Intrastadial developmental resistance of third instar gypsy moths (Lymantria dispar L.) to L. dispar nucleopolyhedrovirus

Gypsy moth larvae become increasingly resistant to lethal infection by the Lymantria dispar M nucleopolyhedrovirus (LdMNPV) as they age within the fourth instar. Newly molted larvae are most sensitive to infection, mid-instars are least sensitive, and late-instars display intermediate sensitivity. This resistance occurs whether the virus is delivered orally or intrahemocoelically. The present study reveals a nearly identical pattern of resistance in third instar larvae. An LD₄₈ dose of polyhedra for newly molted third instars produced 18%, 10%, 8%, 25%, and 24% mortalities in larvae to which virus was orally administered at 12, 24, 48, 72, and 96 hours post-molt (hpm), respectively, which is a 6-fold reduction in mortality between newly molted larvae and mid-instars. An LD₄₄ dose of budded virus for newly molted third instars produced 33%, 23%, 17%, 31%, and 31% mortalities when injected into larvae that were 12, 24, 48, 72, and 96 hpm, respectively, which is a 2.6-fold reduction in mortality between newly molted larvae and mid-instars, indicating that approximately half of this resistance is midgut-based and half is systemically based. Doubling the viral dose did not overcome developmental resistance whether the virus was delivered orally or intrahemocoelically. In addition, time to death was significantly affected by the time post-molt at which the insect was inoculated with the virus. We suggest that intrastadial developmental resistance may affect both the ecology and management of the gypsy moth.     

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD₅₀ in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72 h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72 h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.     

Host specificity of microsporidia pathogenic to the gypsy moth, Lymantria dispar (L.): Field studies in Slovakia

Several species of microsporidia are important chronic pathogens of Lymantria dispar in Europe but have never been recovered from North American gypsy moth populations. The major issue for their introduction into North American L. dispar populations is concern about their safety to native non-target insects. In this study, we evaluated the susceptibility of sympatric non-target Lepidoptera to two species of microsporidia, Nosema lymantriae and Vairimorpha disparis, isolated from European populations of L. dispar and applied in field plots in Slovakia. Application of ultra low volume sprays of the microsporidia maximized coverage of infective spores in a complex natural environment and, thus, exposure of non-target species to the pathogens. Of 653 non-target larvae collected from plots treated with V. disparis in 2002, 18 individual larvae representing nine species in four families were infected. These plots were monitored for two subsequent seasons and V. disparis was not recovered from non-target species. Of 2571 non-target larvae collected in N. lymantriae-treated sites, one larva was found to be infected. Both species of microsporidia, particularly N. lymantriae, appear to have a very narrow host range in the field, even when an inundative technique is used for their introduction. V. disparis infections in L. dispar exceeded 40% of recovered larvae in the treated study sites; infection rates were lower in sites sprayed with N. lymantriae. Several naturally-occurring pathogens were recorded from the non-target species. The most common pathogen, isolated from 21 species in eight families, was a microsporidium in the genus Cystosporogenes.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.     

Effects of increased atmospheric CO2 on nutritional contents in poplar (Populus pseudo-simonii [Kitag.]) tissues and larval growth of gypsy moth (Lymantria dispar)

