Direct Evidence for Infection of Varroa destructor Mites with the Bee-Pathogenic Deformed Wing Virus Variant B, but Not Variant A, via Fluorescence In Situ Hybridization Analysis

Deformed wing virus (DWV) is a bee-pathogenic, single- and positive-stranded RNA virus that has been involved in severe honey bee colony losses worldwide. DWV, when transmitted horizontally or vertically from bee to bee, causes mainly covert infections not associated with any visible symptoms or damage. Overt infections occur after vectorial transmission of DWV to the developing bee pupae through the ectoparasitic mite Varroa destructor. Symptoms of overt infections are pupal death, bees emerging with deformed wings and shortened abdomens, or cognitive impairment due to brain infection. So far, three variants of DWV, DWV-A, DWV-B, and DWV-C, have been described. While it is widely accepted that V. destructor acts as vector of DWV, the question of whether the mite only functions as a mechanical vector or whether DWV can infect the mite, thus using it as a biological vector, is hotly debated because in the literature data can be found that support both hypotheses. In order to settle this scientific dispute, we analyzed putatively DWV-infected mites with a newly established protocol for fluorescence in situ hybridization of mites and demonstrated DWV-specific signals inside mite cells. We provide compelling and direct evidence that DWV-B infects the intestinal epithelium and the salivary glands of V. destructor. In contrast, no evidence for DWV-A infecting mite cells was found. Our data are key to understanding the pathobiology of DWV, the mite's role as a biological DWV vector, and the quasispecies dynamics of this RNA virus when switching between insect and arachnid host species. IMPORTANCE Deformed wing virus (DWV) is a bee-pathogenic, originally rather benign, single- and positive-stranded RNA virus. Only the vectorial transmission of this virus to honey bees by the ectoparasitic mite Varroa destructor leads to fatal or symptomatic infections of individuals, usually followed by collapse of the entire colony. Studies on whether the mite only acts as a mechanical virus vector or whether DWV can infect the mite and thus use it as a biological vector have led to disparate results. In our study using fluorescence in situ hybridization, we provide compelling and direct evidence that at least the DWV-B variant infects the gut epithelium and the salivary glands of V. destructor. Hence, the host range of DWV includes both bees (Insecta) and mites (Arachnida). Our data contribute to a better understanding of the triangular relationship between honey bees, V. destructor, and DWV and the evolution of virulence in this viral bee pathogen

Involvement of secondary metabolites in the pathogenesis of the American foulbrood of honey bees caused by Paenibacillus larvae

Covering: 2011 to end of 2014 The Gram-positive, spore-forming bacterium Paenibacillus larvae (P. larvae) is the causative agent of the epizootic American Foulbrood (AFB), a fatal brood disease of the western honey bee (Apis mellifera). AFB is one of the most destructive honey bee diseases since it is not only lethal for infected larvae but also for the diseased colonies. Due to the high impact of honey bees on ecology and economy this epizootic is a severe and pressing problem. Knowledge about virulence mechanisms and the underlying molecular mechanisms remain largely elusive. Recent genome sequencing of P. larvae revealed its potential to produce unknown secondary metabolites, like nonribosomal peptides and peptide-polyketide hybrids. This article highlights recent findings on secondary metabolites synthesized by P. larvae and discusses their role in virulence and pathogenicity towards the bee larvae

Significant, but not biologically relevant: Nosema ceranae infections and winter losses of honey bee colonies

The Western honey bee Apis mellifera, which provides about 90% of commercial pollination, is under threat from diverse abiotic and biotic factors. The ectoparasitic mite Varroa destructor vectoring deformed wing virus (DWV) has been identified as the main biotic contributor to honey bee colony losses worldwide, while the role of the microsporidium Nosema ceranae is still controversially discussed. In an attempt to solve this controversy, we statistically analyzed a unique data set on honey bee colony health collected from a cohort of honey bee colonies over 15 years and comprising more than 3000 data sets on mite infestation levels, Nosema spp. infections, and winter losses. Multivariate statistical analysis confirms that V. destructor is the major cause of colony winter losses. Although N. ceranae infections are also statistically significantly correlated with colony losses, determination of the effect size reveals that N. ceranae infections are of no or low biological relevance

