Project description:Background: The horn fly, Haematobia irritans (L.), is an obligate blood-feeding parasite of cattle and control of this pest is a continuing problem in the United States and other parts of the world. Worldwide annual economic losses attributable to this pest surpass $1 billion. The fly is becoming resistant to pesticides and new control technologies are needed by cattle producers. Results: Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene. To identify candidate genes to supply this gene promoter, microarrays were designed from a recently developed horn fly EST database and probed to identify female-specific and larval-specific differential gene expression. Analysis of dyeswap experiments found 432 and 417 transcripts which were over- and under-expressed in adult female flies, respectively, compared to adult male flies. Additionally, 419 and 871 transcripts were over- and under-expressed in first instar larvae compared to adult flies. Three transcripts were identified which were over-expressed in adult females flies compared to adult males and which also were over-expressed in the first instar larval lifestage compared to adult flies. Conclusion: We have identified 3 strong candidates for further evaluation as a gene promoter source for the development of a female-specific conditional lethality system in the horn fly. One of these candidates, the putative nanos orthologue, has a high female-to-male expression ratio, has a moderate expression level in first instar larvae, and has been well characterized in D. melanogaster. Further investigations leading from this microarray analysis will include transformation of the horn fly and evaluation of the female conditional lethal system components for applicability to the specific biological parameters of natural populations of the horn fly. Keywords: adult male vs female hornfly gene expression In short, RNA from female horn flies was labeled and hybridized against labeled RNA from male horn flies in a dyeswap design. Similarly, RNA from first instar larvae was labeled and hybridized against labeled a 50:50 mix of RNA from adult male and female flies. All labeling, hybridization and washing procedures were performed according to respective manufacturer protocols. For each sample, 10 ïg of total RNA and 50-fold dilution of Agilent two color spike-in control RNA kit (Agilent Technologies Inc.) were labeled with either CyDye3-dCTP or CyDye5-dCTP (Amersham Biosciences, Piscataway, NJ) using the LabelStar kit (Qiagen Inc., Valencia, CA) and oligo-dT and Random nonamers (Sigma-Aldrich Inc., St. Louis, MO). Labeled cDNA was hybridized to the Agilent microarrays using the Gene Expression Hybridization kit (Agilent Technologies Inc.) following manufacturerâs protocols. Arrays were washed with Gene Expression Wash Buffer kit (Agilent Technologies Inc.) A total of 8 arrays, including a dye swap for each RNA replicate (4 biological replicates), were analyzed to obtain genes that were consistently regulated while limiting false discover rate (FDR) below 2.5% [26].

Project description:Background: The horn fly, Haematobia irritans (L.), is an obligate blood-feeding parasite of cattle and control of this pest is a continuing problem in the United States and other parts of the world. Worldwide annual economic losses attributable to this pest surpass $1 billion. The fly is becoming resistant to pesticides and new control technologies are needed by cattle producers. Results: Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene. To identify candidate genes to supply this gene promoter, microarrays were designed from a recently developed horn fly EST database and probed to identify female-specific and larval-specific differential gene expression. Analysis of dyeswap experiments found 432 and 417 transcripts which were over- and under-expressed in adult female flies, respectively, compared to adult male flies. Additionally, 419 and 871 transcripts were over- and under-expressed in first instar larvae compared to adult flies. Three transcripts were identified which were over-expressed in adult females flies compared to adult males and which also were over-expressed in the first instar larval lifestage compared to adult flies. Conclusion: We have identified 3 strong candidates for further evaluation as a gene promoter source for the development of a female-specific conditional lethality system in the horn fly. One of these candidates, the putative nanos orthologue, has a high female-to-male expression ratio, has a moderate expression level in first instar larvae, and has been well characterized in D. melanogaster. Further investigations leading from this microarray analysis will include transformation of the horn fly and evaluation of the female conditional lethal system components for applicability to the specific biological parameters of natural populations of the horn fly. Keywords: evaluation of 1st instar larvae genes vs pooled adult + egg genes

Project description:Background: The horn fly, Haematobia irritans (L.), is an obligate blood-feeding parasite of cattle and control of this pest is a continuing problem in the United States and other parts of the world. Worldwide annual economic losses attributable to this pest surpass $1 billion. The fly is becoming resistant to pesticides and new control technologies are needed by cattle producers. Results: Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene. To identify candidate genes to supply this gene promoter, microarrays were designed from a recently developed horn fly EST database and probed to identify female-specific and larval-specific differential gene expression. Analysis of dyeswap experiments found 432 and 417 transcripts which were over- and under-expressed in adult female flies, respectively, compared to adult male flies. Additionally, 419 and 871 transcripts were over- and under-expressed in first instar larvae compared to adult flies. Three transcripts were identified which were over-expressed in adult females flies compared to adult males and which also were over-expressed in the first instar larval lifestage compared to adult flies. Conclusion: We have identified 3 strong candidates for further evaluation as a gene promoter source for the development of a female-specific conditional lethality system in the horn fly. One of these candidates, the putative nanos orthologue, has a high female-to-male expression ratio, has a moderate expression level in first instar larvae, and has been well characterized in D. melanogaster. Further investigations leading from this microarray analysis will include transformation of the horn fly and evaluation of the female conditional lethal system components for applicability to the specific biological parameters of natural populations of the horn fly. Keywords: adult male vs female hornfly gene expression

