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.     

Reduction in fitness of female Asian longhorned beetle (Anoplophora glabripennis) infected with Metarhizium anisopliae

Bioassays were conducted to document the effects of Metarhizium anisopliae infection on adult female Asian longhorned beetle (Anoplophora glabripennis) reproduction before death and subsequent survival of offspring. The effect of infection on fecundity was evaluated for females already laying eggs and for newly eclosed females using M. anisopliae isolates ARSEF 7234 and 7711, respectively. Decreased longevity and oviposition compared with controls were observed in females that were already laying eggs when exposed to M. anisopliae ARSEF 7234. Newly eclosed females exposed to M. anisopliae ARSEF 7711 displayed shortened longevity (10.0 ± 0.7 days vs 74.3 ± 6.8 days for controls) and decreased oviposition (1.3 ± 0.7 eggs per ARSEF 7711-exposed female vs 97.2 ± 13.7 eggs per female for controls) compared with controls. Percentages of eggs that did not hatch were greater for both groups of fungal-treated females compared with controls and 60.0% of unhatched eggs contained signs of fungal infection. The percentage of larvae dying within 9 weeks of oviposition was higher for sexually mature females exposed to ARSEF 7234 compared with controls and >40% of dead larvae displayed signs of fungal infection. Thus, for both stages of females and both fungal isolates, fewer surviving larvae were produced after female fungal infection compared with controls. M. anisopliae infection affects female fitness by decreasing female longevity, by decreasing female oviposition before death and through horizontal transmission of M. anisopliae to offspring.     

Vertical transmission and overwintering of microsporidia in the gypsy moth, Lymantria dispar

Vertical transmission and the overwintering success of three different microsporidia infecting Lymantria dispar (Lepidoptera: Lymantriidae) larvae were investigated. Endoreticulatus schubergi, a midgut pathogen, was transmitted to offspring via female and male via the egg chorion (transovum transmission). Between 8% and 29% of the emerging larvae became infected. No spores of E. schubergi were found in surface-washed eggs. Nosema lymantriae, a microsporidium that causes systemic infections, was transovarially transmitted. Between 35% and 72% of the progeny were infected. Vairimorpha disparis, a fat body pathogen, was not vertically transmitted. The infectivity of spores that overwintered in cadavers of infected L. dispar varied by species, placement in the environment, and weather conditions. Spores of E. schubergi were still infective after an eight month exposure period of cadavers on the ground. Spores of N. lymantriae and V. disparis remained highly infective only when cadavers overwintered under a more or less continuous snow cover for four months.     

Identification of a new nucleopolyhedrovirus from naturally-infected Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae) larvae on poplar plantations in South Brazil

A baculovirus was isolated from larvae of Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae), a pest of a forest species known as Poplar (family Salicaceae, genus: Populus) with high economic value. Electron microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra containing multiple nucleocapsids per envelope. This baculovirus was thus named Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV) and characterized by its DNA restriction endonuclease pattern, polyhedral protein, viral protein synthesis, and infectivity in insect cell lines. Restriction endonuclease profiles of viral DNA digested with five restriction enzymes were obtained and the CoveMNPV genome size was estimated to be 81 ± 2.5 kbp. The isolation of the polyhedra (OBs) was done from the crude extract of infected larvae by ultracentrifugation through sucrose gradients. These viral particles were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed a strong band with approximately 33 kDa, corresponding to the main protein of the occlusion bodies (polyhedrin). Also, a similar band was observed for CoveMNPV infected Spodoptera frugiperda cells (SF-21 AE) pulse-labeled with [³⁵S] methionine and fractionated by SDS-PAGE. Of the four insect cell lines tested for susceptibility to CoveMNPV infection, the SF-21 AE was the most susceptible with occlusion bodies produced in most of the inoculated cells. This is the first record of an NPV from C. vestigialis.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

Helicoverpa armigera (Lepidoptera: Noctuidae) larvae that survive sublethal doses of nucleopolyhedrovirus exhibit high metabolic rates

