We report factors that are limiting for Ascosphaera apis growth: water activity (a w ) and temperature. Features of A. apis were a high water activity, 0.85–0.90 a w at 25/30 °C and 0.90–0.95 a w at 35 °C. A minimum water activity of 0.95 a w was required to produce spores. None of these characteristics changed from cycling or descending/ascending between low and high water activity media. Growth rates varied between isolates. This new study provides the biological data for monitoring and early prediction of disease outbreaks. Beekeepers should note that A. apis has a high water activity for spore production. Healthy bees that keep the internal brood area hot and dry can critically limit incidence of infection and transmission via spores.

Nosema ceranae infection is ubiquitous in western honey bees, Apis mellifera, in the United States and the pathogen has apparently replaced Nosema apis in colonies nationwide. Displacement of N. apis suggests that N. ceranae has competitive advantages but N. ceranae was significantly less infective and less virulent than N. apis in commercially available lineages of honey bees in studies conducted in Illinois and Texas. At 5 days post eclosion, the most susceptible age of adult bees tested, the mean ID50 for N. apis was 359 spores compared to 3217 N. ceranae spores, a nearly 9-fold difference. Infectivity of N. ceranae was also lower than N. apis for 24-h and 14-day worker bees. N. ceranae was less infective than reported in studies using European strains of honey bees, while N. apis infectivity, tested in the same cohort of honey bees, corresponded to results reported globally from 1972 to 2010. Mortality of worker bees was similar for both pathogens at a dosage of 50 spores and was not different from the uninfected controls, but was significantly higher for N. apis than N. ceranae at dosages ⩾500 spores. Our results provide comparisons for evaluating research using different ages of bees and pathogen dosages and clarify some controversies. In addition, comparisons among studies suggest that the mixed lineages of US honey bees may be less susceptible to N. ceranae infections than are European bees or that the US isolates of the pathogen are less infective and less virulent than European isolates.

Chronic bee paralysis virus (CBPV) is an important viral disease of adult bees which induces significant losses in honey bee colonies. Despite comprehensive research, only limited data is available from experimental infection for this virus. In the present study winter worker bees were experimentally infected in three different experiments. Bees were first inoculated per os (p/o) or per cuticle (p/c) with CBPV field strain M92/2010 in order to evaluate the virus replication in individual bees. In addition, potential synergistic effects of co-infection with CBPV and Nosema ceranae (N. ceranae) on bees were investigated. In total 558 individual bees were inoculated in small cages and data were analyzed using quantitative real time RT-PCR (RT-qPCR). Our results revealed successful replication of CBPV after p/o inoculation, while it was less effective when bees were inoculated p/c. Dead bees harbored about 1,000 times higher copy numbers of the virus than live bees. Co-infection of workers with CBPV and N. ceranae using either method of virus inoculation (p/c or p/o) showed increased replication ability for CBPV. In the third experiment the effect of inoculation on bee mortality was evaluated. The highest level of bee mortality was observed in a group of bees inoculated with CBPV p/o, followed by a group of workers simultaneously inoculated with CBPV and N. ceranae p/o, followed by the group inoculated with CBPV p/c and the group with only N. ceranae p/o. The experimental infection with CBPV showed important differences after p/o or p/c inoculation in winter bees, while simultaneous infection with CBPV and N. ceranae suggesting a synergistic effect after inoculation.

Background: Chronic infections can profoundly affect the physiology, behavior, fitness and longevity of individuals, and may alter the organization and demography of social groups. Nosema apis and Nosema ceranae are two microsporidian parasites which chronically infect the digestive tract of honey bees (Apis mellifera). These parasites, in addition to other stressors, have been linked to increased mortality of individual workers and colony losses in this key pollinator species. Physiologically, Nosema infection damages midgut tissue, is energetically expensive and alters expression of immune genes in worker honey bees. Infection also accelerates worker transition from nursing to foraging behavior (termed behavioral maturation). Here, using microarrays, we characterized global gene expression patterns in adult worker honey bee midgut and fat body tissue in response to Nosema infection.Results: Our results indicate that N. apis infection in young workers (1 and 2 days old) disrupts midgut development. At 2 and 7 days post-infection in the fat body tissue, N. apis drives metabolic changes consistent with energetic costs of infection. A final experiment characterizing gene expression in the fat bodies of 14 day old workers parasitized with N. apis and N. ceranae demonstrated that Nosema co-infection specifically alters conserved nutritional, metabolic and hormonal pathways, including the insulin signaling pathway, which is also linked to behavioral maturation in workers. Interestingly, in all experiments, Nosema infection did not appear to significantly regulate overall expression of canonical immune response genes, but infection did alter expression of acute immune response genes identified in a previous study. Comparative analyses suggest that changes in nutritional/metabolic processes precede changes in behavioral maturation and immune processes.Conclusions: These genome-wide studies of expression patterns can help us disentangle the direct and indirect effects of chronic infection, and understand the molecular pathways that regulate disease symptoms.

