41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

Venom from the endoparasitic wasp, Pimpla hypochondriaca, contains factors with anti-haemocyte and immunosuppressive properties. The gene for one such factor (vpb) has been cloned and recombinant protein produced. Bio-assays utilising VPB were performed and indicated that introduction of this immunosuppressive protein into the haemocoel of two lepidopteran pests, increases their susceptibility to the biological control agent, Bacillus thuringiensis. The potential for improving the efficacy of Bt through suppression of pest immune responses is discussed.

Venom from the endoparasitoid, Pimpla hypochondriaca contains a mixture of proteins. One of these was previously biochemically isolated and shown to have haemocyte anti-aggregation activity against host haemocytes in vitro. This protein shares significant homology to a second venom protein (VPB) from this parasitoid. The gene for VPB was amplified from a P. hypochondriaca venom gland cDNA library by PCR, cloned and expressed in E. coli. The presence of a fusion tag allowed purification. The purified immunosuppressive protein was found to have anti-haemocyte activity, in vitro, against haemocytes from two lepidopteran pests. This venom protein may have the potential to improve efficacy of biocontrol agents.

Investigations of toxicity of simpler molecules based on the epoxy-alcohol fragment of azadirachtin have revealed insecticidal activity on Galleria mellonella L. larvae. The epoxy-alcohols doses giving 50% mortalities were in the increasing order from 2,3-epoxy-1-propanol (I) (0.22 mg/g), 4.5-epoxy-2-pentanol (II) (0.068 mg/g) and finally, 1,3-bis(2,3-epoxy-1-propyloxyl)-2-propanol (III) (0.147 mg/g). The epoxyalcohols exhibited higher insecticidal activity when compared with the commercial neem product for which the dose giving 50% mortalities was 10.6 mg/g and to azadirachtin that killed the larvae only by injection (dose of 0.20 mg/g of body weight). Following the results of these in vivo experiments, we tested them in vitro on insect cell lines. First, we described the cytotoxic effects on three cell lines (Sf9, A.t. GRIP-1, and Ld-L1) of (I), (II), (III), 3-epoxy-1-propyl butyrate (IV), 2,3-epoxypropyl methyl ether (V), azadirachtin and a neem formulation. We observed, depending on the concentrations, immediate and delayed cytotoxic effects. All molecules tested blocked cell proliferation to the same extent on Sf9 cell line. An antimitotic effect was observed with azadirachtin and the molecules tested on Ld-L1 cells. The cytotoxicity of our molecules emphasizes the importance of the chemical structure between the two half moieties of azadirachtin for the biological activity.