The virulence of the Asian and North American strains of the gypsy moth nucleopolyhedrovirus was studied. Viruses were passaged in gypsy moth larvae, and high-performance sequencing of viral genomes was carried out. It was shown that the virulence of the Asian strain, which was initially 100-fold lower than the North American strain, increased to the level of the latter after passaging. However, the deletion of virulence-associated vef-1 gene, revealed in the genome of the original Asian strain, indicated that it did not recover after passaging. Thus, the increase in virulence was likely determined by other changes in the genome.

The adaptation of pathogens to either their hosts or to environmental conditions is the focus of many current ecological studies. In this work we compared the ability of six spatially-distant Lymantria dispar (gypsy moth) multiple nucleopolyhedrovirus (LdMNPV) strains (three from eastern North America and three from central Asia) to induce acute infection in gypsy moth larvae. We also sequenced the complete genome of one Asian (LdMNPV-27/0) and one North American (LdMNPV-45/0) strain which were used for bioassay. We found that all of the North American virus strains, with the exception of one, demonstrated higher potency than the Asian virus strains, either in North American (Lymantria dispar) larvae or, in Asian (Lymantria dispar asiatica) larvae. Complete genome sequencing revealed two gene deletions in the LdMNPV-27/0 strain: the virus enhancin factor gene (vef-1) and the baculovirus repeated orf gene (bro-p). These deletions were not seen in the LdMNPV-45/0 strain nor in other American strains available in archiving systems. We also found deletions of the bro-e and bro-o genes in LdMNPV-45/0 strain but not in the LdMNPV-27/0 strain. The phylogenetic inference with an alignment of the 37 core gene nucleotide sequences revealed the close relationship of the LdMNPV-45/0 strain with other American strains accessed in GenBank (Ab-a624 and 5-6) while the LdMNPV-27/0 strain was clustered together with the LdMNPV-3054 strain (isolated in Spain) instead of predicted clustering with LdMNPV-3029 (isolated in Asia). Our study demonstrated that first, different LdMNPV isolates from the same metapopulations of L. dispar exhibit little or no difference in the degree of virulence towards host larvae and second, that locality of host population is not an important driver of LdMNPV virulence. Virulence of LdMNPV is determined only by viral genetics. The genetic differences between North American and Central Asian virus strains are discussed.

Host innate immunity is one of the factors that determines the resistance of insects to their entomopathogens. In the research reported here we studied whether or not phenoloxidase (PO), a key enzyme in the melanogenesis component of humoral immunity of insects, plays a role in the protection of Lymantria dispar larvae from infection by L. dispar multiple nucleopolyhedrovirus. We studied two types of viral infection: overt and covert. The following lines of investigation were tested: i) the intravital individual estimation of baseline PO activity in haemolymph plasma followed by virus challenging; ii) the specific inhibition of PO activity in vivo by peroral treatment of infected larvae with phenylthiourea (PTU), a competitive inhibitor of PO; iii) the evaluation of PO activity in the haemolymph plasma after larval starvation. Starvation is a stress that activates the covert infection to an overt form. All of these experiments did not show a relationship between PO activity in haemolymph plasma of L. dispar larvae and larval susceptibility to baculovirus. Moreover, starvation-induced activation of covert viral infection to an overt form occurred in 70 percent of virus-carrying larvae against the background of a dramatic increase of PO activity in haemolymph plasma in the insects studied. Our conclusion is that in L. dispar larvae PO activity is not a predictor of host resistance to baculovirus.

It was established that the virulence of the North American baculovirus strain LdMNPV-45 is almost two orders of magnitude higher than the virulence of the Asian strain LdMNPV-27 and does not depend on the test host population (gypsy moth). The Asian strain carries deletions in bro-p and vef-1 genes (82 and 91%, respectively). In accordance with the published data, the product of the latter can greatly increase the virulence of the virus. This result indicates that the population polymorphism of the virulence of baculoviruses can be explained by the vef-1 gene deletion.

