, depending on insect lifestyle, and life stage. Capa peptide signaling is thus critical for fluid homeostasis and survival, making study of this neuropeptide family attractive for novel routes for insect control. In Dipteran species, including the genetically tractable Drosophila melanogaster, capa peptide action is diuretic; via elevation of nitric oxide, cGMP and calcium in the principal cells of the Malpighian tubules. The identification of the capa receptor (capaR) in several insect species has shown this to be a canonical GPCR. In D. melanogaster, ligand-activated capaR activity occurs in a dose-dependent manner between 10(-6) and 10(-12)M. Lower concentrations of capa peptide do not activate capaR, either in adult or larval Malpighian tubules. Use of transgenic flies in which capaR is knocked-down in only Malpighian tubule principal cells demonstrates that capaR modulates tubule fluid secretion rates and in doing so, sets the organismal response to desiccation. Thus, capa regulates a desiccation-responsive pathway in D. melanogaster, linking its role in osmoregulation and fluid homeostasis to environmental response and survival. The conservation of capa action between some Dipteran species suggests that capa's role in desiccation tolerance may not be confined to D. melanogaster.

  13. Review: Thermal preference in Drosophila

    PubMed Central

    Dillon, Michael E.; Wang, George; Garrity, Paul A.; Huey, Raymond B.

    2009-01-01

    Environmental temperature strongly affects physiology of ectotherms. Small ectotherms, like Drosophila, cannot endogenously regulate body temperature so must rely on behavior to maintain body temperature within a physiologically permissive range. Here we review what is known about Drosophila thermal preference. Work on thermal behavior in this group is particularly exciting because it provides the opportunity to connect genes to neuromolecular mechanisms to behavior to fitness in the wild. PMID:20161211

  14. Safeguarding genetic information in Drosophila.

    PubMed

    Su, Tin Tin

    2011-12-01

    Eukaryotic cells employ a plethora of conserved proteins and mechanisms to ensure genome integrity. In metazoa, these mechanisms must operate in the context of organism development. This mini-review highlights two emerging features of DNA damage responses in Drosophila: a crosstalk between DNA damage responses and components of the spindle assembly checkpoint, and increasing evidence for the effect of DNA damage on the developmental program at multiple points during the Drosophila life cycle.

  15. Identification of Holotrichia oblita midgut proteins that bind to Cry8-like toxin from Bacillus thuringiensis and assembling of H. oblita midgut tissue transcriptome.

    PubMed

    Jiang, Jian; Huang, Ying; Shu, Changlong; Soberón, Mario; Bravo, Alejandra; Liu, Chunqing; Song, Fuping; Lai, Jinsheng; Zhang, Jie

    2017-04-07

    The Bacillus thuringiensis strain HBF-18 (CGMCC2070), containing two cry genes (cry8-like and cry8Ga), is toxic to Holotrichia oblita larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, while Cry8-like is the more toxic protein. To analyze the binding characteristics of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMV) in H. oblita larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. Results of binding saturation assays demonstrated that Cry8-like binds specifically to binding sites on BBMV from H. oblita and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in midgut from H. oblita larvae were identified by pull-down assays, and by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the H. oblita midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like binding proteins. Eight Cry8-like-binding proteins were obtained from pull-down assays conducted with BBMV. The LC-MS/MS data of these proteins successfully matched with the H. oblita transcriptome, and the BLASTX results identified five proteins as serine protease, transferrin-like, ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different with those previously confirmed as receptors for Cry1A proteins in lepidopteran insect species such as aminopeptidase, alkaline phosphatase and cadherin.Importance:Holotrichia oblita is one of main soil-dwelling pests in China. The larvae damage the roots of crops, and resulted in significant yield reduction and economic losses. It is difficult to control principally due to its soil living habits. In recent years, some Cry8 toxins from Bacillus thuringiensis were shown to be active against this pest. The study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control

  16. Cytokines in Drosophila immunity.

    PubMed

    Vanha-Aho, Leena-Maija; Valanne, Susanna; Rämet, Mika

    2016-02-01

    Cytokines are a large and diverse group of small proteins that can affect many biological processes, but most commonly cytokines are known as mediators of the immune response. In the event of an infection, cytokines are produced in response to an immune stimulus, and they function as key regulators of the immune response. Cytokines come in many shapes and sizes, and although they vary greatly in structure, their functions have been well conserved in evolution. The immune signaling pathways that respond to cytokines are remarkably conserved from fly to man. Therefore, Drosophila melanogaster, provides an excellent platform for studying the biology and function of cytokines. In this review, we will describe the cytokines and cytokine-like molecules found in the fly and discuss their roles in host immunity.

  17. Control of apoptosis by Drosophila DCAF12.

    PubMed

    Hwangbo, Dae-Sung; Biteau, Benoit; Rath, Sneha; Kim, Jihyun; Jasper, Heinrich

    2016-05-01

    Regulated Apoptosis (Programmed Cell Death, PCD) maintains tissue homeostasis in adults, and ensures proper growth and morphogenesis of tissues during development of metazoans. Accordingly, defects in cellular processes triggering or executing apoptotic programs have been implicated in a variety of degenerative and neoplastic diseases. Here, we report the identification of DCAF12, an evolutionary conserved member of the WD40-motif repeat family of proteins, as a new regulator of apoptosis in Drosophila. We find that DCAF12 is required for Diap1 cleavage in response to pro-apoptotic signals, and is thus necessary and sufficient for RHG (Reaper, Hid, and Grim)-mediated apoptosis. Loss of DCAF12 perturbs the elimination of supernumerary or proliferation-impaired cells during development, and enhances tumor growth induced by loss of neoplastic tumor suppressors, highlighting the wide requirement for DCAF12 in PCD.

  18. Three-dimensional organization of Drosophila melanogaster interphase nuclei. I. Tissue-specific aspects of polytene nuclear architecture

    PubMed Central

    1987-01-01

    Interphase chromosome organization in four different Drosophila melanogaster tissues, covering three to four levels of polyteny, has been analyzed. The results are based primarily on three-dimensional reconstructions from unfixed tissues using a computer-based data collection and modeling system. A characteristic organization of chromosomes in each cell type is observed, independent of polyteny, with some packing motifs common to several or all tissues and others tissue-specific. All chromosomes display a right-handed coiling chirality, despite large differences in size and degree of coiling. Conversely, in each cell type, the heterochromatic centromeric regions have a unique structure, tendency to associate, and intranuclear location. The organization of condensed nucleolar chromatin is also tissue-specific. The tightly coiled prothoracic gland chromosomes are arrayed in a similar fashion to the much larger salivary gland chromosomes described previously, having polarized orientations, nonintertwined spatial domains, and close packing of the arms of each autosome, whereas hindgut and especially the unusually straight midgut chromosomes display striking departures from these regularities. Surprisingly, gut chromosomes often appear to be broken in the centric heterochromatin. Severe deformations of midgut nuclei observed during gut contractions in living larvae may account for their unusual properties. Finally, morphometric measurements of chromosome and nuclear dimensions provide insights into chromosome growth and substructure and also suggest an unexpected parallel with diploid chromatin organization. PMID:3108264