A Comparison of Different Matrices for the Laboratory Diagnosis of the Epizootic American Foulbrood of Honey Bees

American Foulbrood (AFB) of honey bees caused by the spore-forming bacterium Paenibacillus larvae is a notifiable epizootic in most countries. Authorities often consider a rigorous eradication policy the only sustainable control measure. However, early diagnosis of infected but not yet diseased colonies opens up the possibility of ridding these colonies of P. larvae spores by the shook swarm method, thus preventing colony destruction by AFB or official control orders. Therefore, surveillance of bee colonies for P. larvae infection followed by appropriate sanitary measures is a very important intervention to control AFB. For the detection of P. larvae spores in infected colonies, samples of brood comb honey, adult bees, or hive debris are commonly used. We here present our results from a comparative study on the suitability of these matrices in reliably and correctly detecting P. larvae spores contained in these matrices. Based on the sensitivity and limit of detection of P. larvae spores in samples from hive debris, adult bees, and brood comb honey, we conclude that the latter two are equally well-suited for AFB surveillance programs. Hive debris samples should only be used when it is not possible to collect honey or adult bee samples from brood combs

Production of the Catechol Type Siderophore Bacillibactin by the Honey Bee Pathogen Paenibacillus larvae

The Gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood. This bacterial infection of honey bee brood is a notifiable epizootic posing a serious threat to global honey bee health because not only individual larvae but also entire colonies succumb to the disease. In the recent past considerable progress has been made in elucidating molecular aspects of host pathogen interactions during pathogenesis of P. larvae infections. Especially the sequencing and annotation of the complete genome of P. larvae was a major step forward and revealed the existence of several giant gene clusters coding for non-ribosomal peptide synthetases which might act as putative virulence factors. We here present the detailed analysis of one of these clusters which we demonstrated to be responsible for the biosynthesis of bacillibactin, a P. larvae siderophore. We first established culture conditions allowing the growth of P. larvae under iron-limited conditions and triggering siderophore production by P. larvae. Using a gene disruption strategy we linked siderophore production to the expression of an uninterrupted bacillibactin gene cluster. In silico analysis predicted the structure of a trimeric trithreonyl lactone (DHB-Gly-Thr)3 similar to the structure of bacillibactin produced by several Bacillus species. Mass spectrometric analysis unambiguously confirmed that the siderophore produced by P. larvae is identical to bacillibactin. Exposure bioassays demonstrated that P. larvae bacillibactin is not required for full virulence of P. larvae in laboratory exposure bioassays. This observation is consistent with results obtained for bacillibactin in other pathogenic bacteria

Biological effects of paenilamicin, a secondary metabolite antibiotic produced by the honey bee pathogenic bacterium Paenibacillus larvae

Paenibacillus larvae is the etiological agent of American Foulbrood (AFB) a world-wide distributed devastating disease of the honey bee brood. Previous comparative genome analysis and more recently, the elucidation of the bacterial genome, provided evidence that this bacterium harbors putative functional nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) and therefore, might produce nonribosomal peptides (NRPs) and polyketides (PKs). Such biosynthesis products have been shown to display a wide-range of biological activities such as antibacterial, antifungal or cytotoxic activity. Herein we present an in silico analysis of the first NRPS/PKS hybrid of P. larvae and we show the involvement of this cluster in the production of a compound named paenilamicin (Pam). For the characterization of its in vitro and in vivo bioactivity, a knock-out mutant strain lacking the production of Pam was constructed and subsequently compared to wild-type species. This led to the identification of Pam by mass spectrometry. Purified Pam-fractions showed not only antibacterial but also antifungal and cytotoxic activities. The latter suggested a direct effect of Pam on honey bee larval death which could, however, not be corroborated in laboratory infection assays. Bee larvae infected with the non-producing Pam strain showed no decrease in larval mortality, but a delay in the onset of larval death. We propose that Pam, although not essential for larval mortality, is a virulence factor of P. larvae influencing the time course of disease. These findings are not only of significance in elucidating and understanding host-pathogen interactions but also within the context of the quest for new compounds with antibiotic activity for drug development.DFG, GRK 1121, Genetische und immunologische Determinanten von Pathogen-Wirt-InteraktionenDFG, EXC 314, Unifying Concepts in Catalysi