Gypchek is a gypsy nucleopolyhedrovirus (LdMNPV) product used for management of European gypsy moth (Lymantria dispar dispar L.) in the United States, primarily in areas where the use of broad-spectrum pesticides is not appropriate. Similar LdMNPV products are used in Russia for control of a flighted-female strain of Asian gypsy moth (Lymantria dispar asiatica Vnukovskij), an insect not yet established in the United States. Gypchek is a mixture of LdMNPV genotypic variants and is being developed further toward a single, high- potency genotype product that is effective against both European and Asian strains. We isolated 5 LdMNPV genotypic variants from Gypchek and, through diet incorporation bioassays, assessed their potencies for both a laboratory strain of European gypsy moth and a wild Asian gypsy moth strain. Bioassays conducted in the United States showed that 2 viral isolates, 122b and 122-HP, were about 3 times as potent as Gypchek against European gypsy moth. Bioassays conducted in Russia showed that 122-HP was as effective as a wild Siberian LdMNPV against a wild Asian (Siberian) strain of gypsy moth. Both 122-HP and 122b were shown to be at least as effective as Gypchek in killing European gypsy moth larvae when formulated at a high dose and sprayed on oak foliage in a ground-based field test. Overall results indicated that both 122b and 122-HP are potential candidates for further development as a single-genotype Gypchek product.

Larval gypsy moths, Lymantria dispar (Lepidoptera:Lymantriidae) were co-infected with the L. dispar nucleopolyhedrovirus (LdMNPV) and the Cotesia melanoscela (Hymenoptera:Braconidae) polydnavirus (CmeBV). CmeBV was given along with a parasitoid egg and calyx products in a stinging event, or in the form of an injection of calyx-derived extract. LdMNPV was delivered per os, integrated into artificial diet. Mortality from all sources was recorded over the subsequent three-week period. Neither parasitism nor injections of purified CmeBV with toxin had any effect on the amount of mortality caused by concurrent challenges with LdMNPV.

Bacillus thuringiensis (Bt) Cry1A toxin-binding sites in the Douglas fir tussock moth (DFTM) larval gut were localized using immunofluorescence microscopy. Cry1Aa, Cry1Ab and Cry1Ac all bound strongly to the DFTM peritrophic membrane (PM); weaker binding of the Cry1A toxins was observed along the apical brush border of the midgut epithelium. Comparative analysis of the Cry1A toxin-binding molecules in the PM and brush border membrane vesicles (BBMVs) showed that a similar toxin-binding complex was present in both. The Cry1A toxin-binding substance, a broad band with an apparent size of 180 kDa, consisted of a closely spaced doublet. The doublet was present in peritrophins, proteins tightly bound to the PM. Lectin binding studies of the PM and BBMV toxin-binding components revealed that they are glyconjugates with terminal α-GalNAc residues comprised exclusively of O-linked oligosaccharides in their glycan structures. Mild periodate oxidation, release of O-linked glycans by β-elimination, and enzymatic removal of terminal α-linked GalNAc residues with N-acetyl-α-d-galactosaminidase digestion abolished Cry1A toxin-binding to the PM and BBMV components. These data provide strong evidence that O-linked glycans are the target structures on the toxin-binding glycoconjugates for the Cry1A class of insecticidal proteins in DFTM larvae.

The gypsy moth, Lymantria dispar L., nucleopolyhedrovirus (LdMNPV) product Gypchek is a microbial pesticide produced by the USDA Forest Service. Gypchek is a mixture of LdMNPV genotypes produced in vivo. Commercial interests prefer to develop a stable, high-potency genotype that can be produced at low cost, preferably in vitro. We sprayed 2 LdMNPV strains and Gypchek at various doses, with and without the viral enhancer Blankophor BBH (Burlington Chemical Co., Burlington, NC), on oak foliage under field conditions to determine the relationship between application rate and larval mortality. We used strains previously isolated from Gypchek; strain 203 was produced in vivo and strain 122 was produced in vitro. When applied at a rate of 10(12) viral occlusion bodies (OB) per 379 L of water without enhancer, mortality was 26% with strain 122 and greater than 90% with both strain 203 end Gypchek. In addition, strain 203 killed larvae faster. At an application rate of 10(11) OB with enhancer, larval mortality was greater than 90% with all 3 viral preparations. Strain 122 produced in vitro at a lower cost than is currently possible for Gypchek production would allow compensation for its reduced virulence by the application of a higher dose. Alternatively, if applied with enhancer at a rate of 10(11) OB, there would be no significant differences in efficacy of 122 compared with Gypchek applied at that same rate.

