41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

Venom from the endoparasitic wasp, Pimpla hypochondriaca, contains factors with anti-haemocyte and immunosuppressive properties. The gene for one such factor (vpb) has been cloned and recombinant protein produced. Bio-assays utilising VPB were performed and indicated that introduction of this immunosuppressive protein into the haemocoel of two lepidopteran pests, increases their susceptibility to the biological control agent, Bacillus thuringiensis. The potential for improving the efficacy of Bt through suppression of pest immune responses is discussed.

Venom from the endoparasitoid, Pimpla hypochondriaca contains a mixture of proteins. One of these was previously biochemically isolated and shown to have haemocyte anti-aggregation activity against host haemocytes in vitro. This protein shares significant homology to a second venom protein (VPB) from this parasitoid. The gene for VPB was amplified from a P. hypochondriaca venom gland cDNA library by PCR, cloned and expressed in E. coli. The presence of a fusion tag allowed purification. The purified immunosuppressive protein was found to have anti-haemocyte activity, in vitro, against haemocytes from two lepidopteran pests. This venom protein may have the potential to improve efficacy of biocontrol agents.

Spodoptera litura is a major pest attacking important commercial crops like cotton in India. Commercial Bt cotton hybrids carrying Cry1Ac toxin (Bollgard I type) or Cry1Ac and Cry2Ab toxins (Bollgard II type) give adequate control of the target insect Helicoverpa armigera, however there is a common opinion among farmers that S. litura is an emerging problem in Bt cotton. There is an urgent need to screen for more toxic holotype and/or hybrid Cry proteins against S. litura to minimise the use of chemical insecticides in Bt cotton, the main objective of transgenic cotton technology. In laboratory screening experiments G27, the EEC hybrid toxin producing strain, was more toxic than other holotypes (1Ca and 1Fa) and hybrids (AbAbC and AcAcC) tested.

Parasites can affect the outcome of biological invasions in different ways. Outbreaks of parasites may lead to host population crashes and resultant community change. But parasites do not only act on host population density. We present studies of short-term, behavioural effects of parasites and their effect on invasions. We focus on parasite regulation of crustacean invasions. Using empirical studies and mathematical modelling, we show that two parasites play keystone roles in UK amphipod invasions. Firstly, the microsporidian Pleistophora mulleri may facilitate invasion by two smaller species of amphipod; it has no direct effect on the survival of the native G. d. celticus, but infected animals are less likely to prey on the two smaller invaders. Secondly, the acanthocephalan Echinorynchus truttae may promote coexistence, as infection of the invading species Gammarus pulex reduces its predation on native G. d. celticus. Microsporidia may also drive crayfish invasions. We provide evidence from sequence data that the invading signal crayfish has acquired Thelohania contejeani (porcelain disease) from the native. However, whilst the invader may suffer little from the infection, transmission to the native can cause reduced activity and mortality and so increase the rate of extinction of this species.

RNA viruses are emerging as a serious threat to honey bee (Apis mellifera) health and are suspected as major contributors to the recent malady, Colony Collapse Disorder. Understanding the transmission of these viruses can shed valuable light on the epidemiology of this syndrome. In addition, the recent detection of Deformed Wing Virus in bumble bees as well as some of these viruses in in-hive food reserves of honey bees suggests a possible wider environmental spread of viruses with potential impact on the overall pollinator community. We studied the distribution of viruses in honey bees, their pollen loads and in other non-Apis hymenopteran pollinators collected from flowering plants. All the samples were analyzed with reverse transcriptase-PCR and virus identity was confirmed by sequencing. We report for the first time the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and blackened queen cell virus) in pollen pellets collected directly from forager bees. Furthermore, pollen pellets from some uninfected foragers were detected with virus, indicating a potential role of pollen in viral transmission. These viruses were found in eleven other species of native bees and wasps, expanding the known host range of these viruses and suggesting a possible deeper impact on the health of our ecosystem. Sequence comparisons of viruses isolated from honey bees, pollen and other non-Apis hymenopteran species indicate that the viruses are circulating freely among these species. In addition, the Israeli Acute Paralysis virus was detected in non-Apis pollinators near CCD apiaries but not in those near healthy non-CCD apiaries. Our findings increase the understanding of virus epidemiology and may help explain bee disease patterns and pollinator population decline.

