Quantitative PCR analysis of the salivary gland hypertrophy virus (GpSGHV) in a laboratory colony of Glossina pallidipes

International audienceMany species of tsetse flies can be infected by a virus that causes salivary gland hypertrophy (SGH) and virus isolated from Glossina pallidipes (GpSGHV) has recently been sequenced. Flies having SGH have a reduced fecundity and fertility. To better understand the impact of this virus in a laboratory colony of G. pallidipes, where the majority of flies are infected but asymptomatic, and to follow the development of SGH in symptomatic flies in relation to virus copy number, a quantitative PCR (qPCR) method was developed. The qPCR analyses revealed that in asymptomatic flies virus copy number averaged 1.68E+5, 2.05E+5 and 1.07E+7 log10 in DNA from an excised leg, salivary glands and a whole fly, respectively. In symptomatic flies the virus copy number in the same organs averaged 1.34E+7, 1.42E+10 and 1.5E+9, respectively. Despite these statistically significant differences (p much less-than 0.0001) in virus copy number between asymptomatic and symptomatic flies, there was no correlation between age and virus copy number for either sets in adult flies. A clear correlation between virus copy number in pupae and their mothers was observed. Reverse transcription quantitative PCR (RT-qPCR) of the viral messenger RNA encoding ODV-E66, an envelope protein, revealed a clear correlation between virus copy number and the level of gene expression with values of 2.77 log10 in asymptomatic males and 6.10 log10 in symptomatic males. Taken together these results confirm the close relationship between virus copy number and SGH syndrome. They demonstrate the vertical transmission of GpSGHV from mother to progeny, and suggest that the development of SGH may be correlated to the virus copy number acquired by the larva during its intra-uterine development

Comparative analysis of deformed wing virus (DWV) RNA in Apis mellifera and Varroa destructor

A two step quantitative RT-PCR assay was validated to monitor the deformed wing virus (DWV) RNA loads in Apis mellifera L. and Varroa destructor. A pair of primers hybridising in a conserved domain of the putative DWV RNA polymerase gene region was designed. These primers amplified a 69-nucleotide fragment which was quantified using the SYBR-green chemistry. The experimental validation of the method showed that the RNA extraction and cDNA synthesis steps were responsible for the greatest variability in the results while assays repeated on different PCR plates were reproducible. Quantitative RTPCR analysis on drone bee prepupae showed that DWV RNA loads were higher in cells parasitized by several mother mites. In workers, DWV prevalence was directly correlated to mite infestation and DWV was detected in all the bee developmental stages except in eggs. Very important DWV RNA loads could be recorded even in absence of clinical sign; however bees emerging with deformed wings were predominantly infected by DWV. In mites collected on emerging bees, the DWV RNA yields varied from 10(4) to 10(6) copies per mite but might exceed 10(8) copies in some case

Hyposoter didymator uses a combination of passive and active strategies to escape from the Spodoptera frugiperda cellular immune response

An endoparasitic life style is widespread among Hymenoptera, and various different strategies allowing parasitoids to escape from the host encapsulation response have been reported. Species carrying polydnaviruses (PDVs), such as the ichneumonid Hyposoter didymator, generally rely on the viral symbionts to evade host immune responses. In this work, we show that H. didymator eggs can evade encapsulation by the host in the absence of calyx fluid (containing the viral particles), whereas protection of the larvae requires the presence of calyx fluid. This evasion by the eggs depends on proteins associated with the exochorion. This type of local passive strategy has been described for a few species carrying PDVs. Immune evasion by braconid eggs appears to be related to PDVs or proteins synthesized in the oviducts being associated with the egg. We report that in H. didymator, by contrast, proteins already present in the ovarian follicles are responsible for the eggs avoiding encapsulation. Mass spectrometry analysis of the egg surface proteins revealed the presence of host immune-related proteins, including one with similarities with apolipophorin-III, and also the presence of three viral proteins encoded by IVSPERs (Ichnovirus Structural Protein Encoding Regions)

Polymerase Chain Reaction detection of deformed wing virus (DWV) in Apis mellifera and Varroa destructor

