Project description:Parasitoid wasps of the species Diachasmimorpha longicaudata are associated with a heritable poxvirus, known as DlEPV, that is stored in the venom gland of adult female wasps and transferred to tephritid fly hosts of the wasps during oviposition. We conducted a RNA-seq differential expression analysis to gain insight on how DlEPV can replicate in both wasps and their fly hosts but only cause pathogenic effects during replication in flies. Our analysis revealed that 91.2% (176 of 193) of DlEPV genes showed significant differential expression during peak virus replication in wasp venom glands compared to parasitized flies. Over 80% of DlEPV replication genes were significantly upregulated in wasps, while 79% of DlEPV putative virulence genes were significantly upregulated in fly hosts. These data therefore support a dichotomy of viral function, where virus replication is promoted in wasp tissue and virulence in host tissue. Such a division of viral activity could represent an important adaptation to maintain a stable symbiosis between this virus and its associated parasitoid.
Project description:Diachasmimorpha longicaudata parasitoid wasps carry a symbiotic poxvirus, known as DlEPV, within the female wasp venom gland. We sequenced RNA from venom gland tissue to identify DlEPV orthologs for 3 conserved poxvirus core genes. The DlEPV ORFs identified from this transcriptome were used to design primers for downstream RT-qPCR analysis and RNAi knockdown experiments.
Project description:Parasitoid wasps are one of the most species-rich groups of animals on Earth, due to their ability to successfully develop as parasites of nearly all types of insects. Unlike most known parasitoid wasps that specialize within one or a few host species, Diachasmimorpha longicaudata is a generalist that can survive within multiple genera of tephritid fruit fly hosts, including many globally important pest species. D. longicaudata has therefore been widely released to suppress pest populations as part of biological control efforts in tropical and subtropical agricultural systems. In this study, we investigated the role of a mutualistic poxvirus in shaping the host range of D. longicaudata across three genera of agricultural pest species: two of which are permissive hosts for D. longicaudata parasitism and one that is a nonpermissive host. We found that permissive hosts Ceratitis capitata and Bactrocera dorsalis were highly susceptible to virus infection, displaying rapid virus replication and abundant fly mortality. However, the nonpermissive host Zeugodacus cucurbitae largely overcame virus infection, exhibiting substantially lower mortality and no virus replication. Further investigation of transcriptional dynamics during virus infection demonstrated hindered viral gene expression and limited changes in fly gene expression within the nonpermissive host compared to the permissive species, indicating that the host range of the viral symbiont may dictate the host range of D. longicaudata wasps. These findings also reveal that viral symbiont activity may be a major contributor to the success of D. longicaudata as a generalist parasitoid species and a globally successful biological control agent.
Project description:Goal: Natural populations of Drosophila melanogaster vary widely in their capacity to resist infection by the parasitic wasp Leptopilina boulardi. To date, little is known about the genetic, cellular and molecular basis underpinning the variation in susceptibility to parasitic wasps. In D.melanogaster populations artificially selected for parasitoid resistance, an increase in the number of circulating hemocytes (blood cells) is observed. One possibility is that this is accompanied by changes in hemocyte activation state, resulting in a successful defence response when exposed to parasitic wasps. We tested this possibility by generating populations that are highly resistant and highly susceptible to the parasitic wasps. We generated these populations after 26 generations of experimental evolution, starting from a single interbred wild caught population. Here, we study how selection for resistance to the parasitic wasp L. boulardi changes the number and type of circulating hemocytes in D. melanogaster larvae using scRNA-seq. Methods: Wild caught D. melanogaster females were collected from Cambridge, UK, to establish an outbreed population. From this, three replicated populations were selected for resistance to L. boulardi strain NSRef for 26 generations (Selection). Another three populations were maintained in the laboratory for 26 generations without being exposed to parasitoid-associated selection pressures (No Selection). At generation 26, second instar D. melanogaster larvae (48-63 hours after fertilization) from each population were infected with L. boulardi for three hours (Infection) or maintained without infection (No infection). 48 hours after, circulating hemocytes from third instar D. melanogaster larvae (96-111 hours after fertilization) from each population were collected in PBS and cleaned in OptiPrep solution (1.09g/ml). 10X Single Cell GEX v2 libraries were prepared and sequenced. CellRanger v2 was used to generate sample cell count matrices. Seurat v3 was used to integrate, normalise and cluster the cell types. Results: We identified novel plasmatocyte cell types, immature and mature lamellocytes and crystal cells. We identify a single lineage of cellular differentiation starting from a self-cycling plasmatocyte population, enriched for genes involved in extracellular matrix organisation, and ending in a mature lamellocyte population that has enriched expression of genes involved in signalling, hemostasis and gluconeogenesis. Immature lamellocytes were overrepresented in populations selected for resistance to parasitoids. Infection with parasitoid resulted in an increase in both immature and mature lamellocytes in circulation. Conclusions: Selection for resistance to the parasitic wasps resulted in the constitutive activation of the D. melanogaster immune system, where plasmatocytes differentiate into immature lamellocytes even where they are not exposed to parasitic wasps. We speculate these constitutive changes assist in mounting a successful defence response when exposed to parasitic wasps.
Project description:Venom injected at oviposition is crucial for successful reproduction in most parasitoid wasp species (Moreau & Asgari 2015; Poirié et al. 2014). The venom of the pea aphid parasitoid Aphidius ervi was analyzed previously using a combined transcriptomic and proteomic approach (Colinet et al. 2014) and we applied similar methods here to compare the venom composition in Lysiphlebus fabarum, that also used the pea aphid as host. Venom was extracted from the L. fabarum venom gland and analyzed using 1D gel electrophoresis and mass spectrometry. A total of 35 L. fabarum proteins were identified as putative venom proteins using these results combined with available transcriptomic data (Dennis et al. 2017) and the genomic data.
Project description:Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously we showed that circulating MGHs have high motility and interaction with the parasitoid rapidly triggers encapsulation, structural and molecular mechanisms behind these processes remained elusive. Here, we use detailed ultrastructural analysis of MGHs and also live cell imaging to study encapsulation in Drosophila ananassae after parasitoid wasp infection. We found dynamic structural changes, mainly driven by the formation of diverse vesicular systems and a large variety of newly developed intracytoplasmic membrane organizations, moreover abundant generation of giant cell exosomes (GCE) in the MGHs. Moreover, we used RNA sequencing to study the transcriptomic profile of MGHs and the activated plasmatocytes 72 hours after infection, as well as the uninduced blood cells. This reveals that differentiation of MGHs is accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts mainly related to vesicular function, cytoskeletal organization and adhesion were enriched in MGHs. In addition, transmembrane receptors were upregulated, which may be important for parasitoid recognition. Our results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response.