Project description:Background The generalist dipteran pupal parasitoid Nasonia vitripennis injects 79 venom peptides into the host before egg laying. This venom induces several important changes in the host, including developmental arrest, immunosuppression, and alterations to normal metabolism. It is hoped that diverse and potent bioactivities of N. vitripennis venom provide an opportunity for the design of novel acting drugs. However, currently very little is known about the individual functions of N. vitripennis venom peptides and less than half can be bioinformatically annotated. The paucity of annotation information complicates the design of studies that seek to better understand the potential mechanisms underlying the envenomation response. Although the RNA interference system of N. vitripennis provides an opportunity to functionally characterise venom encoding genes, with 79 candidates this represents a daunting task. For this reason we were interested in determining the expression levels of venom encoding genes in the venom gland, such that this information could be used to rank candidate venoms. To do this we carried out deep sequencing of the transcriptome of the venom gland and neighbouring ovary tissue and used RNA-seq to measure expression from the 79 venom encoding genes. The generation of a specific venom gland transcriptome dataset also provides further opportunities to investigate novel features of this highly specialised organ. Results High throughput sequencing and RNA-seq revealed that the highest expressed venom encoding gene in the venom gland was a serine protease called Nasvi2EG007167, which has previously been implicated in the apoptotic activity of N. vitripennis venom. As expected the RNA-seq confirmed that the N. vitripennis venom encoding genes are almost exclusively expressed in the venom gland relative to the neighbouring ovary tissue. Novel peptides appear to perform key roles in N. vitripennis venom function as only four of the highest 15 expressed venom encoding genes are bioinformatically annotationed. The high throughput sequencing data also provided evidence for the existence of an additional 471 novel genes in the Nasonia genome that are expressed in the venom gland and ovary. Finally, metagenomic analysis of venom gland transcripts identified viral transcripts that may play an important part in the N. vitripennis venom function. Conclusions The expression level information provided here for the 79 venom encoding genes provides an unbiased dataset that can be used by the N. vitripennis community to identify high value candidates for further functional characterisation. These candidates represent bioactive peptides that have value in drug development pipelines.

Project description:The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5’ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5’ and 3’ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression. Whole-genome bisulfite sequencing of 24-hour adult female Nasonia vitripennis whole body samples using Iilumina GAIIx and HiSeq2000.

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.

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Nasonia vitripennis

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