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 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:Research on the ability of certain organisms to reconstruct missing structures of their bodies is still in its infancy, despite numerous efforts performed in multiple species. Using expression profile analyses on different timepoints that cover wound healing and regeneration processes (0, 24 and 72 hours post injury), we studied the regenerative behaviour of fragmented wing imaginal discs of D. melanogaster implanted into adult flies. First, through the comparison between cut and intact discs, we identified the effect of implantation on the regeneration process. Filtering this information, we then constructed the specific early regeneration gene signature of wing discs in which multiple transcription factors, immune response genes and members of the JNK, Notch and WNT signaling pathways seem to play an important role. We next compared the transcriptomes of discs 24 and 72 hours after fragmentation to characterize the regenerative machinery and observed a temporal decrease in the cellular metabolic processes, RNA processing and gene expression machinery, suggesting the discs normal activity was stopped on this first interval of time in response to injury and implantation offences. Based on the expression patterns, we elaborated a catalogue of genes involved in wing disc regeneration divided into four classes (wound healing, regeneration, quick response, constant response). Imaginal discs from third instar larvae were 3/4 fragmented, implanted into the abdomen of adult flies and cultured. In order to subtract the noise due to implantation, we repeated the same protocol using intact discs that were cultivated for 24h. Therefore, a total of 12 microarrays were hybridized: four microarrays (with total RNA from non cut wing discs at 0 and 24 hours) were used to assess the effect of the implantation procedure in intact wing discs and the rest (8) were used to measure changes in gene expression in the first 24 hours after disc dissection and implantation (total RNA from wing discs at 0 and 24 hours was hybridized) and during the period between 24 and 72 hours after the cut (total RNA from wing discs at 24 and 72 hours was hybridized). At least two independent total RNA extractions were carried out (using the RNeasy Protect Mini Kit from Qiagen Inc.) for each condition. The four microarrays hybridized for each pair of conditions were done in biological replicate pairs with dyes swapped to take dye bias into account. The whole set of microarrays was normalized following the same protocol, extracting in each case the list of significant genes (at least 1.5-fold change, FDR adjusted pvalue < 0.05).

Project description:The existence of two separate lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that one of these lineages (lineage I) might be more pathogenic towards human hosts. We have previously shown that the more pathogenic lineage expresses higher levels of Shiga toxin 2 (Stx2) than the non-pathogenic lineage II. To evaluate why lineage 2 isolates do not express appreciable levels of toxin, two lineage 2 isolates (FRIK966 and FRIK2000) were chosen as representatives of lineage 2 and whole genome microarrays were performed using Agilent microarrays using the E. coli O157:H7 EDL933 lineage I clinical type isolate as a reference. Microarray results were utilized to evaluate what genes and pathways might be missing or differentially expressed. Quantitative RT-PCR was utilized to validate the microarray data. Based upon the transcriptome of Escherichia coli O157:H7 EDL933 an oligonucleotide microarray, made up of 60 mers was designed. A total of 4873 genes in an 8 x 15K Agilent microarray design. Designed using a custom script, specifications for gene specific oligos were based upon various design characteristics such as temperature of melting, 3’ location, specificity, lack of repeat nucleotides, etc. (Charbonnier et al., 2005). Arrays were manufactured using Agilent Sure-print technology. Each array consisted of duplicate elements for each gene randomly distributed with Agilent control elements included. All procedures were performed according to respective manufacturer protocols. Lineage I and lineage II strains were grown overnight as described, a total of 10e7 cells were washed twice in fresh media, normalized based upon optical density, inoculated into fresh media, incubated at 37oC shaking 120 x g for 3 hours and then suspended in RNAprotect bacteria reagent (Qiagen Inc., Valencia, CA.). Total RNA was extracted using RNeasy Bacteria Mini Kit (Qiagen Inc., Valencia, CA.) and trace amounts of DNA were removed using RNase-Free DNase Set (Qiagen Inc., Valencia, CA.). RNA was quantified using Nanodrop system (NanoDrop Technologies, Wilmington, DE) and quality confirmed by electrophoresis on a Bio-rad Experion system (BioRad XXX). For each sample, 10 ug of RNA were labeled with either CyDye3-dCTP or CyDye5-dCTP (Perkin Elmer) using the LabelStar kit (Qiagen Inc., Valencia, CA.) and Random nonamers (Integrated DNA Technologies ). Labeled cDNA were hybridized to the microarray using Agilent Hi-RPM hybridization solution in an Agilent Hybridization chamber (Agilent.). A total of 8 arrays, each with duplicate elements for each gene, alternating dye swap for each replicate (4 biological replicates), were analyzed to obtain genes that were consistently and differentially regulated in comparison to EDL933, while limiting false discover rate (FDR) below a stringent 5% (Benjamini and Hochberg, 1995).

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Female adult to male adult comparison and 1st instar larvae to adult comparisons

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