Project description:Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches are developed to control the disease among which the anti-vector Sterile Insect Technique. Another approach in the frame of anti-vector strategies could consist in controlling the fly’s vector competence which needs identifying factors (genes, proteins, biological pathways, …) involved in this process. The present work aims to verify whether protein candidates identified under experimental controlled conditions on insectary-reared tsetse flies have their counterpart in field-collected flies. Glossina palpalis palpalis flies naturally infected with Trypanosoma congolense were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteome from guts of parasite-infected flies were compared to that from uninfected flies in order to identify quantitative and/or qualitative changes associated to infection. A total of 3291 proteins were identified of which 1818 could be quantified. The comparative analysis allowed identifying 175 proteins with significant decreased abundance in infected as compared to uninfected flies, while 61 proteins displayed increased abundance. Among the former are RNA binding proteins, kinases, actin, ribosomal proteins, endocytosis proteins, oxido-reductases, as well as proteins that are unusually found such as tsetse salivary proteins (Tsal) or Yolk proteins. Among the proteins with increased abundance are fructose-1,6-biphosphatase, serine proteases, membrane trafficking proteins, death proteins (or apoptosis proteins), and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed highest increased abundance. Sodalis, Wiggleswothia and Wolbachia proteins are strongly under-represented, particularly when compared to data from similar experimentation conducted under controlled conditions on T. brucei gambiense infected (or uninfected) G. palpalis gambiensis insectary reared flies. Comparing the overall recorded data, 364 proteins identified in gut extracts from field flies were shown to have a homologue in insectary flies. Discrepancies between the two studies may arise from differences in the species of studied flies and trypanosomes as well as in differences in environmental conditions in which the two experiments were carried out. Finally, the present study together with former proteomic and transcriptomic studies on the secretome of trypanosomes, on the gut extracts from insectary reared and on field collected tsetse flies, provide a pool of data and information on which to draw in order to perform further investigations on, for example, mammal host immunization or on fly vector competence modification via para-transgenic approaches.

Project description:This is a prepublication dataset that should change as updates are made to the underling genomes and annotations. Additional samples may be deposited and some samples may fail quality control. Please contact Haiwang Yang <haiwang.yang@nih.gov> for details. The RNA-seq data set contains the transcriptional profiling of eight sexed tissue types (head, thorax, carcass, abdomen w/o gonad, gonad, other reproductive tract, genitalia, and digestive system) and/or whole adult flies from five strains of Drosophila grimshawi, two strains of D. silvestris, and one strain each of D. hemipeza, D. heteroneura, and D. hawaiiensis. Samples were collected in duplicate to quadruplicate from sexually mature sexed adults (4-5 weeks post eclosion), which had been allowed to freely mate. Single-end stranded 76bp PolyA+ RNA-seq was performed on all samples. Libraries were multiplexed with indexed adaptors, and some were also prepared as mixed libraries with RNA from genetically distant species: Drosophila melanogaster (GSE81142) and Anopheles stephensi (GEO entry in preparation). All whole adult samples were prepared from single flies. Tissues were pooled from 3-5 individuals depending on the size of both the tissue and the species. For the G1 strain (reference assembly strain) of D. grimshawi, we profiled seven adult tissues for each sex in addition to whole flies. Samples were whole fly (one fly per sample), head (5 flies), thorax (5 flies), abdomen w/o gonad (5 flies), gonad (testes or ovaries) (5 flies), other reproductive tract (accessory gland and male reproductive duct or female spermatheca and oviduct; 10 flies per sample), genitalia (10 flies), and digestive system (proventriculus, crop, salivary gland, gut, and renal tubule; 5 flies). We prepared sexed single fly whole body samples in quadruplicate for ZA27Z2, ZA27Z3, and Mo010 strains of D. grimshawi. We also prepared sex-specific head, carcass, gonad, other reproductive tract with genitalia with duplicate for Wm1041 strain of D. grimshawi and other species - D. silvestris (Y11R6 and U26B9 strains), D. hemipeza (W40B14 strain), D. heteroneura (W48B6 strain), and D. hawaiiensis (Y17P5 strain). We used reduced number of flies for the non-grimshawi species (i.e., 3 heads, gonads or carcasses per sample, and 6 reproductive tracts plus genitalia per sample).

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Hornfly 1st instar larvae vs pooled adult + egg

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