Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Strongyloides ratti larval stage 2 (L2) direct vs indirect development

ABSTRACT: S. ratti Isofemale line ED321 Heterogonic predominantly undergoes indirect development; isofemale line ED5 Homogonic predominantly undergoes direct development. Therefore, L2 stages of ED321 Heterogonic and of ED5 Homogonic are destined for indirect (i.e. L2 indirect) and direct (i.e. L2 direct) development, respectively; these sources of material were used in this comparison. To do this, for both isofemale lines, rats were infected with ED321 Heterogonic or ED5 Homogonic and faeces collected on days 5, 6, 7 and 8 p.i. and cultured for 24 h at 19oC, after which larvae were prepared with a Baermann funnel held for 6 h at 19oC, larvae were concentrated and cleaned by sucrose flotation, as above. In excess of 75,000 worms were routinely isolated from three infected hosts. The experimental design used, was to have at least three biological replicates for each sample (i.e. three independent preparations of the relevant worm samples and their RNA) and to have at least three technical replicates (i.e. independent, separate cDNA synthesis, amplification and hybridization etc.) for each biological replicate. For each hybridisation a dye-swap was used i.e. each sample to be used in a hybridisation was labelled, separately, with each of the two dyes.

INSTRUMENT(S): Axon- GenePix4000B

ORGANISM(S): Strongyloides ratti

SUBMITTER: Louise Hughes

PROVIDER: E-MEXP-709 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We wished to determine the changes in gene expression that occurred in S. ratti parasitic females as an infection progressed and thus as these stages are exposed to an anti-S. ratti immune response. To do this we compared gene expression in parasitic females recovered 6 days p.i. (i.e. no or very low immune response) with those recovered at 15 days p.i. (i.e. high immune response); for convenience, we refer to these as parasitic females subject to "low immune pressure" and "high immune pressure", respectively. These days were chosen because previous analyses of S. ratti parasitic females have shown significant differences in the size, appearance etc. of worms at these time points. The experimental design used, was to have at least three biological replicates for each sample (i.e. three independent preparations of the relevant worm samples and their RNA) and to have at least three technical replicates (i.e. independent, separate cDNA synthesis, amplification and hybridization etc.) for each biological replicate. For each hybridisation (below) a dye-swap was used i.e. each sample to be used in a hybridisation was labelled, separately, with each of the two dyes (below).<br> <br> 21,085 ESTs were sequenced from various S. ratti stage-specific libraries, of which 14,761 resulted in sequence data above a quality threshold that were then submitted to public databases. 11,551 clones were derived from the S. ratti parasitic libraries of which 7,385 produced sequence data (above a quality threshold); all of these 7,385 clones were arrayed together with a random sample of 1,619 of clones for which no sequence data were available. These 7,385 ESTs are highly redundant since they represent 2,963 contigs and 2,125 clusters, both including 1,220 singletons (i.e. clusters or contigs containing only one EST). Notwithstanding this redundancy, they were used in the microarray construction for two reasons: (i) this approach was less error-prone than attempting to select a unique clone set and (ii) this in-built redundancy provides many replicates of individual contigs and clusters, which can be exploited in quality-control analyses. In addition to these 9,004 S. ratti clones, the following controls were included: 281 EST clones from the mixed iL3/free-living adult library (representing 173 contigs and 167 clusters, 67 of which are singletons) to ensure that gene expression in the parasitic and free-living stages could be differentiated; 230 commercially available controls (Amersham Biosciences UK, Ltd.). 12 poly-A and 457 spotting buffer-only controls. Thus, in total 9,984 spots were arrayed.

Project description:Current commercially available feeds for salmon are predominantly made of plant ingredients, with consequent changes to the composition and contents of a range of nutrients and other components in the diet. There are concerns that, with these major changes in raw materials, new feeds will affect not only the composition and contents of nutrients, but also the bioavailability and, combined with the limited knowledge of micronutrient requirements for Atlantic salmon, this might impact growth performance and health of the fish. The present study investigated the effects of graded levels of a micronutrient package supplemented to feeds formulated with low levels of marine ingredients and fed to diploid and triploid Atlantic salmon throughout the freshwater phase. Specifically, fish were fed three diets containing low levels of FM and FO and identical in formulation other than being supplemented with 3 levels (L1, 100 %; L2, 200 % and L3, 400 %) of a micronutrient mix formulated as a modification of current nutrient levels reported for salmon. Duplicate groups of diploid and triploid parr were fed the experimental diets from around 30 g to seawater transfer and the effects on growth performance, feed efficiency, biochemical composition, liver histology, hepatic gene expression (transcriptome) and smoltification efficiency determined. Microarray analysis revealed that the hepatic transcriptome profile of diploid fish fed diet L2 was more similar to that observed in triploids fed diet L3 than to those fed L2, suggesting that micronutrient requirements of triploid salmon may differ from levels accepted in diploid salmon. Different levels of micronutrient supplementation affected the expression of key genes involved in lipid metabolism. In particular sterol biosynthesis pathways (steroid and terpenoid backbone synthesis) were down-regulated in both L2-fed diploids and L3-fed triploids when compared with diet L1-fed diploids and triploids, respectively. Gene sets analysis showed an up-regulation of genes involved in immune processes in triploid salmon fed diet L3. Another biological category affected by diet in triploid salmon was genetic information processing. In fish fed diet L3 down-regulation of RNA degradation, proteasome, RNA polymerase, spliceosome and ribosome was observed, suggesting a decrease in protein turnover in this group, which may indicate a decrease in energy expenditure. In addition, one-carbon metabolism was affected by diet in diploid and triploid salmon.