To determine the effect of sublethal doses of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) on the metabolic rate of H. armigera, the respiration rates of third instar H. armigera larvae inoculated with sublethal doses of HearSNPV were evaluated. Respiration rates, measured as the rate of CO₂ production (VCO₂), were recorded daily using closed-system respirometry. By 4 days post-inoculation (dpi), the metabolic rates of LD₂₅ or LD₇₅ survivors were significantly higher than that of uninoculated controls. When dose data were pooled, the VCO₂ values of larvae that survived inoculation (0.0288 ml h⁻¹), the uninoculated controls (0.0250 ml h⁻¹), and the larvae that did not survive inoculation (0.0199 ml h⁻¹) differed significantly from one another. At 4 dpi, the VCO₂ of the uninoculated controls were significantly lower than the VCO₂ of inoculation survivors, but significantly higher than the VCO₂ of inoculation non-survivors. Inoculation survivors may have had high metabolic rates due to a combination of viral replication, organ damage, and an energy-intensive induced cellular immune response. The high 4 dpi metabolic rate of inoculation survivors may reflect an effective immune response and may be seen as the metabolic signature of larvae that are in the process of surviving inoculation with HearSNPV.     

Non-target impacts of soybean rust fungicides on the fungal entomopathogens of soybean aphid

Soybean aphid, Aphis glycines, has caused serious economic damage to soybean across the North Central US since its introduction to North America in 2000. The management of another invasive soybean pest, Asian soybean rust, Phakopsora pachyrhizi, using foliar fungicide applications has the potential to impact soybean aphid populations by suppressing beneficial fungal entomopathogens. In 2005 and 2006, we applied recommended soybean rust fungicide treatments, consisting of strobilurin and triazole fungicides, to small soybean plots in two locations to assess if such applications might suppress aphid fungal epizootics. In Lamberton, MN, in 2005, during the epizootic, fungicide-treated plots averaged 2.0 ± 0.7% (mean ± SE) disease prevalence while untreated plots averaged 14.2 ± 5.6%. In 2007, we applied strobilurin and strobilurin-triazole mix fungicides to single-plant microplots either before or after release of Pandora neoaphidis, the most commonly observed aphid pathogen in 2005 and 2006. Treatments that contained a mixture of two active ingredients significantly lowered peak and cumulative aphid disease prevalence in both early and late reproductive stage soybeans indicating that fungicide mixtures used to manage soybean rust can negatively impact an aphid-specific fungal pathogen. However, no consistent soybean aphid population response was observed in these studies of low levels of aphid fungal infection.     

Distribution and residual activity of two insecticidal proteins, avidin and aprotinin, expressed in transgenic tobacco plants, in the bodies and frass of Spodoptera litura larvae following feeding

To understand how a major cosmopolitan pest responds to two very different insecticidal proteins and to determine whether herbivorous insects and their frass could be environmental sources of recombinant proteins from transgenic plants, Spodoptera litura (Fab.) (Lepidoptera, Noctuidae) larvae were fed on tobacco leaves expressing either the biotin-binding protein, avidin, or the protease inhibitor, aprotinin. Control larvae received non-transgenic tobacco. Samples of larvae were taken after 5, 6 or 7 days’ feeding and frass was collected after two 24-h periods at 6 and 7 days. Insects in all treatments grew significantly during the experiment, but the avidin-fed larvae were significantly smaller than the others on Day 7. Avidin was found in all samples of avidin-fed larvae (7.0±0.86 ng mg⁻¹, n=45), at a lower level than in their frass (31.9±5.08 ng mg⁻¹, n=30), and these frass levels were lower than those of the the leaves fed to the larvae (69.0±6.71 ng mg⁻¹, n=45). All of the avidin detected in these samples was capable of binding biotin. On average, between 10 and 28% of avidin was recovered with the methods used, whereas almost full recovery of aprotinin was effected. Aprotinin levels in larvae (8.2±0.53 ng mg⁻¹, n=45) were also lower than aprotinin levels in frass (77.4±6.9 ng mg⁻¹, n=30), which were somewhat lower than those in the leaves fed to the larvae (88.6±2.51 ng mg⁻¹, n=45). Approximately half the trypsin-binding ability of aprotinin was lost in larvae, and in frass, aprotinin had lost about 90% of its ability to bind trypsin.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Modeling horizontal transmission of microsporidia infecting gypsy moth,Lymantria dispar (L.), larvae

