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Managed and wild insect pollinators play a key role in ensuring that mankind is adequately supplied with food. Among the pollinating insects, the managed Western honey bee providing about 90% of commercial pollination is of special importance. Hence, diseases as well as disease causing pathogens and parasites that threaten honey bees, have become the focus of many research studies. The ectoparasitic mite Varroa destructor together with deformed wing virus (DWV) vectored by the mite have been identified as the main contributors to colony losses, while the role of the microsporidium Nosema ceranae in colony losses is still controversially discussed. In an attempt to solve this controversy, we statistically analyzed a unique data set on honey bee colony health comprising data on mite infestation levels, Nosema spp. infections and winter losses continuously collected over 15 years. We used various statistical methods to investigate the relationship between colony mortality and the two pathogens, V. destructor and N. ceranae. Our multivariate statistical analysis confirmed that V. destructor is the major cause of colony winter losses. When using cumulative data sets, we also found a significant relationship between N. ceranae infections and colony losses. However, determining the effect size revealed that this statistical significance was of low biological relevance, because the deleterious effects of N. ceranae infection are normally masked by the more severe effects of V. destructor on colony health and therefore only detectable in the few colonies that are not infested with mites or are infested at low levels.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

The bipartite interactions between insect hosts and their bacterial gut microbiota, or their bacterial pathogens, are empirically and theoretically well-explored. However, direct, and indirect tripartite interactions will also likely occur inside a host. These interactions will almost certainly affect the trajectory of pathogen virulence evolution, an area that is currently under researched. The interactions within tripartite associations can be competitive, that is, exploitative-competition, interference-competition or apparent-competition. Competitive interactions will be significantly influenced by non-competitive effects, for example, immunopathology, immunosuppression, and microbiota-mediated tolerance. Considering a combination of these interactions and effects, will enable an increased understanding of the evolution of pathogen virulence. This new perspective allows us to identify several novel research questions, which we hope will be a useful framework for future research.

American Foulbrood, caused by Paenibacillus larvae, is the most devastating bacterial honey bee brood disease. Finding a treatment against American Foulbrood would be a huge breakthrough in the battle against the disease. Recently, small molecule inhibitors against virulence factors have been suggested as candidates for the development of anti-virulence strategies against bacterial infections. We therefore screened an in-house library of synthetic small molecules and a library of flavonoid natural products, identifying the synthetic compound M3 and two natural, plant-derived small molecules, Acacetin and Baicalein, as putative inhibitors of the recently identified P. larvae toxin Plx2A. All three inhibitors were potent in in vitro enzyme activity assays and two compounds were shown to protect insect cells against Plx2A intoxication. However, when tested in exposure bioassays with honey bee larvae, no effect on mortality could be observed for the synthetic or the plant-derived inhibitors, thus suggesting that the pathogenesis strategies of P. larvae are likely to be too complex to be disarmed in an anti-virulence strategy aimed at a single virulence factor. Our study also underscores the importance of not only testing substances in in vitro or cell culture assays, but also testing the compounds in P. larvae-infected honey bee larvae.

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., 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.