  19. The four serotypes of dengue recognize the same putative receptors in Aedes aegypti midgut and Ae. albopictus cells

    PubMed Central

    Mercado-Curiel, Ricardo F; Esquinca-Avilés, Héctor Armando; Tovar, Rosalinda; Díaz-Badillo, Álvaro; Camacho-Nuez, Minerva; Muñoz, María de Lourdes

    2006-01-01

    Background Dengue viruses (DENV) attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies. Results (1) Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67). (2) Specific antibodies against these two proteins inhibited cell binding and infection. (3) Both proteins were bound by all four serotypes of dengue virus. (4) R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5) In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6) R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut. Conclusion Our results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells. PMID:17014723

  20. Draft Genome Sequences of Two Strains of Serratia spp. from the Midgut of the Malaria Mosquito Anopheles gambiae

    PubMed Central

    Pei, Dong; Hill-Clemons, Casey; Carissimo, Guillaume; Yu, Wanqin; Vernick, Kenneth D.

    2015-01-01

    Here, we report the annotated draft genome sequences of two strains of Serratia spp., Ag1 and Ag2, isolated from the midgut of two different strains of Anopheles gambiae. The genomes of these two strains are almost identical. PMID:25767231

  1. Effects of periplocoside X on midgut cells and digestive enzymes activity of the soldiers of red imported fire ant.

    PubMed

    Li, Yan; Zeng, Xin-Nian

    2013-07-01

    The pathological effects of ingested periplocoside X, an insecticidal component isolated from the root of Periploca sepium Bunge, on the midgut epithelial cells of the soldiers of red imported fire ant were studied and the symptom was described. The results showed that periplocoside X could induce a severe, time-dependent cytotoxicity in the midgut epithelial cells. An optical microscopy showed that epithelial cells swelled firstly and then lysed. Transmission electron microscopy (TEM) showed that numerous swollen lysosomes were appeared, microvilli were disrupted and sloughed off, and the numbers of the rough endoplasmic reticulum and the mitochondria decreased sharply in earlier stage. Numerous vacuoles were observed in the later stage. Finally, periplocoside X resulted in cell death by cytolysis. Assay of main three digestive enzymes activity indicated that amylase activity was significantly inhibited, but no significant changes were seen for lipase activity and total protease activity. So it is suggested that periplocoside X induced mainly to organic damage of midgut epithelium cells of insect. In all, insect midgut is one of targets for periplocoside X.

  2. Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus

    PubMed Central

    Ghanim, Murad; Fattah-Hosseini, Somayeh; Levy, Amit; Cilia, Michelle

    2016-01-01

    Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as Huanglongbing (HLB). CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, in a circulative manner. Two major barriers to transmission within the insect are the midgut and the salivary glands. We performed a thorough microscopic analysis within the insect midgut following exposure to CLas-infected citrus trees. We observed changes in nuclear architecture, including pyknosis and karyorrhexis as well as changes to the actin cytoskeleton in CLas-exposed midgut cells. Further analyses showed that the changes are likely due to the activation of programmed cell death as assessed by Annexin V staining and DNA fragmentation assays. These results suggest that exposure to CLas-infected trees induces apoptotic responses in the psyllid midgut that should be further investigated. Understanding the adaptive significance of the apoptotic response has the potential to create new approaches for controlling HLB. PMID:27630042

  3. Live imaging of baculovirus infection of midgut epithelium cells: a functional assay of per os infectivity factors.

    PubMed

    Mu, Jingfang; van Lent, Jan W M; Smagghe, Guy; Wang, Yun; Chen, Xinwen; Vlak, Just M; van Oers, Monique M

    2014-11-01

    The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.

  4. The effects of Bt Cry1Ie toxin on bacterial diversity in the midgut of Apis mellifera ligustica (Hymenoptera: Apidae)

    PubMed Central

    Jia, Hui-Ru; Geng, Li-Li; Li, Yun-He; Wang, Qiang; Diao, Qing-Yun; Zhou, Ting; Dai, Ping-Li

    2016-01-01

    The honey bee has been regarded as a key species in the environmental risk assessment of biotech crops. Here, the potential adverse effects of Cry1Ie toxin on the midgut bacteria of the worker bees (Apis mellifera ligustica) were investigated under laboratory conditions. Newly emerged bees were fed with different concentrations of Cry1Ie toxin syrups (20 ng/mL, 200 ng/mL, and 20 μg/mL), pure sugar syrup, and 48 ppb of imidacloprid syrups, then sampled after 15 and 30 d. We characterized the dominant midgut bacteria and compared the composition and structure of the midgut bacterial community in all samples using the Illumina MiSeq platform targeting the V3–V4 regions of 16S rDNA. No significant differences in the diversity of the midgut bacteria were observed between the five treatments. This work was the first to show the effects of Cry1Ie toxin on honey bees, and our study provided a theoretical basis for the biosafety assessment of transgenic Cry1Ie maize. PMID:27090812

  5. Amino acids trigger down-regulation of superoxide via TORC pathway in the midgut of Rhodnius prolixus

    PubMed Central

    Gandara, Ana Caroline P.; Oliveira, José Henrique M.; Nunes, Rodrigo D.; Goncalves, Renata L.S.; Dias, Felipe A.; Hecht, Fabio; Fernandes, Denise C.; Genta, Fernando A.; Laurindo, Francisco R.M.; Oliveira, Marcus F.; Oliveira, Pedro L.

    2016-01-01

    Sensing incoming nutrients is an important and critical event for intestinal cells to sustain life of the whole organism. The TORC is a major protein complex involved in monitoring the nutritional status and is activated by elevated amino acid concentrations. An important feature of haematophagy is that huge amounts of blood are ingested in a single meal, which results in the release of large quantities of amino acids, together with the haemoglobin prosthetic group, haem, which decomposes hydroperoxides and propagates oxygen-derived free radicals. Our previous studies demonstrated that reactive oxygen species (ROS) levels were diminished in the mitochondria and midgut of the Dengue fever mosquito, Aedes aegypti, immediately after a blood meal. We proposed that this mechanism serves to avoid oxidative damage that would otherwise be induced by haem following a blood meal. Studies also performed in mosquitoes have shown that blood or amino acids controls protein synthesis through TORC activation. It was already proposed, in different models, a link between ROS and TOR, however, little is known about TOR signalling in insect midgut nor about the involvement of ROS in this pathway. Here, we studied the effect of a blood meal on ROS production in the midgut of Rhodnius prolixus. We observed that blood meal amino acids decreased ROS levels in the R. prolixus midgut immediately after feeding, via lowering mitochondrial superoxide production and involving the amino acid-sensing TORC pathway. PMID:26945025

  6. Biochemical and histological biomarkers in the midgut of Apis mellifera from polluted environment at Beheira Governorate, Egypt.

    PubMed

    El-Saad, Ahmed M Abu; Kheirallah, Dalia A; El-Samad, Lamia M

    2017-01-01

    The aim of this study was to analyze the impact of organophosphorus (OP) pollutants on oxidative stress and ultrastructural biomarkers in the midgut of the honeybee Apis mellifera collected from three locations that differ in their extent of spraying load with OP insecticides: a weakly anthropised rural site, Bolin which is considered as a reference site; moderately spraying site, El Kaza; and a strongly anthropised urban site, Tiba with a long history of pesticide use. Results showed that high concentrations of chlorpyrifos, malathion, diazinon, chlorpyrifos-methyl, and pirimiphos-methyl were detected in midgut at locations with extensive pesticide spraying. Reduced glutathione content, superoxide dismutase, catalase, and glutathione peroxidase displayed lowest activities in the heavily sprayed location (Tiba). Lipid peroxidation level in the midgut of honeybees in the sprayed locations was found to be significantly higher compared to the reference values. Meanwhile, various ultrastructural abnormalities were observed in the epithelial cells of midgut of honeybees collected from El Kaza and Tiba, included confluent and disorganized microvilli and destruction of their brush border, the cytoplasm with large vacuoles and alteration of cytoplasmic organelles including the presence of swollen mitochondria with lysis of matrices, disruption of limiting membranes, and disintegration of cristae. The nuclei with indented nuclear envelope and disorganized chromatin were observed. These investigated biomarkers indicated that the surveyed honeybees are being under stressful environmental conditions. So, we suggest using those biomarkers in the assessment of environmental quality using honeybees in future monitoring of ecotoxicological studies.

  7. Identification of Midgut and Salivary Glands as Specific and Distinct Barriers to Efficient Tick-Borne Transmission of Anaplasma marginale▿

    PubMed Central

    Ueti, Massaro W.; Reagan, James O.; Knowles, Donald P.; Scoles, Glen A.; Shkap, Varda; Palmer, Guy H.

    2007-01-01

    Understanding the determinants of efficient tick-borne microbial transmission is needed to better predict the emergence of highly transmissible pathogen strains and disease outbreaks. Although the basic developmental cycle of Anaplasma and Ehrlichia spp. within the tick has been delineated, there are marked differences in the ability of specific strains to be efficiently tick transmitted. Using the highly transmissible St. Maries strain of Anaplasma marginale in Dermacentor andersoni as a positive control and two unrelated nontransmissible strains, we identified distinct barriers to efficient transmission within the tick. The Mississippi strain was unable to establish infection at the level of the midgut epithelium despite successful ingestion of infected blood following acquisition feeding on a bacteremic animal host. This inability to colonize the midgut epithelium prevented subsequent development within the salivary glands and transmission. In contrast, A. marginale subsp. centrale colonized the midgut and then the salivary glands, replicating to a titer indistinguishable from that of the highly transmissible St. Maries strain and at least 100 times greater than that previously associated with successful transmission. Nonetheless, A. marginale subsp. centrale was not transmitted, even when a large number of infected ticks was used for transmission feeding. These results establish that there are at least two specific barriers to efficient tick-borne transmission, the midgut and salivary glands, and highlight the complexity of the pathogen-tick interaction. PMID:17420231

  8. Ookinete destruction within the mosquito midgut lumen explains Anopheles albimanus refractoriness to Plasmodium falciparum (3D7A) oocyst infection.

    PubMed

    Baton, Luke A; Ranford-Cartwright, Lisa C

    2012-01-01

    Previous studies have shown that the central American mosquito vector, Anopheles albimanus, is generally refractory to oocyst infection with allopatric isolates of the human malaria parasite Plasmodium falciparum. However, the reasons for the refractoriness of A. albimanus to infection with such isolates of P. falciparum are unknown. In the current study, we investigated the infectivity of the P. falciparum clone 3D7A to laboratory-reared A. albimanus and another natural vector of human malaria, Anopheles stephensi. Plasmodium falciparum gametocytes grown in vitro were simultaneously fed to both mosquito species and the progress of malaria infection compared. In 22 independent paired experimental feeds, no mature oocysts were observed on the midguts of A. albimanus 10days after bloodfeeding. In contrast, high levels of oocyst infection were found on the midguts of simultaneously fed A. stephensi. Direct immunofluorescence microscopy and light microscopical examination of Giemsa-stained histological sections were used to identify when the P. falciparum clone 3D7A failed to establish mature oocyst infections in A. albimanus. Similar densities of macrogametes/zygotes, and immature retort-form and mature ookinetes were found within the bloodmeals of both mosquito species. However, in A. albimanus, ookinetes were seldom associated with the peritrophic matrix, and were neither observed in the ectoperitrophic space nor the midgut epithelium. In contrast, ookinetes were frequently observed in these midgut compartments in A. stephensi. Additionally, young oocysts were observed on the midguts of A. stephensi but not A. albimanus 2days after bloodfeeding. Vital staining of the immature retort-form and mature ookinetes found within the luminal bloodmeal, demonstrated that a significantly greater proportion of these malaria parasite stages were non-viable in A. albimanus compared with A. stephensi. Overall, our observations indicate that ookinetes of the P. falciparum clone 3D7

  9. Exploring the mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae)

    PubMed Central

    Anderson, Jennifer M; Sonenshine, Daniel E; Valenzuela, Jesus G

    2008-01-01

    Background Ticks are obligate blood feeders. The midgut is the first major region of the body where blood and microbes ingested with the blood meal come in contact with the tick's internal tissues. Little is known about protein expression in the digestive tract of ticks. In this study, for analysis of global gene expression during tick attachment and feeding, we generated and sequenced 1,679 random transcripts (ESTs) from cDNA libraries from the midguts of female ticks at varying stages of feeding. Results Sequence analysis of the 1,679 ESTs resulted in the identification of 835 distinct transcripts, from these, a total of 82 transcripts were identified as proteins putatively directly involved in blood meal digestion, including enzymes involved in oxidative stress reduction/antimicrobial activity/detoxification, peptidase inhibitors, protein digestion (cysteine-, aspartic-, serine-, and metallo-peptidases), cell, protein and lipid binding including mucins and iron/heme metabolism and transport. A lectin-like protein with a high match to lectins in other tick species, allergen-like proteins and surface antigens important in pathogen recognition and/or antimicrobial activity were also found. Furthermore, midguts collected from the 6-day-fed ticks expressed twice as many transcripts involved in bloodmeal processing as midguts from unfed/2-day-fed ticks. Conclusion This tissue-specific transcriptome analysis provides an opportunity to examine the global expression of transcripts in the tick midgut and to compare the gut response to host attachment versus blood feeding and digestion. In contrast to those in salivary glands of other Ixodid ticks, most proteins in the D. variabilis midgut cDNA library were intracellular. Of the total ESTs associated with a function, an unusually large number of transcripts were associated with peptidases, cell, lipid and protein binding, and oxidative stress or detoxification. Presumably, this is consistent with their role in

  10. Assessing in vivo microRNA function in the germline stem cells of the Drosophila ovary.

    PubMed

    Chan, Kin; Ruohola-Baker, Hannele

    2010-01-01

    A more complete understanding of the biology of adult stem cells could yield important insights toward devising effective cell-based regenerative therapies to treat disease. The germline stem cells (GSCs) in the fruit fly Drosophila melanogaster are an excellent in vivo model for the study of adult stem cell biology. There is increasing evidence from a growing field that microRNAs (miRNAs) play important roles in controlling many aspects of stem-cell biology. Using straightforward genetic manipulations combined with well-established cell biological analysis techniques, we and others have found that the miRNA pathway regulates the cell division rate of Drosophila GSCs as well as the maintenance of the GSCs in their niche. In this chapter, we offer a detailed, self-contained description of a general method to assess the in vivo functions of miRNAs in the GSCs of the Drosophila ovary.

  11. Drosophila Avoids Parasitoids by Sensing Their Semiochemicals via a Dedicated Olfactory Circuit

    PubMed Central

    Ebrahim, Shimaa A. M.; Dweck, Hany K. M.; Stökl, Johannes; Hofferberth, John E.; Trona, Federica; Weniger, Kerstin; Rybak, Jürgen; Seki, Yoichi; Stensmyr, Marcus C.; Sachse, Silke; Hansson, Bill S.; Knaden, Markus

    2015-01-01

    Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. We show that Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes. PMID:26674493

  12. Fluorescence Localization and Comparative Ultrastructural Study of Periplocoside NW from Periploca sepium Bunge in the Midgut of the Oriental Amyworm, Mythimna separata Walker (Lepidoptera: Noctuidae)

    PubMed Central

    Feng, Mingxing; Zhao, Juan; Zhang, Jiwen; Hu, Zhaonong; Wu, Wenjun

    2014-01-01

    Periplocoside NW (PSNW) is a novel insecticidal compound isolated from the root bark of Periploca sepium Bunge and has potent stomach toxicity against some insect pests. Previous studies showed that the Mythimna separata larva is sensitive to PSNW, but the Agrotis ispilon larva is insensitive. In this study, preliminary target localization on the midgut of M. separata larvae was conducted via a fluorescence labeling technique. A comparative ultrastructural study on the effects of PSNW on the midguts of M. separata and A. ispilon larvae was performed. Symptom observation results showed that typical stomach toxicity was induced by PSNW in M. separata larvae. Fluorescence localization results showed that PSNW binds to the midgut cells of M. separata larvae. Ultrastructure observations showed destruction of the microvilli, organelle, and cytomembrane in the midgut cells of M. separata larvae, whereas no obvious changes were observed in midgut cells of A. ispilon larvae. These results were consistent with the insecticidal activity of PSNW. Therefore, PSNW might act on the midgut tissues of the insects, and one or more binding sites of PSNW may exist in M. separata larvae midgut cell cytomembranes. PMID:24831268

  13. Humoral responses in Rhodnius prolixus: bacterial feeding induces differential patterns of antibacterial activity and enhances mRNA levels of antimicrobial peptides in the midgut

    PubMed Central

    2014-01-01

    Background The triatomine, Rhodnius prolixus, is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. It has a strictly blood-sucking habit in all life stages, ingesting large amounts of blood from vertebrate hosts from which it can acquire pathogenic microorganisms. In this context, the production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract. Methods In the present work, we studied the antimicrobial activity of the Rhodnius prolixus midgut in vitro against the Gram-negative and Gram-positive bacteria Escherichia coli and Staphylococcus aureus, respectively. We also analysed the abundance of mRNAs encoding for defensins, prolixicin and lysozymes in the midgut of insects orally infected by these bacteria at 1 and 7 days after feeding. Results Our results showed that the anterior midgut contents contain a higher inducible antibacterial activity than those of the posterior midgut. We observed that the main AMP encoding mRNAs in the anterior midgut, 7 days after a blood meal, were for lysozyme A, B, defensin C and prolixicin while in the posterior midgut lysozyme B and prolixicin transcripts predominated. Conclusion Our findings suggest that R. prolixus modulates AMP gene expression upon ingestion of bacteria with patterns that are distinct and dependent upon the species of bacteria responsible for infection. PMID:24885969

  14. Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus

    PubMed Central

    Hanley, Kathryn A.; Sundararajan, Anitha; Devitt, Nicholas P.; Schilkey, Faye D.; Hansen, Immo A.

    2017-01-01

    Background The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses) may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal. Methodology/Principal findings We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses. Conclusion/Significance Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of

  15. A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus.

    PubMed

    Soares, Tatiane S; Buarque, Diego S; Queiroz, Bruna R; Gomes, Cícera M; Braz, Glória R C; Araújo, Ricardo N; Pereira, Marcos H; Guarneri, Alessandra A; Tanaka, Aparecida S

    2015-05-01

    The triatomine insect, Rhodnius prolixus, is a vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. The parasite must overcome immune response and microbiota to develop inside the midgut of triatomines. In this study, we expressed, purified and characterized a Kazal-type inhibitor from the midgut of R. prolixus, named RpTI, which may be involved in microbiota - T. cruzi interactions. The qPCR showed that the RpTI transcript was primarily expressed in tissues from the intestinal tract and that it was upregulated in the anterior midgut after T. cruzi infection. A 315-bp cDNA fragment encoding the mature protein was cloned into the pPIC9 vector and expressed in Pichia pastoris system. Recombinant RpTI (rRpTI) was purified on a trypsin-Sepharose column and had a molecular mass of 11.5 kDa as determined by SDS-PAGE analysis. This protein inhibited trypsin (Ki = 0.42 nM), whereas serine proteases from the coagulation cascade were not inhibited. Moreover, trypanocidal assays revealed that rRpTI did not interfere in the viability of T. cruzi trypomastigotes. The RpTI transcript was also knocked down by RNA interference prior to infection of R. prolixus with T. cruzi. The amount of T. cruzi in the anterior midgut was significantly lower in RpTI knockdown insects compared to the non-silenced groups. We also verified that the bacterial load is higher in the anterior midgut of silenced and infected R. prolixus compared to non-silenced and infected insects. Our results suggest that T. cruzi infection increases the expression of RpTI to mediate microbiota modulation and is important for parasite immediately after infection with R. prolixus.