Project description:Ants provide remarkable examples of equivalent genotypes developing into divergent and discrete phenotypes. Diploid eggs can develop either into queens, which specialize in reproduction, or workers, which participate in cooperative tasks such as building the nest, collecting food, and rearing the young. In contrast, the differentiation between males and females generally depends upon whether eggs are fertilized, with fertilized (diploid) eggs giving rise to females and unfertilized (haploid) eggs giving rise to males. To obtain a comprehensive picture of the relative contributions of gender (sex), caste, developmental stage, and species divergence to gene expression evolution, we investigated gene expression patterns in pupal and adult queens, workers, and males of two species of fire ants, Solenopsis invicta and Solenopsis richteri. Microarray hybridizations revealed that variation in gene expression profiles is influenced more by developmental stage than by caste membership, sex, or species identity. The second major contributor to variation in gene expression was the combination of sex and caste. Although workers and queens share equivalent diploid nuclear genomes, they have highly distinctive patterns of gene expression in both the pupal and the adult stages, as might be expected given their extraordinary level of phenotypic differentiation. Overall, the difference in the proportion of differentially expressed genes was greater between workers and males than between workers and queens or queens and males, consistent with the fact that workers and males share neither gender nor reproductive capability. Moreover, between-species comparisons revealed that the greatest difference in gene expression patterns occurred in adult workers, a finding consistent with the fact that adult workers most directly experience the distinct external environments characterizing the different habitats occupied by the two species. Thus, much of the evolution of gene expression in ants may occur in the worker caste, despite the fact that these individuals are largely or completely sterile. Analyses of gene expression evolution revealed a combination of positive selection and relaxation of stabilizing selection as important factors driving the evolution of such genes. Transcriptome analysis is a powerful tool for unveiling the distribution and magnitude of genetic incompatibilities between hybridizing taxa. The nature of such incompatibilities is closely associated with the evolutionary histories of the parental species and may differ across tissues and between the sexes. In eusocial insects, the presence of distinct castes that experience divergent selection regimes may result in additional distinct patterns of caste-specific hybrid incompatibilities. We analyzed levels of expression of >14,000 genes in two life stages of each caste and sex in the fire ants Solenopsis invicta and Solenopsis richteri and in their hybrids. We found strong contributions of both developmental stage and caste to gene expression patterns. In contrast, variability in expression was only weakly associated with species, with hybrid scores falling between those of the two parental species in both life stages and all castes. Hybrid incompatibilities were surprisingly modest, with only 16 genes in pupae and 18 genes in adults being mis-expressed, and indicating low levels of disruption in gene regulation in hybrids. The castes differed in numbers of mis-expressed genes, with males and workers each mis-expressing at least seven times as many genes as queens in both life stages. Interestingly, homologues of many of the mis-expressed genes have been implicated in behavioral variation in Drosophila melanogaster. General expression profiles of hybrids consistently were more similar to those of Solenopsis richteri than Solenopsis invicta, presumably because Solenopsis richteri trans-regulatory elements tend to be dominant to those of Solenopsis invicta and/or because there is an overall bias in the genetic composition of the hybrids towards Solenopsis richteri. Altogether, our results suggest that selection acting on each caste may contribute differently to interspecific divergence and speciation in this group of ants. We analyzed samples from two species, their hybrids: a total of 140 samples, consisting of either workers, queens or males (1-5 pooled individuals) and collected either as larvae, pupae or adults. Each sample was tested once (i.e. single biological replicate) against a common reference (dye-swap across experiments)

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Transcription profiling of Strongyloides ratti larval stage 2 (L2) direct vs indirect development

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