We have developed a specific assay for the detection of deformed wing virus (DWV) in Apis mellifera L. and Varroa destructor based on the reverse transcriptase polymerase chain reaction (RT-PCR) technology. Primers were designed from the sequence of a 4700 nucleotides cDNA fragment located in the 3'-end of the DWV genome. This fragment encodes a single open reading frame of 1565 amino acids showing similarity to viral RNA dependent RNA polymerase consensus motif. RT-PCR assays from DWV infected individual mite or bee produced a 395 nucleotide DNA fragment clearly identifiable by agarose gel electrophoresis. The signal in bees having deformed wings was significantly higher than in normal ones. A search for DWV in 40 colonies showed that DWV is broadly distributed in bee colonies and mites. As an average, greater virus prevalence of virus was detected in bees collected in autumn compared to bees collected in spring or during the summer perio

Lepidopteran transcriptome analysis following infection by phylogenetically unrelated polydnaviruses highlights differential and common responses

International audienceThe Polydnaviridae is a family of double-stranded DNA viruses that are symbionts of parasitoid wasps. The family is currently divided into two genera, the Ichnovirus (IV) and Bracovirus (BV), which are associated with wasps in the families Ichneumonidae and Braconidae, respectively. IVs and BVs have similar immunosuppressive and developmental effects on parasitized hosts but their encapsidated genomes largely encode different genes. To assess whether IV and BV infection has similar or disparate effects on the transcriptome of shared hosts, we characterized the effects of Hyposoter didymator Ichnovirus (HdIV) and Microplitis demolitor Bracovirus (MdBV) on the fat body and hemocyte transcriptome of Spodoptera frugiperda (Lepidoptera: Noctuidae). Our results indicated that HdIV and MdBV infection alters the abundance of a relatively low proportion of S. frugiperda transcripts at 24 h post-infection. A majority of the transcripts affected by infection also differed between MdBV and HdIV. However, we did identify some host transcripts that were similarly affected by both viruses. A majority of these genes were transcribed in the fat body and most belonged to functional classes with roles in immunity, detoxification, or cell structure. Particularly prominent in this suite of transcripts were genes encoding for predicted motor-related and collagen IV-like proteins. Overall, our data suggest that the broadly similar effects that HdIV and MdBV have on host growth and immunity are not due to these viruses inducing profound changes in host gene expression. Given though that IVs and BVs encode few shared genes, the host transcripts that are similarly affected by HdIV and MdBV could indicate convergence by each virus to target a few processes at the level of transcription that are important for successful parasitism of hosts by H. didymator and M. demolitor

Venom gland extract is not required for successful parasitism in the polydnavirus-associated endoparasitoid Hyposoter didymator (Hym. Ichneumonidae) despite the presence of numerous novel and conserved venom proteins

Corrigendum to “Venom gland extract is not required for successful parasitism in the polydnavirus-associated endoparasitoid Hyposoter didymator (Hym. Ichneumonidae) despite the presence of numerous novel and conserved venom proteins” [Insect Biochem. Mol. Biol. 43 (2013), 292–307] http://dx.doi.org/10.1016/j.ibmb.2013.07.001(UT:000329772300006)The venom gland is a conserved organ in Hymenoptera that shows adaptations associated with life-style diversification. Few studies have investigated venom components and function in the highly diverse parasitic wasps and all suggest that the venom regulates host physiology. We explored the venom of the endoparasitoid Hyposoter didymator (Campopleginae), a species with an associated polydnavirus produced in the ovarian tissue. We investigated the effects of the H. didymator venom on two physiological traits of the host Spodoptera frugiperda (Noctuidae): encapsulation response and growth rate. We found that H. didymator venom had no significant effect on host cellular immunity or development, suggesting that it does not contribute to parasitism success. The host physiology seemed to be modified essentially by the ovarian fluid containing the symbiotic polydnaviruses. Proteomic analyses indicated that the H. didymator venom gland produces a large variety of proteins, consistent with the classical hymenopteran venom protein signature, including: reprolysin-like, dipeptidyl peptidase IV, hyaluronidase, arginine kinase or allergen proteins. The venom extracts also contained novel proteins, encoded by venom genes conserved in Campopleginae ichneumonids, and proteins with similarities to active molecules identified in other parasitoid species, such as calreticulin, reprolysin, superoxide dismutase and serpin. However, some of these proteins appear to be produced only in small amounts or to not be secreted. Possibly, in Campopleginae carrying polydnaviruses, the host-modifying activities of venom became. redundant following the acquisition of polydnaviruses by the lineage