Expression of a neurotoxin gene improves the insecticidal activity of Spodoptera litura nucleopolyhedrovirus (SpltNPV).

SpltNPV-C3 is a novel nucleopolyhedrovirus (NPV) screened from 189 wild Spodoptera litura nucleopolyhedrovirus (SpltNPV) clones collected throughout Japan and has high potential insecticidal activity against S. litura. In this study, we constructed two recombinant SpltNPVs, SpltNPV-Δegt and SpltNPV-Δegt-BmK, which were characterized by the disruption of the UDP-glucosyltransferase gene (egt) only and by replacing the egt locus with the Buthus martensi insect-specific toxin gene (BmK ITa1), respectively. The insecticidal activity of these two recombinant viruses was evaluated by 50% lethal time (LT(50)) and 50% lethal concentration (LC(50)) using third instar S. litura larvae. Bioassays showed that the LT(50) of SpltNPV-Δegt was reduced by 25% at a dose of 10(9) Occlusion bodies (OBs)/ml compared to the wild-type SpltNPV. However, the LC(50) values did not change. The SpltNPV-Δegt-BmK reduced LT(50) by 28% at the highest dose of OBs (10(9) OBs/ml) compared to the wild-type SpltNPV, and the LC(50) was nearly an order of magnitude lower than those of the wild-type SpltNPV and SpltNPV-Δegt. However, there was no discernable difference in the LT(50) between SpltNPV-Δegt-BmK and SpltNPV-Δegt when the 3rd instars of S. litura were fed a dose of 10(8) or 10(9) OBs/ml. The results suggest that these two recombinant forms of SpltNPV provide further opportunities to develop these viruses into commercially viable products to control S. litura populations.

[Construction and toxicity the recombinant SpltMNPV expressing the scorpion toxin gene].

OBJECTIVE: To develop a high toxic recombinant Spodoptera litura multicapsid nucleopolyhedroviruse (SpltMNPV) insecticide. METHODS: We constructed a recombinant transfer vector that was characterized by disrupting of ecdysterioid UDP-glucosyltransferase (egt) gene and expressing the mature peptide of the Chinese scorpion, B. martensi Karsch (BmK ITal) gene at the control of ie-1 promoter. The transfer vector and the SpltMNPV II DNA cotransfected the SpLi cells. Recombinant viruses were purified by the end point dilution and fluorescent spot purification. RESULTS: We successfully screened the recombinant SpltMNPV-deltaegt-Pph-egfp-ie-1-BmK ITal of which the egt gene was knocked out and expressed the mature peptide of the BmK ITal gene at the control of ie-1 promoter. Bioassays showed that, compared to the wide-type SpltMNPV, the speed of the recombinant virus killing the S. litura (LT50) increased by 0.7-0.8 days. CONCLUSION: The insecticidal effect of SpltNPV could be increased by inserting the foreign gene, which provided a further opportunity to develop the SpltNPV into commercially viable products to control the S. litura.

Selectivity and neuroendocrine regulation of the precursor uptake by pheromone glands from hemolymph in geometrid female moths, which secrete epoxyalkenyl sex pheromones.

Macrolepidopteran female moths in families such as Geometridae produce epoxyalkenyl sex pheromones, which are biosynthesized via epoxidation of polyunsaturated hydrocarbons in their pheromone glands. The precursors, however, are expected to be produced outside of the pheromone glands, probably in oenocytes or in the fat body, and transported to the glands via hemolymph. Based on these facts, the selectivity of the epoxidation substrates and of the precursor uptake by pheromone glands was examined with two geometrid species, Hemerophila artilineata and Ascotis selenaria cretacea, using binary mixtures of deuterated precursors and their analogs, which were topically applied to the pheromone glands or injected into the abdomen. GC-MS measurements of pheromone extracts showed equal epoxidation of two polyenes, indicating a low selectivity for both processes, while the epoxidation proceeded at only one double bond specific to each species. This result makes it possible to conclude that the formation of species-specific epoxyalkenyl pheromones results from the rigid formation of polyunsaturated precursors and their epoxidation at a fixed position. Next, the neuroendocrine regulation of these processes was studied with in vivo and in vitro experiments using decapitated females. The epoxy pheromones disappeared completely within 36 h of decapitation, and epoxidation of the injected precursors was not detected in the decapitated females, which restarted the reaction by treatment with a pheromone biosynthesis-activating neuropeptide (PBAN). The precursors topically applied to glands of the decapitated females, however, were converted into epoxy pheromones without PBAN, indicating that this neuropeptide hormone accelerated the precursor uptake by pheromone glands but not the epoxidation already underway in the glands.

Characterization of geometrid sex pheromones by electrospray ionization time-of-flight mass spectrometry.

The utility of liquid chromatography combined with time-of-flight mass spectrometry (LC/TOFMS) was demonstrated for studies on chiral unsaturated epoxy compounds, sex pheromones produced mainly by female moths in the family Geometridae. By electrospray ionization (ESI), each synthetic epoxyalkadiene derived from (Z,Z,Z)-3,6,9-triene with a C(18)-C(23) straight chain showed three ion series, [M + NH(4)](+), [M + H](+) and [M - OH](+), with high resolution and good sensitivity, indicating its molecular formula. In addition to these, characteristic fragment ions at m/z M - 57 and M - 71 for the 3,4-epoxides and at m/z M - 123 and 123 for the 9,10-epoxides were detected, whereas the 6,7-epoxides did not produce fragment ions that reflected their structures. Monitoring these diagnostic ions during the LC/MS analysis of a gland extract, the natural sex pheromone of the mulberry looper was confirmed to be (Z,Z)-cis-9,10-epoxy-3,6-octadecadiene, which was separable from the other positional isomers on an ODS column. Furthermore, (Z,Z)-cis-3,4-epoxy-6,9-nonadecadiene secreted by the Japanese giant looper was analyzed with a chiral column, and the stereochemistry was determined directly.