Pollination provided by managed honey bees as well as by all the wild bee species is a crucial ecosystem service contributing to the conservation of biodiversity and human food security. Therefore, it is not only the health status of honey bees but also the health status of wild bees that concerns us all. In this context, recent field studies suggesting interspecies transmission of the microsporidium parasite Nosema ceranae from honey bees (Apis mellifera) to bumblebees (Bombus spp.) were alarming. On the basis of these studies, N. ceranae was identified as an emerging infectious agent (EIA) of bumblebees, although knowledge of its impact on its new host was still elusive. In order to investigate the infectivity, virulence, and pathogenesis of N. ceranae infections in bumblebees, we performed controlled laboratory exposure bioassays with Bombus terrestris by orally inoculating the bees with infectious N. ceranae spores. We comprehensively analyzed the infection status of the bees via microscopic analysis of squash preparations, PCR-based detection of N. ceranae DNA, histology of Giemsa-stained tissue sections, and species-specific fluorescence in situ hybridization. We did not find any evidence for a true infection of bumblebees by N. ceranae. Through a series of experiments, we ruled out the possibility that spore infectivity, spore dosage, incubation time, or age and source of the bumblebees caused these negative results. Instead, our results clearly demonstrate that no infection and production of new spores took place in bumblebees after they ingested N. ceranae spores in our experiments. Thus, our results question the classification of N. ceranae as an emerging infectious agent for bumblebees. IMPORTANCE Emerging infectious diseases (EIDs) pose a major health threat to both humans and animals. EIDs include, for instance, those that have spread into hitherto naive populations. Recently, the honey bee-specific microsporidium Nosema ceranae has been detected by molecular methods in field samples of bumblebees. This detection of N. ceranae DNA in bumblebees led to the assumption that N. ceranae infections represent an EID of bumblebees and resulted in speculations on the role of this pathogen in driving bumblebee declines. In order to address the issue of whether N. ceranae is an emerging infectious agent for bumblebees, we experimentally analyzed host susceptibility and pathogen reproduction in this new host-pathogen interaction. Surprisingly, we did not find any evidence for a true infection of Bombus terrestris by N. ceranae, questioning the classification of N. ceranae infections as EIDs of bumblebees and demonstrating that detection of microsporidian DNA does not equal detection of microsporidian infection.

Pollination is an indispensable ecosystem service provided by many insects, especially by wild and managed bee species. Hence, reports on large scale honey bee colony losses and on population declines of many wild bees were alarming and resulted in increased awareness of the importance of bee health and increased interest in bee pathogens. To serve this interest, this review will give a comprehensive overview on bacterial bee pathogens by covering not only the famous pathogens (Paenibacillus larvae, Melissococcus plutonius), but also the orphan pathogens which have largely been neglected by the scientific community so far (spiroplasmas) and the pathogens which were only recently discovered as being pathogenic to bees (Serratia marcescens, Lysinibacillus sphaericus).

The toxin Plx2A is an important virulence factor of Paenibacillus larvae, the etiological agent of American Foulbrood, the most destructive bacterial disease of honey bees. Biochemical and functional analyses as well as the crystal structure of Plx2A revealed that it belongs to the C3 mono-ADP-ribosylating toxin subgroup. RhoA was identified as the cellular target of Plx2A activity. The kinetic parameters (KM , kcat ) were established for both the transferase and glycohydrolase reactions. When expressed in yeast, Plx2A was cytotoxic for eukaryotic cells and catalytic variants confirmed that the cytotoxicity of Plx2A depends on its enzymatic activity. The crystal structure of Plx2A was solved to 1.65 A and confirmed that it is a C3-like toxin, although with a new molecular twist, it has a B-domain. A molecular model of the 'active' enzyme conformation in complex with NAD+ was produced by computational methods based on the recent structure of C3bot1 with RhoA. In murine macrophages, Plx2A induced actin cytoskeleton reorganization while in insect cells, vacuolization and the occurrence of bi-nucleated cells was observed. The latter is indicative of an inhibition of cytokinesis. All these cellular effects are consistent with Plx2A inhibiting the activity of RhoA by covalent modification.