Changes in the concentrations of phytochemical compounds usually occur when plants are grown under elevated atmospheric CO₂. CO₂-induced changes in foliar chemistry tend to reduce leaf quality and may further affect insect herbivores. Increased atmospheric CO₂ also has a potential influence on decomposition because it causes variations in chemical components of plant tissues. To investigate the effects of increased atmospheric CO₂ on the nutritional contents of tree tissues and the activities of leaf-chewing forest insects, samples of Populus pseudo-simonii [Kitag.] grown in open-top chambers under ambient and elevated CO₂ (650 μmol mol^-1) conditions were collected for measuring concentrations of carbon, nitrogen, C : N ratio, soluble sugar and starch in leaves, barks, coarse roots (>2 mm in diameter) and fine roots (<2 mm in diameter). Gypsy moth (Lymantria dispar) larvae were reared on a single branch of experimental trees in a nylon bag with 1 mm 1 mm grid. The response of larval growth was observed in situ. Elevated CO₂ resulted in significant reduction in nitrogen concentration and increase in C : N ratio of all poplar tissues. In all tissues, total carbon contents were not affected by CO₂ treatments. Soluble sugar and nonstructural carbohydrate (TNC) in the poplar leaves significantly increased with CO₂ enrichment, whereas starch concentration increased only on partial sampling dates. Carbohydrate concentration in roots and barks was generally not affected by elevated CO₂, whereas soluble sugar contents in fine roots decreased in response to elevated CO₂. When second instar gypsy moth larvae consuming poplars grew under elevated CO₂ for the first 13 days, their body weight was 30.95% lower than that of larvae grown at ambient CO₂, but no significant difference was found when larvae were fed in the same treatment for the next 11 days. Elevated atmospheric CO₂ had adverse effects on the nutritional quality of Populus pseudo-simonii [Kitag.] tissues and the resultant variations in foliar chemical components had a significant but negative effect on the growth of early instar gypsy moth larvae.     

Physiological and Nutritional Effects of a Fluorescent Brightener on Nuclear Polyhedrosis Virus-Infected Lymantria dispar (L.) Larvae (Lepidoptera: Lymantriidae)

Recent studies with gypsy moths (Lymantria dispar L.) show that the fluorescent brightener, Tinopal LPW, acts synergistically with the L. dispar nuclear polyhedrosis virus (LdMNPV), lowering the 50% lethality concentration of the virus (LC₅₀) by 1000-fold. As a continuation of this work, we investigated the effects of ingestion of LdMNPV/Tinopal on larval midgut pH, hemolymph pH, and nutritional indices. Larval feeding for 48 h on diet treated with either LdMNPV or Tinopal did not affect midgut luminal pH, which averaged 10.3. In contrast, after 48 h of larval feeding on LdMNPV/ Tinopal diet, midgut pH averaged 8.5, and frass production, weight gain, and larval nutritional indices were significantly reduced. These effects appeared to be irreversible after the first 24 h of feeding ad libitum on LdMNPV/Tinopal diet, and the reduction in midgut pH was not due to feeding cessation per se. No significant effects on hemolymph pH were detected with any treatment. No treatment effects were observed when "SITS," a related fluorescent brightener, was substituted for Tinopal. The severe biological perturbations resulting from LdMNPV/Tinopal consumption are interpreted in light of current understanding of larval lepidopteran midgut physiology.     

The Lymantria dispar nucleopolyhedrovirus enhancins are components of occlusion-derived virus

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of approximately 84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.     

Quantifying horizontal transmission of Nosema lymantriae, a microsporidian pathogen of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in field cage studies

Nosema lymantriae is a microsporidian pathogen of the gypsy moth, Lymantria dispar that has been documented to be at least partially responsible for the collapse of L. dispar outbreak populations in Europe. To quantify horizontal transmission of this pathogen under field conditions we performed caged-tree experiments that varied (1) the density of the pathogen through the introduction of laboratory-infected larvae, and (2) the total time that susceptible (test) larvae were exposed to these infected larvae. The time frame of the experiments extended from the early phase of colonization of the target tissues by the microsporidium to the onset of pathogen-induced mortality or pupation of test larvae. Upon termination of each experiment, the prevalence of infection in test larvae was evaluated. In the experiments performed over a range of pathogen densities, infection of test larvae increased with increasing density of inoculated larvae, from 14.2 ± 3.5% at density of 10 inoculated per 100 larvae to 36.7 ± 5.7% at 30 inoculated per 100 larvae. At higher densities, percent infection in test larvae appeared to level off (35.7 ± 5.5% at 50 inoculated per 100 larvae). When larval exposure to the pathogen was varied, transmission of N. lymantriae did not occur within the first 15 d post-inoculation (dpi) (11 d post-exposure of test larvae to inoculated larvae). We found the first infected test larvae in samples taken 20 dpi (16 d post-exposure). Transmission increased over time; in the cages sampled 25 dpi (21 d post-exposure), Nosema prevalence in test larvae ranged from 20.6% to 39.2%.     