Paenibacillus larvae Chitin-Degrading Protein PlCBP49 Is a Key Virulence Factor in American Foulbrood of Honey Bees

Paenibacillus larvae, the etiological agent of the globally occurring epizootic American Foulbrood (AFB) of honey bees, causes intestinal infections in honey bee larvae which develop into systemic infections inevitably leading to larval death. Massive brood mortality might eventually lead to collapse of the entire colony. Molecular mechanisms of host-microbe interactions in this system and of differences in virulence between P. larvae genotypes are poorly understood. Recently, it was demonstrated that the degradation of the peritrophic matrix lining the midgut epithelium is a key step in pathogenesis of P. larvae infections. Here, we present the isolation and identification of PlCBP49, a modular, chitin-degrading protein of P. larvae and demonstrate that this enzyme is crucial for the degradation of the larval peritrophic matrix during infection. PlCBP49 contains a module belonging to the auxiliary activity 10 (AA10, formerly CBM33) family of lytic polysaccharide monooxygenases (LPMOs) which are able to degrade recalcitrant polysaccharides. Using chitin-affinity purified PlCBP49, we provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for members of the AA10 family. Using P. larvae mutants lacking PlCBP49 expression, we analyzed in vivo biological functions of PlCBP49. In the absence of PlCBP49 expression, peritrophic matrix degradation was markedly reduced and P. larvae virulence was nearly abolished. This indicated that PlCBP49 is a key virulence factor for the species P. larvae. The identification of the functional role of PlCBP49 in AFB pathogenesis broadens our understanding of this important family of chitin-binding and -degrading proteins, especially in those bacteria that can also act as entomopathogens

honeybee workers exhibit conserved molecular responses to diverse pathogens

Background Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses. Results We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non- differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses. Conclusions Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions

Genomic Potential and Virulence Mechanisms of Paenibacillus larvae

Paenibacillus larvae, a Gram positive bacterial pathogen, causes American Foulbrood (AFB), which is the most serious infectious disease of honey bees. In order to investigate the genomic potential of P. larvae, two strains belonging to two different genotypes were sequenced and used for comparative genome analysis. The complete genome sequence of P. larvae strain DSM 25430 (genotype ERIC II) consisted of 4,056,006 bp and harbored 3,928 predicted protein-encoding genes. The draft genome sequence of P. larvae strain DSM 25719 (genotype ERIC I) comprised 4,579,589 bp and contained 4,868 protein- encoding genes. Both strains harbored a 9.7 kb plasmid and encoded a large number of virulence-associated proteins such as toxins and collagenases. In addition, genes encoding large multimodular enzymes producing nonribosomally peptides or polyketides were identified. In the genome of strain DSM 25719 seven toxin associated loci were identified and analyzed. Five of them encoded putatively functional toxins. The genome of strain DSM 25430 harbored several toxin loci that showed similarity to corresponding loci in the genome of strain DSM 25719, but were non-functional due to point mutations or disruption by transposases. Although both strains cause AFB, significant differences between the genomes were observed including genome size, number and composition of transposases, insertion elements, predicted phage regions, and strain-specific island-like regions. Transposases, integrases and recombinases are important drivers for genome plasticity. A total of 390 and 273 mobile elements were found in strain DSM 25430 and strain DSM 25719, respectively. Comparative genomics of both strains revealed acquisition of virulence factors by horizontal gene transfer and provided insights into evolution and pathogenicity

Cold case: The disappearance of Egypt bee virus, a fourth distinct master strain of deformed wing virus linked to honeybee mortality in 1970’s Egypt

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes