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.

The complete Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome contained 139,342 bp with a G1C content of 42.7%, and 141 putative open reading frames (ORFs) or genes of 150 nucleotides or greater that showed minimal overlap. Ninety-six ORFs had homologues in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), 16 had homologues in other baculoviruses, and 29 were unique to SpltMNPV. The homologues of ubiquitin and gp37 are fused in SpltMNPV. The genome lacked a homologue of the major budded virus glycoprotein gene gp64, but it contained a homologue of ORF130 of Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV). There were two homologues of AcMNPV ORF2 (bro gene), and a DnaJ protein gene (SpltORF39) in which the N-terminus showed homologies with the J domain of DnaJ family proteins. Seventeen homologous regions (hrs) were identified, each containing 2

A typical apoptosis of BTI-Tn-5B1-4 (Hi5) cells induced by Heliothis armigera single capsid nucleopolyhedrovirus (HaSNPV) infection was completely suppressed by coinfection with Trichoplusia ni multicapsid nucleopolyhedrovirus polyhedron-negative recombinant (TnMNPV-SVING) (OCCN) at a low multiplicity of infection (6

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