Colony Collapse Disorder (CCD) was first recognized in 2006 in several beekeeping operations and presented symptoms previously not known.  Collapse of the colony occurred rapidly over a short time, with loss of most workers leaving behind the queen, a small number of newly emerged workers, and brood.  Analysis of remaining bees revealed large numbers of known pathogens in individual bees, without any one pathogen being linked to the symptoms.  This presentation will discuss the collaborative efforts to identify pathogens involved in CCD using a metagenomic analysis of bees taken colonies having identified CCD versus historical and healthy colonies. This analysis resulted in the identification of four pathogens that were strongly linked to CCD, the Israeli Acute Paralysis Virus, the Kashmir Bee Virus, Nosema ceranae, and Nosema apis.  Further examination of IAPV reveals that multiple lineages exist, with at least two being present in CCD colonies in the United States and Canada.  Additional studies will be described in which colonies were exposed to IAPV in containment greenhouses and symptoms observed.  Current questions will be discussed concerning the role of pathogens in Colony Collapse Disorder and bee health worldwide.

The use of transgenic Bt-maize is increasing yearly (last year accounting for about 19% of the total maize planted area in the world) because of the efficient control of the corn borers, in especial Ostrinia nubilalis . Resistance to Bacillus thuringiensis (Bt) insecticidal toxins has been linked to the 12-domain cadherin locus in 3 lepidopteran species. The O. nubilalis cadherin gene has been revealed as a complex gene of about 20 kbp in length, with 34 introns. In the present work, we have studied the size polymorphism of the gene in a Spanish population, by amplifying the genomic sequence of the gene in 16 overlapping regions. The variability observed was not uniformly distributed, with a maximum in region 14 and a minimum (no polymorphism) in region 4. All this size variability must be due to changes in the intronic regions because we found no detectable size differences in mRNA. This variability can be useful to select appropriate polymorphic regions to be used as markers of this gene in experiments such us to determine the genetic linkage of the cadherin to Bt resistance traits.

The Western Corn Root Worm (Diabrotica v. virgifera) first time occurred in Germany in July 2007. Four different maize cultivars including Diabrotica-resistant MON88017, were assessed in respect to its effects on saprophagous Diptera and predators out of Carabidae and Staphylinidae. The methodological approach comprised a hierarchic order of different ecological scale levels (agro-ecosystem, population, organisms). Abundance and species composition of both Diptera and their predators were recorded in the field. Most saprophagous Diptera belong to Sciaridae (fungus gnats), of which the predominant Lycoriella castanescens was used for feeding trials. It was tested whether mortality, pupation, hatching rates, duration of larval development and pupation were affected by uptake of Cry3Bb1-contminated plant tissues. Species of Carabidae and Staphylinidae were fed with Sciaridae-larvae reared on Bt- and non-Bt-maize-litter respectively. In a similar way Diabrotica-larvae were offered as prey. Toxin analyses of saprophagous Diptera and predators reared with Bt-plant parts or feeding on Bt-contaminated prey contained Bt-toxin up to 1.6% (decomposers) and 14.0% (predators) of the toxin level recorded in the source material. Predators collected from Bt-maize fields stated these findings. Thus, Bt-toxin is transferred into the food chain. Predators feeding on prey containing Cry3Bb1-toxin showed a significant delay in accepting the prey in comparison to prey free of Bt-toxin, but this didn't result in higher mortality or less longevity. However, predators which were fed with Sciaridae-larvae containing Bt-toxin produced significantly less offspring than those feeding on prey reared with non-Bt-maize litter. Thus, an uptake of Cry3Bb1-toxin by carnivorous beetles doesn't lead to a higher mortality, but results in subtle effects like lower fertility of the females.

Viral disease is a major component of the cyclic population dynamics of some Lepidoptera including western tent caterpillars. Epizootics of nucleopolyhedrovirus and host population subdivision provide an arena in which selection on virulence of virus and resistance of hosts could act. Theory predicts that epizootics should select for host resistance and that viral isolates should respond to this change on a population-by-population basis. Experiments provide evidence that these interactions are occurring but that patterns are weak as compared to other factors that determine the cyclic population dynamics. In addition there is no evidence for induced immunity or selection within a generation of tent caterpillars. The factors that promote the rapid development of NPV epizootics remain a mystery and are the topic of future research.

The value in having Bt products with consistent performance and confirmed safety is an increase in consumer confidence, ultimately leading to increased use and demand of Bt products and potentially other biological control agents. However, poor quality control in even one company can damage the reputation of Bt’s and microbials in general. Assuring biopotency of products by using reliable standards is important, but is only one aspect of QC. At Valent BioSciences, quality control measures are applied at all stages of manufacturing, from strain identity to packaging and distribution of the final product. Recently, Bt products have been appearing in the worldwide market that demonstrate an obvious lack of quality control, even though biopotency may be met. In some cases the products have been misrepresented or adulterated. Thus, in addition to implementing high standards of quality control, it is in the interest of the entire biopesticide industry to provide stewardship for all products on the market.