Transcription profiling of Strongyloides ratti free living vs parasitic females
ABSTRACT: In previous studies we have shown that the two adult females morphs of S. ratti have very different lifespans. This experiment was designed to try to identify differentially expressed genes in these two adult morphs that may account for these differing lifespans. The genes expressed by S. ratti parasitic females at day 6 p.i. were compared to the genes expressed by S. ratti free living females at 3 days 19 degrees C. This comparison was done using a microarray chip that is spotted with PCR fragments from the libraries that were generated from parasitic females extracted at day 6 and day 15 p.i., and a microarray chip that is spotted with PCR fragments from the libraries that were generated from free-living larval stages L1, L2 and infective L3s and from free-living males and females.
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:Isofemale line ED321 Heterogonic was used. Fresh faeces were collected from S. ratti-infected rats at days 5, 6, 7 and 8 p.i. and L1s prepared with a Baermann funnel held for 6 h at 19oC. The larvae were concentrated by centrifugation and then cleaned by flotation on 60% v/v sucrose. Infective L3s were harvested from 14 day-old faecal cultures that had been maintained at 19oC. The iL3s were cleaned by sucrose flotation as for the L1s, above. In excess of 150,000 larvae of either stage were routinely isolated from 6 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.
Project description: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.
Project description:Linking the evolution of the phenotype to the underlying genotype is a key aim of evolutionary genetics and is crucial to our understanding of how natural selection shapes a trait. Here we consider the genetic basis of sex allocation behaviour in the parasitoid wasp Nasonia vitripennis using a transcriptomics approach. Females allocate offspring sex in line with Local Mate Competition (LMC) theory. Female-biased sex ratios are produced when one or few females lay eggs on a patch. As the number of females contributing offspring to a patch increases, less female-biased sex ratios are favoured. We contrasted the transcriptomic responses of females as they oviposit under conditions known to influence sex allocation: foundress number (a social cue) and the state of the host (parasitised or not). We found, that when females encounter other females on a patch, or assess host quality with their ovipositors, the resulting changes in sex allocation is not associated with significant changes in whole-body gene expression. We also found that the gene expression changes produced by females, as they facultatively allocate sex in response to a host cue and a social cue, are very closely correlated. We expanded the list of candidate genes associated with oviposition behaviour in Nasonia, some of which may be involved in fundamental processes underlying the ability to facultatively allocate sex, including sperm storage and utilisation. 2 x 3 factorial design. Females were placed into 1 of 2 "foundress number" groups: 1) alone or 2) in the presence of 9 other females (co-foundresses). Females were further subdivided into host treatment groups: i) given no host, ii) given a fresh host and iii) given a pre-parasitised host. This gives a total of 6 possible treatment combinations. For each of these 6 groups, 7 pools of 10 females were sequenced giving 42 libraries altogether.
Project description:Strongyloides ratti is a parasitic nematode of rats and a laboratory model for nematode infection more generally. The response of two lines of S. ratti were compared in contrasting immunological environments: (i) day 5 post infection (p.i.) in naive rats; (ii) day 12 p.i. in naive rats; day 5 p.i. in rats previously immunised with 10 iL3s; and day 12 p.i. in rats previously immunised with 10 iL3s. The gene expression response of parasitic females were assayed using cDNA microarrays. Large numbers of responding genes were found (but with modest fold changes) and clusters of co-expressed genes identified with differences observed between worms taken from naive and previously exposed hosts and from the two time points.
Project description:We used H83M2 P element to ectopically expressed MSL2 protein in females to test if the novel formed MSL complex is the directly reason of dosage compensation Compare the MSL2 females with normal females and normal males; MSL2 males also included.
Project description:For many behaviours studied at the phenotypic level, we have little or no idea of where to start searching for “candidate” genes: the transcriptome provides such a starting point. Here we consider transcriptomic changes associated with oviposition in the parasitoid wasp Nasonia vitripennis. Oviposition is a key behaviour, as females are faced with a variety of decisions that will impact offspring fitness. These include choosing between hosts of differing quality, as well as deciding on clutch size and offspring sex ratio. We compared the whole-body transcriptomes of resting or ovipositing female Nasonia using a “DEEP-Sage” gene expression approach on the Illumina sequencing platform. Single 2-day old mated Nasonia vitripennis females (ASymC strain) were isolated in a glass vial and provided with a single host to produce F1 daughters. Eight 2-day old mated F1 females were subsequently provided with three hosts to produce the F2 test females. We randomly selected one host from each F1 female, and isolated 16 2-day old mated F2 test females in glass tubes, of which eight were randomly allocated to the oviposition treatment and eight to the resting treatment. We provided the test females with a single host for 24 hours as pre-treatment to facilitate egg development. We then discarded the pre-treatment hosts and gave each female a piece of chromatography paper soaked in honey solution for a further 24 hours. For the experiment, we transferred the females to 1.5 mL Eppendorf tubes that contained a single host for the oviposition experiment, or were empty for the resting treatment. After 60 mins, females were flash-frozen in liquid nitrogen and stored on dry-ice until the addition of RNAlater-ICE (Ambion, Austin, TX, USA) after which they were transferred to -20C. All females in the oviposition treatment were observed to have commenced ovipositing. We pooled the F2 test females from each F1 mother according to treatment, generating a total of eight pooled samples per treatment (consisting of 8 females per pool) for RNA isolation and sequencing.
Project description:The genus Strongyloides spp. include important human parasites. There is also a well studied rodent model, S. ratti. Uniquely among parasitic nematodes, the Strongyloides life-cycle includes both a parasitic female stage and a genetically identical free-living female stage. Differences between these two female forms must be epigenetic, presumably controlled by altered transcription and translation. This is a project to compare the proteome and transcriptome of the parasitic and free-living females of S. ratti. From this we will define the genes and gene products of the parasitic female stage. This approach exploits the currently advanced S. ratti genome sequencing project. This work will give an understanding of the molecular basis of nematode parasitism, and so define new potential drug targets. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Strongyloides ratti is a parasitic nematode of rats and a laboratory model for nematode infection more generally. The aim of this experiment was to determine the gene expression response of parasitic females to abiotic factors in its environment ex vivo that may be relevant to its natural environment in the gut in vivo. Thus, we used cDNA arrays to assay transcriptional responses to high and low salt, to RPMI versus PBS media and to 37C versus 40C. A moderate number of gene expression changes were observed.