  16. Salinity alters snakeskin and mesh transcript abundance and permeability in midgut and Malpighian tubules of larval mosquito, Aedes aegypti.

    PubMed

    Jonusaite, Sima; Donini, Andrew; Kelly, Scott P

    2017-03-01

    This study examined the distribution and localization of the septate junction (SJ) proteins snakeskin (Ssk) and mesh in osmoregulatory organs of larval mosquito (Aedes aegypti), as well as their response to altered environmental salt levels. Ssk and mesh transcripts and immunoreactivity were detected in tissues of endodermal origin such as the midgut and Malpighian tubules of A. aegypti larvae, but not in ectodermally derived hindgut and anal papillae. Immunolocalization of Ssk and mesh in the midgut and Malpighian tubules indicated that both proteins are concentrated at regions of cell-cell contact between epithelial cells. Transcript abundance of ssk and mesh was higher in the midgut and Malpighian tubules of brackish water (BW, 30% SW) reared A. aegypti larvae when compared with freshwater (FW) reared animals. Therefore, [(3)H]polyethylene glycol (MW 400Da, PEG-400) flux was examined across isolated midgut and Malpighian tubule preparations as a measure of their paracellular permeability. It was found that PEG-400 flux was greater across the midgut of BW versus FW larvae while the Malpighian tubules of BW-reared larvae had reduced PEG-400 permeability in conjunction with increased Cl(-) secretion compared to FW animals. Taken together, data suggest that Ssk and mesh are found in smooth SJs (sSJs) of larval A. aegypti and that their abundance alters in association with changes in epithelial permeability when larvae reside in water of differing salt content. This latter observation suggests that Ssk and mesh play a role in the homeostatic control of salt and water balance in larval A. aegypti.

  17. Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

    PubMed Central

    Pan, Lixia; Xie, Wenbing; Li, Kai-Le; Yang, Zhihao; Xu, Jiang; Zhang, Wenhao; Liu, Lu-Ping; Ren, Xingjie; He, Zhimin; Wu, Junyu; Sun, Jin; Wei, Hui-Min; Wang, Daliang; Xie, Wei; Li, Wei; Ni, Jian-Quan; Sun, Fang-Lin

    2015-01-01

    Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors. PMID:26508632

  18. Genotoxicity of copper oxide nanoparticles in Drosophila melanogaster.

    PubMed

    Carmona, Erico R; Inostroza-Blancheteau, Claudio; Obando, Veroska; Rubio, Laura; Marcos, Ricard

    2015-09-01

    Copper oxide nanoparticles (CuONPs) are used as semiconductors, catalysts, gas sensors, and antimicrobial agents. We have used the comet and wing-spot assays in Drosophila melanogaster to assess the genotoxicity of CuONPs and ionic copper (CuSO4). Lipid peroxidation analysis was also performed (Thiobarbituric Acid Assay, TBARS). In larval hemocytes, both CuONPs and CuSO4 caused significant dose-dependent increases in DNA damage (comet assay). In the wing-spot assay, an increase in the frequency of mutant spots was observed in the wings of the adults; CuONPs were more effective than was CuSO4. Both agents induced TBARS; again, CuONPs were more active than was CuSO4. The results indicate that CuONPs are genotoxic in Drosophila, and these effects may be mediated by oxidative stress. Most of the effects appear to be related to the presence of copper ions.

  19. Heterochromatin remodeling by CDK12 contributes to learning in Drosophila.

    PubMed

    Pan, Lixia; Xie, Wenbing; Li, Kai-Le; Yang, Zhihao; Xu, Jiang; Zhang, Wenhao; Liu, Lu-Ping; Ren, Xingjie; He, Zhimin; Wu, Junyu; Sun, Jin; Wei, Hui-Min; Wang, Daliang; Xie, Wei; Li, Wei; Ni, Jian-Quan; Sun, Fang-Lin

    2015-11-10

    Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.

  20. Drosophila Vps13 Is Required for Protein Homeostasis in the Brain

    PubMed Central

    Vonk, Jan J.; Lahaye, Liza L.; Kanon, Bart; van der Zwaag, Marianne; Velayos-Baeza, Antonio; Freire, Raimundo; van IJzendoorn, Sven C.; Grzeschik, Nicola A.; Sibon, Ody C. M.

    2017-01-01

    Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis. PMID:28107480

  1. The ultrastructure of the midgut glands in Ligia italica (Isopoda) under different nutritional conditions

    NASA Astrophysics Data System (ADS)

    Štrus, J.; Burkhardt, P.; Storch, V.

    1985-12-01

    After a period of food deprivation, Ligia italica were refed for 2 days with different diets and their midgut glands were examined under the electron microscope with special reference to the large cells. The predominant features are the following: extended glycogen fields after sucrose-diet; numerous lipid droplets and peroxisome-like vesicles after lipid-diet (butter); swollen mitochondria and a great number of pinocytotic vesicles after protein diet (curds); electron dense vesicles and myelin bodies after the uptake of Escherichia coli. In contrast to amphipods, the intertidal isopod L. italica is not able to digest cellulose, as the cell ultrastructure exhibits all features of starved animals, as well as that following feeding with lignin.

  2. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae.

    PubMed

    Elpidina, E N; Tsybina, T A; Dunaevsky, Y E; Belozersky, M A; Zhuzhikov, D P; Oppert, B

    2005-08-01

    A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

  3. Alkalinization in the Isolated and Perfused Anterior Midgut of the Larval Mosquito, Aedes aegypti

    PubMed Central

    Onken, Horst; Moffett, Stacia B.; Moffett, David F.

    2008-01-01

    In the present study, isolated midguts of larval Aedes aegypti L. (Diptera: Culicidae) were mounted on perfusion pipettes and bathed in high buffer mosquito saline. With low buffer perfusion saline, containing m-cresol purple, transepithelial voltage was monitored and luminal alkalinization became visible through color changes of m-cresol purple after perfusion stop. Lumen negative voltage and alkalinization depended on metabolic energy and were stimulated in the presence of serotonin (0.2 µmol l-1). In some experiments a pH microelectrode in the lumen recorded pH values up to 10 within minutes after perfusion stop. The V-ATPase inhibitor concanamycin (50 µmol l-1) on the hemolymph side almost abolished Vte and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 µmol l-1), on either the luminal or hemolymph-side, or the inhibitor of anion transport, DIDS (1 mmol l-1) on the luminal side, had no effect on Vte or alkalinization. Cl- substitution in the lumen or on both sides of the tissue affected Vte, but the color change of m-cresol purple was unchanged from control conditions. Hemolymph-side Na+ substitution or addition of the Na+/H+ exchange inhibitor, amiloride (200 µmol l-1), reduced Vte and luminal alkalinization. Luminal amiloride (200 µmol l-1) was without effects on Vte or alkalinization. High K+ (60 mmol l-1) in the lumen reduced Vte without affecting alkalinization. These results indicate that strong luminal alkalinization in isolated and perfused anterior midgut of larval A. aegypti depends on basolateral V-ATPase, but is apparently independent of carbonic anhydrase, apical Cl-/HCO3- exchange or apical K+/2H+ antiport. PMID:20307229

  4. Interaction between Host Complement and Mosquito-Midgut-Stage Plasmodium berghei

    PubMed Central

    Margos, Gabriele; Navarette, Sandra; Butcher, Geoff; Davies, Alex; Willers, Christine; Sinden, Robert E.; Lachmann, Peter J.