Bacteria representing several genera were isolated from integument and alimentary tracts of live Asian longhorned beetle, Anaplophora glabripennis (Motschulsky), larvae and adults. Insects examined were from infested tree branches collected from sites in New York and Illinois. Staphylococcus sciuri (Kloos) was the most common isolate associated with adults, from 13 of 19 examined, whereas members of the Enterobacteriaceae dominated the isolations from larvae. Leclercia adecarboxylata (Leclerc), a putative pathogen of Colorado potato beetle, Leptinotarsa decemlineata (Say), was found in 12 of 37 larvae examined. Several opportunistic human pathogens, including S. xylosus (Schleifer and Kloos), S. intermedius (Hajek), S. hominis (Kloos and Schleifer), Pantoea agglomerans (Ewing and Fife), Serratia proteamaculans (Paine and Stansfield) and Klebsiella oxytoca (Flugge) also were isolated from both larvae and adults. One isolate, found in 1 adult and several larvae, was identified as Tsukamurella inchonensis (Yassin) also an opportunistic human pathogen and possibly of Korean origin.. We have no evidence that any of the microorganisms isolated are pathogenic for the Asian longhorned beetle.

ABSTRACT Nucleopolyhedroviruses (NPVs) can initiate devastating disease outbreaks in populations of defoliating Lepidoptera, a fact that has been exploited for the purposes of biological control of some pest insects. A key part of the horizontal transmission process of NPVs is the degradation of the larval integument by virus-coded proteins called chitinases, such as V-CHIA produced by the v-chiA genes. We used recombinant and naturally occurring strains of the Lymantria dispar NPV (LdMNPV) to test horizontal transmission in the field, release of virus from dead larvae under laboratory conditions, and cell lysis and virus release in cell culture. In the field, strains of LdMNPV lacking functional v-chiA genes showed reduced horizontal transmission compared to wild-type or repaired strains. These findings were mirrored by a marked reduction in released virus in laboratory tests and cell culture when the same strains were used to infect larvae or cells. Thus, this study tests the pivotal role of liquefaction and the v-chiA gene in field transmission for the first time and uses complementary laboratory data to provide a likely explanation for our findings.

This handbook is an update of handbook FHTET-2012-01, Gypchek - Environmentally Safe Viral Insecticide for Gypsy Moth Control, printed in May, 2012. This update contains information on virus production, safety evaluations, results of efficacy and deposition evaluations, commercial production, and a copy of the revised registration label, safety data sheet, and technical bulletin.

Gypchek is a gypsy moth (Lymantria dispar L.) - specific biopesticide whose primary use is for treating areas where environmental concerns outweigh the use of broad-spectrum pesticides for gypsy moth management. Gypchek is a lyophilized powder produced from larvae that have been infected with the gypsy moth nucleopolyhedrovirus (LdMNPV). The product contains a mixture of closely related LdMNPV genotypes that, in combination, act as the active ingredient.

Gypchek is a baculovirus-based insecticide produced by the U.S. Forest Service. This biopesticide is species-specific to the gypsy moth and contains as an active ingredient the Lymantria dispar nucleopolyhedrovirus, also known as the gypsy moth virus or LdMNPV. Currently, Gypchek is a mixture of many strains of LdMNPV, produced in vivo, and refined into a usable product at our facility in Ansonia, CT. From 2003 to 2006, we conducted a number of field experiments designed to determine if a single strain of LdMNPV might be suitable to use as a replacement for the current mixture. Additionally, research has been conducted in Delaware, OH (J. Slavicek) toward producing the virus in vitro. Central to our testing methodology was the bugsin-bags experiments in which virus was applied to branches as infected first-instar larvae or as a sprayed product. Branches with approximately 40 leaves were selected on oak trees in the Cedar Swamp State Wildlife Management Area near Smyrna, DE. These branches were then enclosed in mesh bags with 25 third-instar test larvae representing bugs that would be eating contaminated foliage in the field. After 1 week in the field, branches were cut off trees and returned to the lab. Test larvae were removed to individual diet cups where they were reared for 3 weeks and necropsied if they died.

Periodic intrusions of Asian strains of gypsy moth, Lymantria dispar L., into North America have occurred over the past several years. Preventative measures in the countries of origin and around ports of entry in North America have lowered the risk of invasion and establishment but the threat remains current. Asian strains have a wider host range than the established North American (European) strain and, unlike North American female moths, Asian females fly.