We developed a simulation model that describes the horizontal transmission of three different microsporidia, Endoreticulatus schubergi, Nosema lymantriae and Vairimorpha disparis and their insect host, the gypsy moth, Lymantria dispar. The model describes the stage specific development and mortality of uninfected, latently infected or infectious hosts, the food consumption, the infection by spore-laden feces of E. schubergi and N. lymantriae and by spore-laden cadaver of N. lymantriae and V. disparis. Model results were compared to percent infection of L. dispar test larvae published in earlier studies using caged oak trees and potted oak-plants. When feces were selected as the source of spores for transmission of E. schubergi or N. lymantriae, the model estimated a percent infection in susceptible larvae that was in the range of the experimental studies. When spore-laden cadavers were the source of spores of N. lymantriae or V. disparis, the model did not correctly predict the experimentally measured percent infection in susceptible larvae. The most critical points of the simulation model are exact calculation of spore release, mortality and exact determination of the transmission coefficients when cadavers were included as a source for microsporidian infection.     

Effectiveness and immunomodulation of chemotherapeutants against scuticociliate Philasterides dicentrarchi in olive flounder

Philasterides dicentrarchi is a histophagous scuticociliate causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis due to P. dicentrarchi infections was increased from May to July (40 ± 3.1% to 79.4 ± 1.7%) and it decreased from August to November (63 ± 2.3% to 30 ± 2.6%) in olive flounder farms at Jeju Island, South Korea during 2000-2006. The prevalence of mixed infection along with Vibrio spp. bacterial infection was 49 ± 7.2% than that of other mixed infection. At present no effective control measure for P. dicentrarchi infection has been described and large production losses continue. In the present study, formalin, hydrogen peroxide and Jenoclean chemotheraputants were used for bath treatment. Among Jenoclean at a low concentration of 50 ppm proved effective. The results were confirmed with in vitro motility assessments and morphological changes scoring system in P. dicentrarchi. On the other hand, similar trend was noted following hydrogen peroxide treatment at this concentration, but formalin was only moderately effective. Either hydrogen peroxide or Jenoclean are the promising compounds effective at low concentrations with short application time for P. dicentrarchi. Therefore, these substances were evaluated on day 10, 20 and 30 for their ability to enhance innate immune response and disease resistance against P. dicentrarchi in olive flounder after chemotheraputants bath treatment with 100 ppm for 30 min per day. All the tested immune parameters were enhanced by treatment with Jenoclean, but not formalin and hydrogen peroxide. These findings suggest that Jenoclean bath treatment can be used for ensuring the heath of cultured marine fish against internal parasites such as P. dicentrarchi.     

Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus

The potential of a strain of Beauveria bassiana (Ascomycota: Clavicipitaceae) obtained from a naturally infected Rhynchophorus ferrugineus (Coleoptera: Curculionidae) pupa as a biological control agent against this weevil was evaluated both in the laboratory and in semi-field assays. Laboratory results indicate that this strain of B. bassiana can infect eggs, larvae and adults of R. ferrugineus (LC₅₀ from 6.3 × 10⁷ to 3.0 × 10⁹ conidia per ml). However, mortality was not the only indicator of treatment efficacy because adults of either sex inoculated with the fungus efficiently transmitted the disease to untreated adults of the opposite sex, with male-to-female and female-to-male rates of transmission of 55% and 60%, respectively. In addition, treatment with B. bassiana significantly reduced fecundity (up to 62.6%) and egg hatching (32.8%) in pairing combinations with fungus-challenged males, females or both sexes. Likewise, 30-35% increase in larval mortality was observed in larvae obtained from eggs from fungus-challenged females or from untreated females coupled with inoculated males, resulting in an overall 78% progeny reduction. Semi-field preventive assays on potted 5-year old Phoenix canariensis palms, with efficacies up to 85.7%, confirmed the potential of this strain as a biological control agent against R. ferrugineus.     