    2001-01-01

    After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement on Plasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules. PMID:11447187

  5. Avoidance of antinutritive plant defense: Role of midgut pH in Colorado potato beetle.

    PubMed

    Felton, G W; Workman, J; Duffey, S S

    1992-04-01

    The fate of the tomato foliar phenolic, chlorogenic acid, in the digestive systems of Colorado potato beetleLeptinotarsa decemlineata (Coleoptera: Chrysomelidae) andHelicoverpa tea (Lepidoptera: Noctuidae) is compared. In larvalH. zea and other lepidopteran species previously examined, approximately 35-50% of the ingested chlorogenic acid was oxidized in the digestive system by foliar phenolic oxidases (i.e., polyphenol oxidase and peroxidase) from the tomato plant. The oxidized form of chlorogenic acid, chlorogenoquinone, is a potent alkylator of dietary protein and can exert a strong antinutritive effect upon larvae through chemical degradation of essential amino acids. In contrast, inL. decemlineata less than 4% of the ingested dose of chlorogenic acid was bound to protein. In vitro experiments to determine the influence of pH on covalent binding of chlorogenic acid to protein showed that 30-45% less chlorogenic acid bound to protein at pHs representative of the beetle midgut (pH 5.5-6.5) than at a pH representing the lepidopteran midgut (pH 8.5). At an acidic pH, considerably more of the alkylatable functional groups of amino acids (-NH2, -SH) are in the nonreactive, protonated state. Hence, polyphenol oxidases are unlikely to have significant antinutritive effects against the Colorado potato beetle and may not be a useful biochemical source of resistance against this insect. The influence of feeding by larval Colorado potato beetle on foliar polyphenol oxidase activity in tomato foliage and its possible significance to interspecific competition is also considered.

  6. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells

    PubMed Central

    Lara, Flavio Alves; Pohl, Paula C.; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H. F.; Almeida, Igor C.; Vaz, Itabajara da Silva; Oliveira, Pedro L.

    2015-01-01

    In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new

  7. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

    PubMed

    Lara, Flavio Alves; Pohl, Paula C; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H F; Almeida, Igor C; Vaz, Itabajara da Silva; Oliveira, Pedro L

    2015-01-01

    In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new

  8. A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

    PubMed Central

    Cheng, Yang; Wang, Xue-Yang; Hu, Hao; Killiny, Nabil; Xu, Jia-Ping

    2014-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV. PMID:25502928

  9. Genome of Drosophila suzukii, the Spotted Wing Drosophila

    PubMed Central

    Chiu, Joanna C.; Jiang, Xuanting; Zhao, Li; Hamm, Christopher A.; Cridland, Julie M.; Saelao, Perot; Hamby, Kelly A.; Lee, Ernest K.; Kwok, Rosanna S.; Zhang, Guojie; Zalom, Frank G.; Walton, Vaughn M.; Begun, David J.

    2013-01-01

    Drosophila suzukii Matsumura (spotted wing drosophila) has recently become a serious pest of a wide variety of fruit crops in the United States as well as in Europe, leading to substantial yearly crop losses. To enable basic and applied research of this important pest, we sequenced the D. suzukii genome to obtain a high-quality reference sequence. Here, we discuss the basic properties of the genome and transcriptome and describe patterns of genome evolution in D. suzukii and its close relatives. Our analyses and genome annotations are presented in a web portal, SpottedWingFlyBase, to facilitate public access. PMID:24142924

  10. The role of reduced oxygen in the developmental physiology of growth and metamorphosis initiation in Drosophila

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rearing oxygen level is known to affect final body size in a variety of insects, but the physiological mechanisms by which oxygen affects size are incompletely understood. In Manduca and Drosophila, the larval size at which metamorphosis is initiated largely determines adult size, and metamorphosis ...

  11. Activities of natural methyl farnesoids on pupariation and metamorphosis of Drosophila melanogaster

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methyl farnesoate (MF) and juvenile hormone (JH III), which respectively bind to the receptors USP and MET, and bisepoxy JH III (bisJHIII) were assessed for several activities during Drosophila larval development, and during prepupal development to eclosed adults. Dietary MF and JH III were similar...

  12. Microarray Analysis of Juvenile Hormone Response in Drosophila melanogaster S2 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A microchip array encompassing probes for 14,010 genes of Drosophila melanogaster was used to analyze the effect of juvenile hormone (JH) on genome-wide gene expression. JH is a member of a key group of insect hormones involved in regulating larval development and adult reproductive processes. Altho...

  13. Learning and Memory Deficits upon TAU Accumulation in "Drosophila" Mushroom Body Neurons

    ERIC Educational Resources Information Center

    Mershin, Andreas; Pavlopoulos, Elias; Fitch, Olivia; Braden, Brittany C.; Nanopoulos, Dimitri V.; Skoulakis, Efthimios M. C.

    2004-01-01

    Mutations in the neuronal-specific microtubule-binding protein TAU are associated with several dementias and neurodegenerative diseases. However, the effects of elevated TAU accumulation on behavioral plasticity are unknown. We report that directed expression of wild-type vertebrate and "Drosophila" TAU in adult mushroom body neurons, centers for…

  14. Thermal Tolerances of the Spotted-Wing Drosophila Drosophila suzukii (Diptera: Drosophilidae).

    PubMed

    Ryan, Geraldine D; Emiljanowicz, Lisa; Wilkinson, Francesca; Kornya, Melanie; Newman, Jonathan A

    2016-04-01

    The spotted-wing drosophila (Drosophila suzukii Matsumura) is an invasive species of Asian origin that is now widely distributed in North America and Europe. Because of the female’s serrated ovipositor, eggs are laid in preharvest fruit, causing large economic losses in cultivated berries and stone fruit. Modeling D. suzukii population dynamics and potential distribution will require information on its thermal tolerance. Large summer populations have been found in regions with severe winter conditions, though little is known about responses to prolonged low-temperature exposure. We used controlled chambers to examine D. suzukii fecundity, development rate, and mortality across a range of temperatures encompassing the upper and lower thresholds (5–35°C). Optimal temperatures (Topt) were found to be 28.2°C for the development of the egg-to-adult stage, and 22.9°C for reproductive output. No adult eclosion occurred below 8.1°C (Tlower) or above 30.9°C (Tupper). We also investigated survival outcomes following prolonged (42-d) low-temperature exposure to a simulated cold winter (−5, −3, −1, 1, 3, and 5°C). Adult survival was dependent on temperature, with a mean LT50 of 4.9°C. There were no effects of sex, mating status, geographic strain, and photoperiod preexposure on overwintering survival. Thirty-eight percent of females that were mated prior, but not after, prolonged low-temperature exposure produced viable offspring, suggesting that this species may undergo sperm storage. This study provides data on the thermal tolerances of D. suzukii, which can be used for models of D. suzukii population dynamics, degree-day, and distribution models.