Four terpene synthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa

After herbivore damage, many plants increase their emission of volatile compounds, with terpenes usually comprising the major group of induced volatiles. Populus trichocarpa is the first woody species with a fully sequenced genome, enabling rapid molecular approaches towards characterization of volatile terpene biosynthesis in this and other poplar species. We identified and characterized four terpene synthases (PtTPS1-4) from P. trichocarpa which form major terpene compounds of the volatile blend induced by gypsy moth (Lymantria dispar) feeding. The enzymes were heterologously expressed and assayed with potential prenyl diphosphate substrates. PtTPS1 and PtTPS2 accepted only farnesyl diphosphate and produced (−)-germacrene D and (E,E)-α-farnesene as their major products, respectively. In contrast, PtTPS3 and PtTPS4 showed both mono- and sesquiterpene synthase activity. They produce the acyclic terpene alcohols linalool and nerolidol but exhibited opposite stereospecificity. qRT-PCR analysis revealed that the expression of the respective terpene synthase genes was induced after feeding of gypsy moth caterpillars. The TPS enzyme products may play important roles in indirect defense of poplar to herbivores and in mediating intra- and inter-plant signaling.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

Comparison of adherence, cytotoxicity, and Gal/GalNAc lectin gene structure in Entamoeba histolytica and Entamoeba dispar

The recent development of axenic culture for E. dispar allowed us to examine this ameba's ability to bind and lyse target cells and compare it to E. histolytica which has been axenically cultured for years. We found that under axenic conditions, E. dispar's adherence to target cells, ligand binding, and cytotoxicity were less than that of E. histolytica. These events were Gal/GalNAc-inhibitable supporting the idea that E. dispar expresses a lectin similar to E. histolytica. Genetic analysis showed that E. dispar had at least two members of the lectin heavy subunit family and four members of the lectin light subunit family that hybridized to ehhgl and ehlgl gene probes. A library screen produced clones which were isolated and sequenced. Derived amino acid sequences showed that the E. dispar heavy and light subunit clones were 86% and 79% identical, respectively, to their E. histolytica counterparts. In particular, the region which contains the epitopes for two adherence-enhancing monoclonal antibodies and a complement-sensitizing monoclonal antibody (amino acids 882–959 of the lectin heavy subunit) were conserved between the species.     

Comparative studies on the nuclear polyhedrosis viruses of Lymantria monacha and L. dispar

Immunodiffusion and tube precipitation tests, polyacrylamide gel electrophoresis of virus polypeptides, and cross-transmission experiments suggest that two nuclear polyhedrosis viruses, one from Lymantria monacha and one from L. dispar, are partially related to each other, but not identical. The virus particle proteins seem to be more specific than the polyhedron proteins.     

A new nucleopolyhedrovirus strain (LdMNPV-like virus) with a defective fp25 gene from Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan

A new multiple nucleopolyhedrovirus strain was isolated from casuarina moth, Lymantria xylina Swinhoe, (Lepidoptera: Lymantriidae) in Taiwan. This Lymantria-derived virus can be propagated in IPLB-LD-652Y and NTU-LY cell lines and showed a few polyhedra (occlusion bodies) CPE in the infected cells. The restriction fragment length polymorphism (RFLP) profiles of whole genome indicated that this virus is distinct from LyxyMNPV and the virus genome size was approximately 139 kbps, which was smaller than that of LyxyMNPV. The molecular phylogenetic analyses of three important genes (polyhedrin, lef-8 and lef-9) were performed. Polyhedrin, LEF-8 and LEF-9 putative amino acid analyses of this virus revealed that this virus belongs to Group II NPV and closely related to LdMNPV than to LyxyMNPV. The phylogenetic distance analysis was further clarified the relationship to LdMNPV and this virus provisionally named LdMNPV-like virus. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. In ultrastructural observations, the nuclei of the infected LD host cells contained large occlusion bodies (OBs), and few OBs, which presented as one or two OBs in a nucleus that was otherwise filled with free nuclocapsids and virions. We concluded that this LdMNPV-like virus is a new LdMNPV strain from L. xylina.     

