Isolation and characterization of genes related to the suppression of host immune response by Bracon hebetor (Say) (Hymenoptera: Braconidae)

Abstract

Host-parasitoid interactions in insects offer a great opportunity to identify new genes and molecules responsible for the alteration of the host physiology. To circumvent the host immune response, female of the parasitic wasp injects a blend of secretions containing mainly ovarian proteins, calyx fluid, polydnaviruses and venom in their host insects. Crude venom extract containing a wide range of bioactive proteinaceous compounds inhibits the motility of larval hemocytes and prevents the formation of cell aggregates upon artificial envenomation in vitro. In the present research work, a host parasitoid relationship of ectoparasitic wasp, Bracon hebetor (Say) (Hymenoptera, Braconidae) and host insect, greater wax moth, Galleria mellonella (Lepidoptera, Pyralidae) was studied for comprehensive analysis of the venom glands Transcriptome of the wasp B. hebetor through different Bioinformatics tools for fishing out immunosuppressive genes which reveals the occurance of a number of novel genes with immune suppressive acitivities in the venom blend of the wasp suggesting their toxic/ insecticidal potential. Out of them, Venom acid phosphatase, Tryptase -2 and CTD nuclear envelope phosphatase were selected for more detailed studies on the basis of high sequence homology/identity. The selected contigs were partial sequences of the immunosuppressive genes and their 5’ ends were isolated by Rapid Amplification of cDNA Ends repair kit (RACER kit, Invitrogen) and then full-length genes were isolated by III RT module of Gene Racer kit. Isolated genes were cloned in pGEMTeasy vector in DH5-Alpha competent cells. Screening of blue white colonies of E. coli and colony PCR were also performed for the confirmation of inserts along with the plasmid extraction of the desired colony by Plasmid DNA mini prep. Cloned genes were sequenced, analysed and characterized by different bioinformatics tools i.e., Expasy translate tool, Clustal Omega and SignalP etc. Deduced amino acid sequences of immune suppressive genes were multialighned with other parasitoid species in the database which revealed significant homologies with other braconid species. Functional bioassays of the crude venom and recombinant proteins were performed through artificial microinjection techniques in which different concentrations of the crude venom and different combinations of the recombinant proteins were injected into the last instar larvae of the host and mortality data was recorded. The effects of parasitization by the wasp on cellular immune response of the host larvae were also investigated by following an approach of hemocytes count methodology. It may be concluded that B. hebetor venom and its recombinant proteins have significant impact on survival of the host larvae by inducing biochemical and physological alterations in the parasitized host insects. Analysis of the venom glands Transcriptome of the B. hebetor has shown the natural occurance of a number of novel venom genes with potential insecticidal activity involved significantly in the suppression of host immune response in the venom blend of the wasp which may be exploited further for indepth studies through integrated approaches of functional genomics and proteomics for the development of innovative plant protection strategies for sustainable control of diverse range of insect pests of Pyralidae of agriculture crops. Further, this preliminary research work has a significant contribution towards sustainable programmes for food security and food safety by controlling the insects through natural means and reducing the toxic residues of the synthetic chemicals in food chain supply system