  15. Morphogenesis of the somatic musculature in Drosophila melanogaster

    PubMed Central

    Schulman, Victoria K.; Dobi, Krista C.; Baylies, Mary K.

    2015-01-01

    In Drosophila melanogaster, the somatic muscle system is first formed during embryogenesis, giving rise to the larval musculature. Later during metamorphosis, this system is destroyed and replaced by an entirely new set of muscles in the adult fly. Proper formation of the larval and adult muscles is critical for basic survival functions such as hatching and crawling (in the larva), walking and flying (in the adult), and feeding (at both larval and adult stages). Myogenesis, from mononucleated muscle precursor cells to multinucleated functional muscles, is driven by a number of cellular processes that have begun to be mechanistically defined. Once themesodermal cells destined for themyogenic lineage have been specified, individual myoblasts fuse together iteratively to form syncytial myofibers. Combining cytoplasmic contents demands a level of intracellular reorganization that, most notably, leads to redistribution of the myonuclei to maximize internuclear distance. Signaling from extending myofibers induces terminal tendon cell differentiation in the ectoderm, which results in secure muscle-tendon attachments that are critical formuscle contraction. Simultaneously, muscles become innervated and undergo sarcomerogenesis to establish the contractile apparatus that will facilitate movement. The cellular mechanisms governing these morphogenetic events share numerous parallels to mammalian development, and the basic unit of all muscle, the myofiber, is conserved from flies to mammals. Thus, studies of Drosophila myogenesis and comparisons to muscle development in other systems highlight conserved regulatory programs of biomedical relevance to general muscle biology and studies of muscle disease. PMID:25758712

  16. A novel mode of induction of the humoral innate immune response in Drosophila larvae

    PubMed Central

    Kenmoku, Hiroyuki

    2017-01-01

    ABSTRACT Drosophila adults have been utilized as a genetically tractable model organism to decipher the molecular mechanisms of humoral innate immune responses. In an effort to promote the utility of Drosophila larvae as an additional model system, in this study, we describe a novel aspect of an induction mechanism for innate immunity in these larvae. By using a fine tungsten needle created for manipulating semi-conductor devices, larvae were subjected to septic injury. However, although Toll pathway mutants were susceptible to infection with Gram-positive bacteria as had been shown for Drosophila adults, microbe clearance was not affected in the mutants. In addition, Drosophila larvae were found to be sensitive to mechanical stimuli with respect to the activation of a sterile humoral response. In particular, pinching with forceps to a degree that might cause minor damage to larval tissues could induce the expression of the antifungal peptide gene Drosomycin; notably, this induction was partially independent of the Toll and immune deficiency pathways. We therefore propose that Drosophila larvae might serve as a useful model to analyze the infectious and non-infectious inflammation that underlies various inflammatory diseases such as ischemia, atherosclerosis and cancer. PMID:28250052

  17. Why Drosophila to Study Phototransduction?

    PubMed Central

    Pak, William L.

    2010-01-01

    This review recounts the early history of Drosophila phototransduction genetics, covering the period between approximately 1966 to 1979. Early in this period, the author felt that there was an urgent need for a new approach in phototransduction research. Through inputs from a number of colleagues, he was led to consider isolating Drosophila mutants that are defective in the electroretinogram. Thanks to the efforts of dedicated associates and technical staff, by the end of this period, he was able to accumulate a large number of such mutants. Particularly important in this effort was the use of the mutant assay protocol based on the “prolonged depolarizing afterpotential.” This collection of mutants formed the basis of the subsequent intensive investigations of the Drosophila phototransduction cascade by many investigators. PMID:20536286

  18. Modelling the Drosophila embryo.

    PubMed

    Jaeger, Johannes

    2009-12-01

    I provide a historical overview on the use of mathematical models to gain insight into pattern formation during early development of the fruit fly Drosophila melanogaster. It is my intention to illustrate how the aims and methodology of modelling have changed from the early beginnings of a theoretical developmental biology in the 1960s to modern-day systems biology. I show that even early modelling attempts addressed interesting and relevant questions, which were not tractable by experimental approaches. Unfortunately, their validation was severely hampered by a lack of specificity and appropriate experimental evidence. There is a simple lesson to be learned from this: we cannot deduce general rules for pattern formation from first principles or spurious reproduction of developmental phenomena. Instead, we must infer such rules (if any) from detailed and accurate studies of specific developmental systems. To achieve this, mathematical modelling must be closely integrated with experimental approaches. I report on progress that has been made in this direction in the past few years and illustrate the kind of novel insights that can be gained from such combined approaches. These insights demonstrate the great potential (and some pitfalls) of an integrative, systems-level investigation of pattern formation.

  19. Micromechanics of Drosophila Audition

    NASA Astrophysics Data System (ADS)

    Göpfert, M. C.; Robert, D.

    2003-02-01

    An analysis is presented of the auditory micromechanics of the fruit fly Drosophila melanogaster. In this animal, the distal part of the antenna constitutes a resonantly tuned sound receiver, the vibrations of which are transduced by a chordotonal sense organ in the antenna's base. Analyzing the mechanical behavior of the antennal receiver by means of microscanning laser Doppler vibrometry, we show that the auditory system of wild-type flies exhibits a hardening stiffness nonlinearity and spontaneously generates oscillations in the absence of external stimuli. According to the deprivation of these mechanical properties in mechanosensory mutants, the receiver's nonlinearity and oscillation activity are introduced by chordotonal auditory neurons. Requiring the mechanoreceptor-specific extracellular linker protein No-mechanoreceptor-potential-A (NompA), NompC mechanosensory transduction channels, Beethoven (Btv), and Touch-insensitive-larva-B (TilB), nonlinearity and oscillation activity of the fly's antennal receiver depend on prominent components of the auditory transduction machinery and seem to originate from motility of auditory receptor cilia.