Identification and Knockdown of the Olfactory Receptor (OrCo) in Gypsy Moth,Lymantria dispar

The gypsy moth, Lymantria dispar, is an important economic pest that causes large-scale damage to forests worldwide. Because of its important role in initiating and controlling insect behavior, olfaction—and olfaction-based pest management—has drawn increasing attention from entomologists. In this study, we identified the gene that encodes the olfactory receptor co-receptor (OrCo). Through amino acid sequence alignment, we found that LdisOrCo shares high identity with other OrCo proteins from different insect orders. Next, we performed RNA-interference (RNAi) to assess the role of OrCo in olfaction. Electroantennographic assays showed that after RNAi, the average value of males'' response to sex pheromones was 0.636 mV, significantly lower than that of the positive control (average = 1.472 mV). Females showed no response to sex pheromones before or after RNAi. Finally, quantitative PCR showed a strong decrease in the expression of OrCo after RNAi, by ~74% in males and by 23% in females relative to the positive controls. These results indicate that OrCo is not only critical to odor recognition, but it may also represent a new target for development of semiochemicals that can influence insect behavior.     

Dose dependency of time to death in single and mixed infections with a wildtype and egt deletion strain of Helicoverpa armigera nucleopolyhedrovirus

Recombinant insect nucleopolyhedroviruses lacking the egt gene generally kill their hosts faster than wild-type strains, but the response of insects to mixtures of virus genotypes is less well known. Here, we compared the survival time, lethal dose and occlusion body yield in third instar larvae of Helicoverpa armigera (Hübner) after challenge with wild-type H. armigera SNPV (HaSNPV-wt), a strain with a deletion of the egt gene, HaSNPV-LM2, and a 1:1 mixture of these two virus strains. A range of doses was used to determine whether the total number of OBs influenced the response to challenge with a mixture of virus strains versus single strains. At high virus doses, HaSNPV-LM2 killed H. armigera larvae significantly faster (ca. 20 h) than HaSNPV-wt, but at low doses, there was no significant difference in survival time between the viruses. The survival time after challenge with mixed virus inoculum was significantly different from and intermediate between that of the single viruses at high doses, and not different from that of the single viruses at low doses. No differences in lethal dose were found between single and mixed infections or between virus genotypes. The number of occlusion bodies produced per larva increased with time to death and decreased with virus dose, but no significant differences among virus types were found.     

Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera)

We developed a protocol for obtaining high yields (10-15 mg per 1100 ml of culture supernatant) of highly purified (up to 95%) Vip3Aa protein from HD-1 cultures. The protocol is based on acetone precipitation of supernatant protein, followed by HPLC fractionation (DEAE-5PW column) and several concentration steps. Our protocol resulted in higher yields and purity of Vip3Aa than a previously published method [Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a 353 novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of 354 activities against lepidopteran insects. Proc. Nat. Acad. Sci. USA 93, 5389-5394.]. This was achieved by using acetone rather than ammonium sulfate for precipitation of proteins from culture supernatants, and a shallow rather than a steep NaCl gradient for elution of the toxin, and by conducting all the purification steps at low temperature to prevent toxin degradation. In bioassays of the purified protein, Choristoneura fumiferana and Lymantria dispar larvae were less susceptible than Spodopteraexigua (10- and ∼100-fold, respectively). A B. thuringiensis var. kurstaki strain HD-1 from which the vip3Aa gene had been deleted (EG12414) showed reduced toxicity to S. exigua relative to the unmodified parental strain (EG2001), but not to L. dispar or C. fumiferana. We interpret these results as indicating that the Vip3Aa toxin does not contribute measurably to pathogenicity of HD-1 in these species.