Katherine A. Aronstein

Immune pathways and defence mechanisms in honey bees Apis mellifera.

Social insects are able to mount both group‐level and individual defences against pathogens. Here we focus on individual defences, by presenting a genome‐wide analysis of immunity in a social insect, the honey bee Apis mellifera. We present honey bee models for each of four signalling pathways associated with immunity, identifying plausible orthologues for nearly all predicted pathway members. When compared to the sequenced Drosophila and Anopheles genomes, honey bees possess roughly one‐third as many genes in 17 gene families implicated in insect immunity. We suggest that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens.

Chalkbrood disease in honey bees

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.

Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis

Genome sequences offer a broad view of host–pathogen interactions at the systems biology level. With the completion of the sequence of the honey bee, interest in the relevant pathogens is heightened. Here we report the genome sequences of two of the major pathogens of honey bees, the bacterium Paenibacillus larvae (causative agent for American foulbrood disease) and the fungus Ascosphaera apis. (causative agent for chalkbrood disease). Ongoing efforts to characterize the genomes of these species can be used to understand and mitigate the effects of two important pathogens, and will provide a contrast with pathogenic, benign and freeliving relatives.

SID-I is implicated in systemic gene silencing in the honey bee

Summary RNA interference (RNAi) has become a powerful functional genomics tool that can be used to effectively silence gene expression. The implications for analysis of loss-of-function phenotypes through systemic or localized silencing are enormously significant in the application of this technology. The Sid-I gene was implicated in the cellular import of RNAi signal that enables passive uptake of dsRNA. Here we demonstrate that RNAi in the honey bee (Apis mellifera) is systemic and our data suggest that honey bee SID-I homologue, a putative transmembrane protein encoded by AmSid-I, is necessary for the uptake of systemically administered dsRNA and subsequent gene silencing. The honey bee SID-I homologue shares strong similarities with human (NP-060169; 44.3%), mouse (NM-198034; 43.9%), and Caenorhabditis elegans (Q9GZC8; 19%).AmSid-I was expressed in the entire set of honey bee tissues examined with the highest abundance in adult head followed by egg tissue. To test the role of AmSid-I in the systemic effect of RNAi, we induced systemic gene silencing of the honey bee Toll-related receptor 18W by a feeding-soaking delivery method of dsRNA and measured expression levels of AmSid-I and Am18w using real time PCR. A 3.4–fold increase in expression of AmSid-I was observed at 26 h. In contrast, Am18w gene expression was decreased about 60–fold at 30 h. High mortality and morphological abnormalities were also seen due to gene silencing. The presence of SID-I in honey bees and its function as a transmembrane channel that facilitates uptake of dsRNA are discussed.

Transcriptional responses in Honey Bee larvae infected with chalkbrood fungus

BackgroundDiseases 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.ResultsWe used cDNA-AFLP ®Technology to profile transcripts in infected and uninfected bee larvae. From 64 primer combinations, over 7,400 transcriptionally-derived fragments were obtained A total of 98 reproducible polymorphic cDNA-AFLP fragments were excised and sequenced, followed by quantitative real-time RT-PCR (qRT-PCR) analysis of these and additional samples.We have identified a number of differentially-regulated transcripts that are implicated in general mechanisms of stress adaptation, including energy metabolism and protein transport. One of the most interesting differentially-regulated transcripts is for a chitinase-like enzyme that may be linked to anti-fungal activities in the honey bee larvae, similarly to gut and fat-body specific chitinases found in mosquitoes and the red flour beetle. Surprisingly, we did not find many components of the well-characterized NF-κB intracellular signaling pathways to be differentially-regulated using the cDNA-AFLP approach. Therefore, utilizing qRT-PCR, we probed some of the immune related genes to determine whether the lack of up-regulation of their transcripts in our analysis can be attributed to lack of immune activation or to limitations of the cDNA-AFLP approach.ConclusionsUsing a combination of cDNA-AFLP and qRT-PCR analyses, we were able to determine several key transcriptional events that constitute the overall effort in the honey bee larvae to fight natural fungal infection. Honey bee transcripts identified in this study are involved in critical functions related to transcriptional regulation, apoptotic degradation of ubiquitinated proteins, nutritional regulation, and RNA processing. We found that immune regulation of the anti-fungal responses in honey bee involves highly coordinated activation of both NF-κB signaling pathways, leading to production of anti-microbial peptides. Significantly, activation of immune responses in the infected bee larvae was associated with down-regulation of major storage proteins, leading to depletion of nutritional resources.

How Varroa Parasitism affects the immunological and nutritional status of the honey bee, Apis mellifera

We investigated the effect of the parasitic mite Varroadestructor 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.

Transformation of the Gram-positive honey bee pathogen, Paenibacillus larvae, by electroporation

In this study we developed an electrotransformation method for use with the Gram-positive bacterium Paenibacillus larvae-a deadly pathogen of honey bees. Combining multiple Bacillus electrotransformation methods to generate an initial protocol, we then optimized the following parameters for use with P. larvae: cell density of culture at harvest time, contents of the washing/electroporation solution, field strength of the electrical pulse, recovery growth medium, and recovery time period. With the optimized method, we achieved an average transformation efficiency of 1.9x10(5) transformants/mug DNA. The method is substantially different from the only other electrotransformation method for a Paenibacillus species found in the literature. This work should facilitate the study of the several previously discovered natural plasmids of P. larvae, and is a step toward developing a genetic system for this species.

Infectivity and virulence of Nosema ceranae and Nosema apis in commercially available North American honey bees

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.

Oxytetracycline-resistance in the honey bee pathogen Paenibacillus larvae is encoded on novel plasmid pMA67

Summary The gram-positive bacterium, Paenibacillus larvae, causes a serious honey bee disease, American Foulbrood. For several decades, commercial and hobbyist beekeepers have controlled this disease with the antibiotic oxytetracycline. However, in recent years there have been reports of oxytetracycline-resistant P. larvae. In this study, we report that the reason for the oxytetracycline resistance in P. larvae is the presence of a novel plasmid carrying a tetracycline resistance gene–tetL. We tested 36 strains of P. larvae from the USA and Canada and found this plasmid in all 21 oxytetracycline-resistant strains and in none of the 15 oxytetracycline-sensitive strains. We cloned and expressed the P. larvae tetL gene in Escherichia coli and showed it was functional. Sequencing of the entire plasmid, which we named pMA67, revealed that it is likely a mobilizable rolling-circle replication plasmid. This work provides the first sequence information for any P. larvae plasmid, a new tetL ortholog with significant sequence divergence from tetL genes found in other species, and the first tetracycline-resistance gene found in the entire Paenibacillus genus.

Variation in and Responses to Brood Pheromone of the Honey Bee (APIS mellifera L.)

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.