American foulbrood is one of the most devastating diseases of the honey bee. It is caused by the spore-forming, Gram-positive rod-shaped bacterium Paenibacillus larvae. The recent updated genome assembly and annotation for this pathogen now permits in-depth molecular studies. In this paper, selected techniques and protocols for American foulbrood research are provided, mostly in a recipe-like format that permits easy implementation in the laboratory. Topics covered include: working with Paenibacillus larvae, basic microbiological techniques, experimental infection, and "'omics" and other sophisticated techniques. Further, this chapter covers other technical information including biosafety measures to guarantee the safe handling of this pathogen.

We developed a new method for detection of the intracellular parasite, Nosema ceranae, one of the most economically devastating pathogens of the honeybee.

We investigated the effect of the parasitic mite Varroa destructor on the immunological and nutritional condition of honey bees, Apis mellifera, from the perspective of the individual bee and the colony. Pupae, newly-emerged adults and foraging adults were sampled from honey bee colonies at one site in S. Texas, USA. Varroa‑infested bees displayed elevated titer of Deformed Wing Virus (DWV), suggestive of depressed capacity to limit viral replication. Expression of genes coding three anti-microbial peptides (defensin1, abaecin, hymenoptaecin) was either not significantly different between Varroa-infested and uninfested bees or was significantly elevated in Varroa-infested bees, varying with sampling date and bee developmental age. The effect of Varroa on nutritional indices of the bees was complex, with protein, triglyceride, glycogen and sugar levels strongly influenced by life-stage of the bee and individual colony. Protein content was depressed and free amino acid content elevated in Varroa-infested pupae, suggesting that protein synthesis, and consequently growth, may be limited in these insects. No simple relationship between the values of nutritional and immune-related indices was observed, and colony-scale effects were indicated by the reduced weight of pupae in colonies with high Varroa abundance, irrespective of whether the individual pupa bore Varroa.

This chapter was inspired by rapid developments in the field of RNA interference (RNAi), an evolutionarily-conserved cellular mechanism that directs protection against nucleic-acid invaders (e.g., viruses and repetitive DNA sequences including transposable elements) in plants, animals,protozoans and fungi. It is now evident that prokaryotes also possess an RNA-based defense system, though completely distinct from that in eukaryotes. The recent discovery of post-transcriptional gene silencing (PTGS) has generated tremendous interest in basic and applied research, including the development of in vitro and in vivo therapeutic approaches to reduce expression of disease-associated genes. This cutting-edge technology has already been successfully used in genomic manipulations of insect disease vectors, such as development of Dengue-resistant mosquitoes (Blair et al., 2006; Franz et al., 2006 ; Mathur et al., 2010). Our aim in this chapter is to provide an overview of the profound knowledge accumulated in recent years from invertebrate RNAi studies, but with a focus on agriculturally important arthropods. We start with a brief discussion of the RNAi mechanism to introduce readers to key concepts that underlie the practical application of RNAi discussed in the remainder of the chapter. Our discussion will include a number of important issues that should be carefully considered when working with multi-cellular organisms, including bidirectional transport of silencing signals, processing of dsRNA, homology-driven mRNA degradation and subsequent gene silencing. We will look at examples of gene silencing in different arthropod systems to illustrate commonalities and differences found in their silencing machinery. Arthropods are a diverse group of organisms, including ticks, mites, spiders, crabs, and insects, many of which are economically significant pests of agricultural crops, parasites of commercially managed pollinators and vectors of livestock diseases. Considering the huge economic impact of honey bees on crop production, and particularly on pollination of specialty crops, a substantial part of this chapter will be dedicated to honey bee research. Recently, catastrophic losses of honey bee colonies drew national media attention and ignited a renewed interest in basic and applied bee research (Aronstein et al., 2006; Campbell et al., 2010; Dearden et al., 2009; Maori et al., 2009a, 2009b; Nelson et al., 2007). The latest achievements in basic honey bee research provide a better understanding of bee physiology and behavior. Analysis of gene function by selective gene silencing has been

Nosema ceranae, a honey bee pathogen now known worldwide, may be detected quickly by either light microscopy or by antibody tests. While these tests are less sensitive than polymerase chain reaction, they may be more practical for routine diagnosis. Phase contrast light microscopy allows one to distinguish between primary, environmental, and germinated spores. Fluorescent stains may demonstrate the maturity of spores and the integrity of the spore membranes. Polar filaments from the spores are also seen by proper microscopic techniques. Antibodies are also helpful in identifying Nosema spores and can be specific to the Nosema ceranae species. The antibodies attach to protein in the wall of the spore. This test is able to detect an infestation as few as 1,000 spores, a tiny fraction of the spores present in a highly infected bee. We hope to see this test commercialized so that it is available to beekeepers.

A new genomic antibody (Ab) has been developed against a spore-wall protein SWP-32 of the honey bee intracellular pathogen, Nosema ceranae. In dot blots and Western blots this Ab specifically recognized N. ceranae spore antigens and did not cross-react with N. apis spore lysates, unless blots were overdeveloped. The detection sensitivity depends on both the concentration of the anti-SWP-32 Ab and the concentration of Nosema spores in the lysates. To avoid non-specific staining, we suggest using this new Ab at 1: 5000 dilution for detection of 1x10(3) and higher spore numbers per assay. Considering that a single infected bee can produce over 50 x 10(6) spores, this level of sensitivity will allow detection of a very low level of Nosema infection in bee colonies.

Diseases and other stress factors working synergistically weaken honey bee health and may play a major role in the losses of bee populations in recent years. Among a large number of bee diseases, chalkbrood has been on the rise. We present here the experimental identification of honey bee genes that are differentially expressed in response to infection of honey bee larvae with the chalkbrood fungus, Ascosphaera apis.

Chalkbrood is a fungal disease of honey bee brood caused by Ascosphaera apis. This disease is now found throughout the world, and there are indications that chalkbrood incidence may be on the rise. In this review we consolidate both historic knowledge and recent scientific findings. We document the worldwide spread of the fungus, which is aided by increased global travel and the migratory nature of many beekeeping operations. We discuss the current taxonomic classification in light of the recent complete reworking of fungal systematics brought on by application of molecular methods. In addition, we discuss epidemiology and pathogenesis of the disease, as well as pathogen biology, morphology and reproduction. New attempts at disease control methods and management tactics are reviewed. We report on research tools developed for identification and monitoring, and also include recent findings on genomic and molecular studies not covered by previous reviews, including sequencing of the A. apis genome and identification of the mating type locus.

The 10 fatty acid ester components of brood pheromone were extracted from larvae of different populations of USA and South African honey bees and subjected to gas chromatography-mass spectrometry quantitative analysis. Extractable amounts of brood pheromone were not significantly different by larval population; however, differences in the proportions of components enabled us to classify larval population of 77% of samples correctly by discriminant analysis. Honeybee releaser and primer pheromone responses to USA,Africanized and–European pheromone blends were tested. Texas-Africanized and Georgia-European colonies responded with a significantly greater ratio of returning pollen foragers when treated with a blend from the same population than from a different population. There was a significant interaction of pheromone blend by adult population source among Georgia-European bees for modulation of sucrose response threshold, a primer response. Brood pheromone blend variation interacted with population for pollen foraging response of colonies, suggesting a self recognition cue for this pheromone releaser behavior. An interaction of pheromone blend and population for priming sucrose response thresholds among workers within the first week of adult life suggested a more complex interplay of genotype, ontogeny, and pheromone blend.

Entomopathogenic fungi infect a wide array of insec t from most orders and they are among the natural enemies that contribute to the regulation o f i sect populations. However, only a limited number of studies have focused on the impact of fun gal pathogens on populations of non-pest insects. Effects of entomopathogenic fungi on non-p est host populations should receive more attention based on the increasing interest in conse rvation biological control. In this strategy, founded on competition theory, non-pest host popula tions adjacent to cropping systems will in principle affect pest populations through shared na tural enemies. We present examples of selected non-pest host-fungus systems from temperate ecosyst ems that are relevant for the expected ecosystem service provided by entomopathogenic fung i. Predators are among the non-pest hosts that are infected by fungi. Recent advances in our understanding of the effect of pathogens on the behaviour of predators may shed light on the signif icance of entomopathogenic fungi for the regulation of predator populations. We discuss what we can learn about host-pathogen interactions from behavioural ecology and which life history par ameters in the host that may be important for the impacts of fungal pathogens on their host popul ations.

Entomopathogenic fungi infect a wide array of insec t from most orders and they are among the natural enemies that contribute to the regulation o f i sect populations. However, only a limited number of studies have focused on the impact of fun gal pathogens on populations of non-pest insects. Effects of entomopathogenic fungi on non-p est host populations should receive more attention based on the increasing interest in conse rvation biological control. In this strategy, founded on competition theory, non-pest host popula tions adjacent to cropping systems will in principle affect pest populations through shared na tural enemies. We present examples of selected non-pest host-fungus systems from temperate ecosyst ems that are relevant for the expected ecosystem service provided by entomopathogenic fung i. Predators are among the non-pest hosts that are infected by fungi. Recent advances in our understanding of the effect of pathogens on the behaviour of predators may shed light on the signif icance of entomopathogenic fungi for the regulation of predator populations. We discuss what we can learn about host-pathogen interactions from behavioural ecology and which life history par ameters in the host that may be important for the impacts of fungal pathogens on their host popul ations.