When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel. In the present study a novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV. Polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25%. This recombinant lacked 10593 bp of sequence information, located between 13

Direct evidence of in vivo apoptosis of Spodoptera litura larvae was demonstrated by haemocoel inoculation with wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virus (BV). In sharp contrast to natural infection, cadavers did not melt, liquefy and melanize. Typical morphological changes of apoptosis in insect haemocytes post-infection, including blebbing of the cell surface, chromatin margination and condensation, vacuolization of the cytoplasm and formation of apoptotic bodies, were observed by light and electron microscopy. Total DNAs extracted from virus-infected haemocytes showed DNA ladders. Cleavage of chromatin DNA by endogenous endonucleases were detected in the cells of most tissues cells, including epithelial cells and fat body cells, using terminal dUTP nick end labelling assays. Virogenic stroma and viral nucleocapsids could be seen in the nuclei of a few haemocytes. Yields of BV and OV (occluded virus) produced from the infected S. litura larvae were much lower than from the infected S. exigua larvae. These data suggest that host apoptotic responses to virus infection reduce AcMNPV spread at the level of the organism and that apoptosis could be host-range limiting factor for baculovirus infections.

Full-length (72K) and truncated (61K) CrylVD mosquitocidal proteins of Bacillus thuringiensis (Bt) were expressed in Spodoptera frugiperda cells and larvae of Trichoplusia ni using a baculovirus vector to investigate the role of CrylVD peptides in toxicity as well as to evaluate further the baculovirus/lepidopteran system for expressing Bt proteins. The crylVD genes were inserted into the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) under control of the polyhedrin promoter by recombination in S. frugiperda cells between a transfer vector carrying the Bt genes and vDA26Z, a recombinant AcMNPV carrying the Escherichia coli fl-galactosidase gene under control of the DA26 promoter. Recombinant AcMNPVs carrying the genes were detected as blue occlusion body-negative plaques in monolayers of S. frugiperda cells grown in the presence of X-Gal. Infection of S. frugiperda cells and T. ni larvae with plaque-purified recombinant virus, expressing either the full-length or truncated CrylVD protein, resulted in the synthesis of proteins of the expected size, as confirmed by immunoblot analyses, and their crystallization into cuboidal inclusions in the cytoplasm. Infected cells and purified inclusions from the virus (AcCrylVD) expressing the full-length protein were highly toxic to mosquito larvae, but similar preparations from the virus (AcCrylVD-C) expressing the truncated protein with a 9-6K deletion at the N terminus were non-toxic. Proteolysis with trypsin of CrylVD proteins produced by Bt and the recombinant AcMNPVs yielded peptides corresponding in size, showing that synthesis of mosquitocidal Bt proteins in lepidopteran cells occurred. The lack of toxicity of the truncated CryIVD protein, which like the toxic fulllength protein yielded a 34K protein on proteolysis that has been implicated in toxicity, indicates that by itself this protein is non-toxic. These results demonstrate the utility of the baculovirus system for expression of mosquitocidal Bt proteins and for investigation of their mode of action.

A new species of entomopathogenic nematode, Steinernema guangdongense sp. n. was recovered from a soil sample collected from Jijia town in the western part of Guangdong province, the Peoples Republic of China during a survey for entomopathogenic nematodes in 2001. The nematode can be separated from other described species of Steinernema, by morphological, morphometrical characteristics of different stages of the nematode, by crossbreeding tests and by characterizations and phylogeny of DNA sequences of either a partial 28S or the internal transcribed spacer regions of rDNA. This nematode is closest to S. longicaudum. It can be distinguished from that nematode by characteristics of different stages. For infective juveniles, although the body length is almost similar (1055 μm compared to 1063μm), body diameter of the new species is larger; values of EP (length from anterior end to excretory pore), NR (length from anterior end to nerve ring) and a body length/body width ratio are smaller, and tail with dorsal constriction. For male, the new species has longer spicule, not well curved, spicule head shorter, shaft not prominent or absent and spicule tip not suddenly tapered as shown in S. longicaudum. Also, the ratios SW (spicule length/anal body width) and GS (gubernaculums/spicule) are smaller. For female, the presence of a small double flapped epiptygma, a small projection on dorsal side of the tail tips and prominent post-anal swelling is typical for the new species.

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) gp41 gene is 993 bp long and the protein encoded by this gene has 6–66% amino acid identities with other known baculovirus GP41 proteins. Slgp41 transcripts were detected from 12 to 96 h post-infection (p.i.) and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a 41 kDa protein, and this protein was present in the nucleus of infected cells from 12 to 96 h p.i., whereas in the cytoplasm from 24 to 96 h p.i. Structural localization confirmed that SlGP41 is associated with the envelope of occlusionderived virus (ODV). Lectin-binding assay showed that three lectins erythrina cristaglli lectin (ECL), lycopersicon esculentum lectin (LEL), and bandeiraea simlicifolia lectin (BSL) recognizing N-acetylglucosamine were specifically bound to SlGP41. It was proposed that SlGP41 is an O-glycoprotein.