Stage dependent habitat use under conflicting predation pressure: An experimental test with larval and juvenile two-spotted gobies, Gobiusculus flavescens Fabricius

The effect of size, predator types and presence of multiple predators on the microhabitat use of larvae and juveniles of a sublittoral, semipelagically schooling fish, the two-spotted goby (Gobiusculus flavescens), was tested in two experiments. Larvae (15 and 25 days old, Experiment I) and juveniles (mean ± 1 S.E.: small, 15.9 mm ± 1.28; medium, 19.2 mm ± 1.43; and large, 23.4 mm ± 1.67, Experiment II) were allowed to choose between two sections of the tanks; an upper, representing a water column habitat, and a lower, artificially vegetated, representing the hyperbenthic habitat. Position of larvae or juveniles and the activity level of juveniles were recorded. Predator treatments were: (I) no predators (control), (II) a pelagic predator, the jellyfish Aurelia aurita L., (III) a hyperbenthic predator, the mysid Praunus flexuosus O.F. Müller or (VI) both predator types simultaneously. In Experiment I predators were restricted to the habitat which they were chosen to represent, while goby larvae could move freely. In Experiment II both predators and juvenile gobies were allowed to move freely between compartments.Increasing age caused larval gobies, but not juveniles to shift downwards. Only 25-day-old larvae and small juveniles avoided the mysid by shifting upwards. Larval response to A. aurita was also size dependant: 25-day-old larvae avoided medusae by shifting downwards, while 15 day olds did not. Emergent multiple predator effects were found for the vertical distribution of 15-day-old larvae and small juveniles. Larger juveniles were more active than smaller, both in the upper and the lower sections of tanks. P. flexuosus caused juvenile gobies in their vicinity (i.e. in the lower section) to increase their activity level, while small juveniles (but not medium-sized or large) increased their activity level even when further away (i.e. in the upper section). The presence of A. aurita led to a reduction in activity of small juveniles in its vicinity (i.e. in the upper section), while no response was observed among older juveniles or juveniles further away from the predator (i.e. in the lower section). Emergent multiple predator effects on the activity level of juveniles were not observed.     

Ultrastructure and molecular characterization of a microsporidium, Tubulinosema hippodamiae, from the convergent lady beetle, Hippodamia convergens Guérin-Méneville

Hippodamia convergens, the convergent lady beetle, is available for aphid control in home gardens and in commercial food production systems throughout the United States and Canada. Beetles received from commercial insectaries for biological control are occasionally infected with a microsporidium. The objective of this study was to describe the pathogen by means of ultrastructure, molecular characterization and tissue pathology. All stages of the microsporidium were in direct contact with the host cell cytoplasm. Early developmental stages were proximal to mature spores and both were observed throughout the tissue sections that were examined. Merogony resulted from binary fission. Early-stage sporoblasts were surrounded by a highly convoluted plasma membrane and contained an electron-dense cytoplasm and diplokaryon. Ovoid to elongated late-stage sporoblasts were surrounded by a relatively complete spore wall. The polar filament, polaroplast, and anchoring disk were readily observed within the cell cytoplasm. Mature spores were typical of terrestrial microsporidia, with a thickened endospore surrounded by a thin exospore. Spores contained well-defined internal structures, including a diplokaryon, lamellar polaroplast and a slightly anisofilar polar filament with 10-14 coils arranged in a single or double row. A prominent indentation was evident at the apical end of the spore wall proximal to the anchoring disk. Aberrant spores were also observed. These had a fully developed endospore and exospore but lacked any discernable internal spore structures, and were, instead, filled with lamellar or vesicular structures. Typical and aberrant spores measured 3.58 ± 0.2 × 2.06 ± 0.2 μm (n = 10) and 3.38 ± 0.8 × 2.13 ± 0.2 μm (n = 10), respectively. Spores were observed in longitudinal muscle surrounding the midgut and within the fat body, Malpighian tubules, pyloric valve epithelium, ventral nerve cord ganglia, muscles and ovaries. The hindgut epithelium was often infected but the connective tissues were rarely invaded. The life cycle and pathology of the microsporidium bears some resemblance to Nosema hippodamiae, the only microsporidium reported from H. convergens by Lipa and Steinhaus in 1959. Molecular characterization of the pathogen genomic DNA revealed that it is 99% similar to Tubulinosema acridophagus and T. ratisbonensis, two pathogens that infect Drosophila melanogaster and 98% similar to T. kingi from D. willistoni. Based on similarities in pathogen ultrastructure and the molecular information gained during this study, we propose that the microsporidium in H. convergens be given the name Tubulinosema hippodamiae.     

