Molecular and functional characterization of bioactive peptides from the venom of Bracon hebetor (Say.) (Hymenoptera: Braconidae)

Abstract

Venom is a key-factor in the regulation of host physiology by parasitic Hymenoptera and a potentially rich source of novel bioactive substances for biotechnological applications. The limited study of venom of ectoparasitic wasp Bracon hebetor (Say.) (Hymenoptera, Braconidae) has not described the full complement and composition of these biomolecules or the biotechnological potential that they could represent. To provide a more complete insight, the transcripts expressed in the venom glands of the wasp B. hebetor were described using a high-throughput approach with Illumina technology. The transcriptome of the B. hebetor venom glands was analysed by high throughput sequencing using Illumina technology followed by de novo assemblies of the 18,143,814 sequence reads yielded 22,425 contigs of which 10,578 have significant BLASTx homologies in the available databases. The majority of sequence homology was with the ectoparasitoid, Diachasma alloeum, a wasp from the same taxonomic family. Homology was also observed with other wasps, bees, and ants. Gene ontology was used to group sequences by molecular functions in which catalytic activity with 42.2% was the most highly represented, cellular components comprised 33.8% of homologies and biological and metabolic processes represented 30% of the relationships. In this study, we highlight the most abundant sequences, and those that are likely to be functional components of the venom for parasitization. Specifically, we focused on genes encoding proteins that are reported in the literature to be involved in host developmental arrest, disrupting the host immune system, host paralysis, and transcripts that support these functions. The full length ORFs of Calreticulin, the Venom Acid Phosphatase Acph-1 like protein and Arginine Kinase proteins were isolated and their tissue specific expression was studied by RT-PCR. The data showed that these putative venom genes were not expressed in tissues other than venom glands and whole females. Cloning of these genes was also done successfully and Calreticulin was further processed for expression analysis. Functional analysis of the venom of the wasp was performed by artificial microinjections of both crude and treated venom (heat and proteinase) of the wasp containing 0.3 μl and 0.5 μl in non-parasitized and synchronized host insects. Last instars of the host greater wax moth Galleria mellonella (Lepidoptera, Pyralidae) were selected for venom injection bioassays and mortality data of the host insects was recorded after 1, 12, and 24 hours of venom injections while host receiving saline injections were acted as control. Our findings demonstrate that maximum mortality of the host insects (98%) was obtained by microinjections containing volume (0.5 μl) of crude venom. On the other hand heat and proteinase treated venom did not show any significant mortality of the host insect. This is the first report of large-scale analysis of genes transcribed by the venomous gland of the wasp B. hebetor. In addition, results also demonstrate that de novo transcriptome assembly allows useful venom gene expression analysis in a species lacking a genome sequence database, which ultimately provides useful information for devising control tools for insect pests of Pyralidae and other applications.