John D. Podgwaite

Properties of two Lymantria dispar nuclear polyhedrosis virus isolates obtained from the microbial pesticide Gypchek

Abstract Two Lymantria dispar nuclear polyhedrosis virus isolates, 5–6 and A2-1, differing in the phenotypic characteristic of the number of viral occlusions in infected cells, were obtained from a production lot of the microbial pesticide Gypchek and several of their replication properties were investigated and compared. Budded virus titer produced in cell culture, polyhedral inclusion body production in cell culture and in vivo, the number of virions present within occlusion body cross sections, and potency determinations suggest that isolate 5–6 is a few polyhedra plaque variant and that A2-1 is a many polyhedra wild-type isolate.

Pathogen clumping: an explanation for non-linear transmission of an insect virus

Abstract.  1. Previous work has shown that transmission of some insect pathogens is a non‐linear process. A number of hypotheses have been put forward as explanations for this phenomenon; however, none have proven wholly satisfactory. Here we test the effects on transmission of spatial distribution of an insect virus by testing whether or not experimental manipulations of pathogen clumping lead to different values of a clumping parameter. The gypsy moth nucleopolyhedrovirus (LdMNPV) was used, which is transmitted when larvae consume virus released from previously infected larvae that have died on foliage.

Bacteria associated with larvae and adults of the Asian longhorned beetle (Coleoptera: Cerambycidae)

Abstract 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.

Biological Activity of Bacillus thuringiensis and Associated Toxins against the Asian Longhorned Beetle (Coleoptera: Cerambycidae)

Bacillus fhuringiensis Berliner var. tenebrionis and 5. thuringiensis toxins were as- sayed against larval and adult Asian longhorned beetles, Anoplophora glabripennis (A. glabrip- ennis). Preliminary in vitro assays showed some toxins to be active on whole midgut prepara- tions in voltage clamp assays and in assays on brush border membrane vesicles formed from midgut epithelial cells. For in vivo tests, a commercially-available product (Novodo@) was incorporated into artificial diet, upon which larvae were allowed to feed ad lib. In other tests, droplets of solubilized B. thuringiensis toxins were fed to larval and adult beetles using a mi- cropipette. None of the in vivo assays showed significant negative effects on either larvae or adults. We believe that some aspect of A. glabripennis midgut chemistry may be incompatible with toxin activation or mode of action. The Asian longhorned beetle, Anoplophora glabripennis (Motschulsky), is a large wood-boring cerambycid native to parts of mainland China and Korea. Anoplophora glabripennis is a polyphagous feeder with a marked preference for poplar, maple and willow. Adult beetles feed and mate on the leaves, branches, and bole of trees, and female beetles chew slits in the bark in which to deposit a single egg. After hatching, larvae burrow and consume wood under the bark surface, eventually penetrating deeply into trees. The damage resulting from larval feeding can be sufficient to render large trees unsound, creating hazardous conditions in populated areas. Anoplophora glabripennis was accidentally introduced into the United States in solid wood packing material from China, discarded in areas adjacent to airports. There were likely a number of such introductions in the 1980s and 1990s. Populations of A. glabripennis now exist in and near New York City, NY and Chicago, IL. These populations have been the targets of intense efforts aimed at eradication, usually in the form of removal and burning of infested trees. Less intrusive methods of control are currently being sought, especially biological control methods using parasites and microbial pesticides. Bacillus thuringiensis Berliner, an aerobic soil-dwelling bacterium, has a long his- tory of use in biopesticides. Bacillus thuringiensis and its subspecies produce insec- ticidal proteins during sporulation, the primary of which is S-endotoxin. Because the 6-endotoxins are derived from bacterial Cry genes, they are also known as Cry toxins (e.g., Cryl or Cry Ma). Some Cry toxins, notably Cryl b, have been shown to have

A strain of Serratia marcescens pathogenic for larvae of Lymantria dispar: Infectivity and mechanisms of pathogenicity

The ED50 of a strain of Serratia marcescens for microinjected instar III and IV gypsy moth larvae was 7.5 and 14.5 viable cells, respectively. Percentage and rate of mortality were found to be highly variable among replicates of the same instar and between instars in free-feeding bioassays. Mortality in second instar larvae occurred before ecdysis, whereas practically no mortality occurred in third and fourth instars until the molting period. Neither Boivin endotoxin preparations nor culture filtrates were toxic to instar III larvae when administered per os or by microinjection. Histological evidence indicated that the microorganism invaded the hemocoel of healthy or predisposed insects through the gut wall. The rapid multiplication of the bacterium in the hemocoel of infected insects, followed by death in the absence of extensive tissue damage, indicated mortality was due to a septicemia. The histological and biological evidence presented indicated that the microorganism would be less than effective if utilized as a conventional microbial insecticide.

Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field

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.

Bacillus thuringiensis Cry1A toxin-binding glycoconjugates present on the brush border membrane and in the peritrophic membrane of the Douglas-fir tussock moth are peritrophins

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 180kDa, 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.

Efficacy of Aerially-Applied Gypchek against Gypsy Moth (Lepidoptera: Lymantriidae) in the Appalachian Highlands

Gypsy moth, Lymantria dispar L., populations in six northern Virginia plots were aerially treated with the nucleopolyhedrosis virus product, Gypchek. Two applications of an aqueous Orzan LS-Pro Mo-Rhoplex B60A tank mix, each at 18.7 liters and 1.25 × 1012 polyhedral inclusion bodies per ha, reduced larvae by more than 92% and egg masses by more than 94% in all but one of the treated plots. Defoliation averaged 22% in Gypchek-treated plots compared to 67% in control plots.

Comparison of aerially-applied Gypchek strains against gypsy moth (Lepidoptera: Lymantriidae) in the presence of an Entomophaga maimaiga epizootic

The standard strain (LDP-226) of Gypchek®, a nucleopolyhedrovirus product registered by the USDA Forest Service against the gypsy moth, Lymantria dispar (L.), was compared against a strain, LdMNPV-203NL (NL = nonliquefying), that was developed for production in cell culture. Both strains were applied by air to U.S. government property in Prince Georges Co., MD, in early May 2003 at the rate of 1 x 1012 occlusion bodies per ha. The two goals of the study were (1) to compare the first and second wave effects of the two strains against gypsy moth populations; and (2) to delineate the combined effects of the applied virus and the expected epizootic of the gypsy moth specialist fungal entomopathogen Entomophaga maimaiga Humber, Shimazu, and Soper. Heavy rainfall in May and June preceded a massive epizootic of E. maimaiga, whose effects did not mask the first wave of viral mortality. When the effect of application sequence was considered, it was concluded that the two strains were equivalent in their first-wave impacts. High fungal-induced mid and late-season gypsy moth larval mortality suppressed the second wave of virus at all evaluation sites. There were no obvious differences in the second waves engendered by the two LdNPV strains in the greatly reduced late-instar larval population.

A comparison of the adaptations of strains of Lymantria dispar multiple nucleopolyhedrovirus to hosts from spatially isolated populations

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.