Nutritional value and intake of aquatic ferns (Azolla filiculoides Lam. and Salvinia molesta Mitchell.) in sows

Aquatic ferns (AFs) such as Azolla filiculoides and Salvinia molesta are grown on swine lagoons in the tropics and used in diets for pigs. The present work is aimed at evaluating their potential as feed ingredients for sows. When presented with ad libitum AFs, gilts weighing 110 ± 14 kg (mean ± SD), were able to ingest 9.1–9.7 kg fresh AF per day (from 597 to 630 g dry matter (DM) per day) and from 1240 to 1428 g DM per day when presented in a dry, ground form. A digestibility study was conducted, using sows weighing 213 ± 9 kg (mean ± SD), which were fed diets containing maize, soybean meal and 0, 150 or 300 g AF kg⁻¹ diet. The presence of AFs had a negative impact on the faecal digestibility of the crude protein, NDF and energy content of the whole diet (P<0.001) and on the ileal protein digestibility, especially with 300 g AFs kg⁻¹ diet. The level of AFs in the diet had no effect on stomach weight (P>0.05) but increased the weight of the rest of the gastrointestinal tract (P<0.001). The rate of AF fibre fermentation in the pig large intestine was measured using an in vitro gas test. The rates were much lower than tropical tree foliage, which can also be used in pig diets in the tropics. This could partly explain the low apparent digestibility of AFs in pigs. In conclusion, the inclusion level of AFs in rations for sows should be limited to 150 g AFs kg⁻¹ diet due to the low digestibility and energy density, as well as the negative impact on the digestibility of the whole diet.     

Entomopathogenic fungi in cornfields and their potential to manage larval western corn rootworm Diabrotica virgifera virgifera

Entomopathogenic ascomycete fungi are ubiquitous in soil and on phylloplanes, and are important natural enemies of many soil-borne arthropods including larval western corn rootworm, Diabrotica virgifera virgifera, which is a major pest of corn. We measured the prevalence of Beauveria bassiana and Metarhizium anisopliae sensu lato in ten cornfields in Iowa, USA by baiting with larval insects. B. bassiana and M. anisopliae s.l. were present in 60% ± 6.3% and 55% ± 6.4% of soil samples, respectively. Subsequent laboratory bioassays found that some M. anisopliae s.l. strains collected from cornfields killed a greater proportion of D.v. virgifera larvae than a standard commercial strain.     

Response of Lymantria dispar L. (Lepidoptera: Lymantriidae) to Bacillus thuringiensis subsp. kurstaki at different ingested doses and temperatures

We examined mortality and feeding inhibition response of Lymantria dispar L. (Lepidoptera: Lymantriidae) larvae to ingested doses of Bacillus thuringiensis subsp. kurstaki as a function of dose, instar and temperature. We developed generalized (logistic) linear mixed models and a mixture survival model, commonly used in medical statistics, to analyze the complex data set. We conducted bioassays of Foray 48B with larvae from the NJSS laboratory stock, using droplet imbibing or force-feeding to ensure dose ingestion. The dose causing mortality in 50% of the test population (LD₅₀) under standard test conditions (22 °C) ranged from 0.019 International Units (IU)/larva for first instar larvae (L₁) to 1.6 IU/larva for L₄. Temperature affected larval mortality in two ways. Mortality occurred sooner and progressed more rapidly with increasing temperature (13-25 °C) at each dose level and instar, while the maximum level of mortality attained by each instar decreased with increasing rearing temperature. The mechanisms underlying this effect are being investigated. Larvae that survived exposure to B. thuringiensis resumed feeding after a period that was dependent on instar, dose, and temperature. The equations describing observed mortality and feeding recovery responses were used to construct a simulation model, which was able to predict both processes, and which forms the basis for a process-oriented model that can be used as a decision support tool in aerial sprays.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.