  20. Analysis of neurotransmitter tissue content of Drosophila melanogaster in different life stages.

    PubMed

    Denno, Madelaine E; Privman, Eve; Venton, B Jill

    2015-01-21

    Drosophila melanogaster is a widely used model organism for studying neurological diseases with similar neurotransmission to mammals. While both larva and adult Drosophila have central nervous systems, not much is known about how neurotransmitter tissue content changes through development. In this study, we quantified tyramine, serotonin, octopamine, and dopamine in larval, pupal, and adult fly brains using capillary electrophoresis coupled to fast-scan cyclic voltammetry. Tyramine and octopamine content varied between life stages, with almost no octopamine being present in the pupa, while tyramine levels in the pupa were very high. Adult females had significantly higher dopamine content than males, but no other neurotransmitters were dependent on sex in the adult. Understanding the tissue content of different life stages will be beneficial for future work comparing the effects of diseases on tissue content throughout development.

  1. Evaluation of in vitro and in vivo effects of semipurified proteinase inhibitors from Theobroma seeds on midgut protease activity of Lepidopteran pest insects.

    PubMed

    Paulillo, Luis Cesar Maffei Sartini; Sebbenn, Alexandre Magno; de Carvalho Derbyshire, Maria Tereza Vitral; Góes-Neto, Aristóteles; de Paula Brotto, Marco Aurélio; Figueira, Antonio

    2012-09-01

    We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.

  2. Basal tolerance to heat and cold exposure of the spotted wing drosophila, Drosophila suzukii

    PubMed Central

    Enriquez, Thomas

    2017-01-01

    The spotted wing Drosophila, Drosophila suzukii, is a new pest in Europe and America which causes severe damages, mostly to stone fruit crops. Temperature and humidity are among the most important abiotic factors governing insect development and fitness. In many situations, temperature can become stressful thus compromising survival. The ability to cope with thermal stress depends on basal level of thermal tolerance. Basic knowledge on temperature-dependent mortality of D. suzukii is essential to facilitate management of this pest. The objective of the present study was to investigate D. suzukii basal cold and heat tolerance. Adults and pupae were subjected to six low temperatures (−5–7.5 °C) and seven high temperatures (30–37 °C) for various durations, and survival-time-temperature relationships were investigated. Data showed that males were globally more cold tolerant than females. At temperature above 5 °C, adult cold mortality became minor even after prolonged exposures (e.g., only 20% mortality after one month at 7.5 °C). Heat tolerance of males was lower than that of females at the highest tested temperatures (34, 35 and 37 °C). Pupae appeared much less cold tolerant than adults at all temperatures (e.g., Lt50 at 5° C: 4–5 d for adults vs. 21 h for pupae). Pupae were more heat tolerant than adults at the most extreme high temperatures (e.g., Lt50 at 37 °C: 30 min for adults vs. 4 h for pupae). The pupal thermal tolerance was further investigated under low vs. high humidity. Low relative humidity did not affect pupal cold survival, but it reduced survival under heat stress. Overall, this study shows that survival of D. suzukii under heat and cold conditions can vary with stress intensity, duration, humidity, sex and stage, and the methodological approach used here, which was based on thermal tolerance landscapes, provides a comprehensive description of D. suzukiithermal tolerance and limits. PMID:28348931

  3. Thorsellia anophelis is the dominant bacterium in a Kenyan population of adult Anopheles gambiae mosquitoes.

    PubMed

    Briones, Aurelio M; Shililu, Josephat; Githure, John; Novak, Robert; Raskin, Lutgarde

    2008-01-01

    Anopheles gambiae mosquitoes are not known to harbor endosymbiotic bacteria. Here we show, using nucleic acid-based methods, that 16S rRNA gene sequences specific to a recently described mosquito midgut bacterium, Thorsellia anophelis, is predominant in the midgut of adult An. gambiae s.l. mosquitoes captured in residences in central Kenya, and also occurs in the aquatic rice paddy environment nearby. PCR consistently detected T. anophelis in the surface microlayer of rice paddies, which is also consistent with the surface-feeding behavior of A. gambiae s.l. larvae. Phylogenetic analysis of cloned environmental 16S rRNA genes identified four major Thorsellia lineages, which are closely affiliated to an insect endosymbiont of the genus Arsenophonus. Physiological characterizations support the hypothesis that T. anophelis is well adapted to the female anopheline midgut by utilizing blood and tolerating the alkaline conditions in this environment. The results suggest that aquatically derived bacteria such as T. anophelis can persist through mosquito metamorphosis and become well-established in the adult mosquito midgut.

  4. Drosophila larvae: Thermal ecology in changing environments

    NASA Astrophysics Data System (ADS)

    Wang, George

    Temperature affects almost all aspects of life. Although much work has been done to assess the impact of temperature on organismal performance, relatively little is known about how organisms behaviorally regulate temperature, how these behaviors effect population fitness, or how changing climate may interact with these behaviors. I explore these questions with the model system Drosophila larvae. Larvae are small, with a low thermal mass and limited capacity for physiological thermoregulation. Mortality is generally high in larvae, with large potential impacts on population growth rate. Thus behavioral thermoregulation in larvae should be of critical selective importance. I present a review of the current knowledge of Drosophila thermal preference. I describe quantifiable thermoregulatory behaviors ( TMV and TW) unique to larvae. I show interspecific variation of these behaviors in Drosophila melanogaster and several close relatives, and intraspecific variation between populations collected from different environments. I also investigate these behaviors in two mutant lines, ssa and biz, to investigate the genetic basis of these behaviors. I show that larval thermoregulatory systems are independent of those of adults. Further these thermoregulatory behaviors differ between two sister species, D. yakuba and D. santomea. Although these two species readily hybridize in laboratory conditions, very few hybrids are observed in the field. The surprising result that hybrids of D. yakuba and D. santomea seem to inherit TMV from D. yakuba suggests a novel extrinsic isolation mechanism between the two species. I explore how fitness is the result of the interaction between genetics and the environment. I utilize Monte Carlo simulation to show how non-linear norms of reaction generate variation in populations even in the absence of behavior or epigenetic evolutionary mechanisms. Finally I investigate the global distribution of temperatures in which these organisms exist using

  5. Host species and environmental effects on bacterial communities associated with Drosophila in the laboratory and in the natural environment.

    PubMed

    Staubach, Fabian; Baines, John F; Künzel, Sven; Bik, Elisabeth M; Petrov, Dmitri A

    2013-01-01

    The fruit fly Drosophila is a classic model organism to study adaptation as well as the relationship between genetic variation and phenotypes. Although associated bacterial communities might be important for many aspects of Drosophila biology, knowledge about their diversity, composition, and factors shaping them is limited. We used 454-based sequencing o