C3larvinA is a putative virulence factor produced by Paenibacillus larvae enterobacterial-repetitive-intergenic-consensus (ERIC) III/IV (strain 11-8051). Biochemical, functional and structural analyses of C3larvinA revealed that it belongs to the C3-like mono-ADP-ribosylating toxin subgroup. Mammalian RhoA was the target substrate for its transferase activity suggesting that it may be the biological target of C3larvinA. The kinetic parameters of the NAD+ substrate for the transferase (KM = 75 ± 10 µM) and glycohydrolase (GH) (KM = 107 ± 20 µM) reactions were typical for a C3-like bacterial toxin, including the Plx2A virulence factor from Paenibacillus larvae ERIC I. Upon cytoplasmic expression in yeast, C3larvinA caused a growth-defective phenotype indicating that it is an active C3-like toxin and is cytotoxic to eukaryotic cells. The catalytic variant of the Q187-X-E189 motif in C3larvinA showed no cytotoxicity toward yeast confirming that the cytotoxicity of this factor depends on its enzymatic activity. A homology consensus model of C3larvinA with NAD+ substrate was built on the structure of Plx2A, provided additional confirmation that C3larvinA is a member of the C3-like mono-ADP-ribosylating toxin subgroup. A homology model of C3larvinA with NADH and RhoA was built on the structure of the C3cer-NADH-RhoA complex which provided further evidence that C3larvinA is a C3-like toxin that shares an identical catalytic mechanism with C3cer from Bacillus cereus. C3larvinA induced actin cytoskeleton reorganization in murine macrophages, whereas in insect cells, vacuolization and bi-nucleated cells were observed. These cellular effects are consistent with C3larvinA disrupting RhoA function by covalent modification that is shared among C3-like bacterial toxins.

Paenibacillus larvae is the causative agent of the notifiable epizootic American foulbrood, a fatal bacterial disease of honey bee larvae. The species P. larvae has been classified into four differentially virulent and prevalent genotypes (ERIC I-IV), which also differ in their virulence factor equipment. Recently, a novel P. larvae toxin, the C3-like C3larvin, has been described. Genome analysis now revealed that the C3larvin gene is actually a part of a toxin locus encompassing two genes encoding a binary AB toxin with the A subunit being C3larvin (C3larvinA) and a putative B subunit (C3larvinB) encoded by the second gene. Sequence and structural analyses demonstrated that C3larvinB is a homologue of the Bacillus anthracis protective antigen (PA), the B subunit of anthrax toxin. The C3larvinAB toxin locus was interrupted by point mutations in all analysed P. larvae ERIC I and ERIC II strains. Only one P. larvae ERIC III/IV strain harboured an uninterrupted toxin locus comprising full-length genes for C3larvinA and B. Exposure bioassays did not substantiate a role as virulence factor for C3larvinAB in P. larvae ERIC I/II. However, the PA homologue C3larvinB had an influence on the virulence of the unique P. larvae strain expressing the functional C3larvinAB locus.

Damage confirmed Early studies of the impacts of neonicotinoid insecticides on insect pollinators indicated considerable harm. However, lingering criticism was that the studies did not represent field-realistic levels of the chemicals or prevailing environmental conditions. Two studies, conducted on different crops and on two continents, now substantiate that neonicotinoids diminish bee health (see the Perspective by Kerr). Tsvetkov et al. find that bees near corn crops are exposed to neonicotinoids for 3 to 4 months via nontarget pollen, resulting in decreased survival and immune responses, especially when coexposed to a commonly used agrochemical fungicide. Woodcock et al. , in a multicounty experiment on rapeseed in Europe, find that neonicotinoid exposure from several nontarget sources reduces overwintering success and colony reproduction in both honeybees and wild bees. These field results confirm that neonicotinoids negatively affect pollinator health under realistic agricultural conditions. Science , this issue p. 1395 , p. 1393 ; see also p. 1331

The health of the Western honey bee is threatened by a global epidemic of deformed wing virus (DWV) infections driven by the ectoparasitic mite Varroa destructor acting as mechanical and biological virus vector. Three different variants of DWV, DWV-A, -B and -C exist. Virulence differences between these variants and their relation to V. destructor are still controversially discussed. We performed laboratory experiments to analyze the virulence of DWV directly isolated from crippled bees (DWVP0 ) or after one additional passage in bee pupae (DWVP1 ). We demonstrated that DWVP0 was more virulent than DWVP1 for pupae, when pupal mortality was taken as virulence marker, and for adult bees, when neurotropism and cognitive impairment were taken as virulence markers. Phylogenetic analysis supported that DWV exists as quasispecies and showed that DWVP0 clustered with DWV-B and DWVP1 with DWV-A when the phylogeny was based on the master sequences of the RNA-dependent RNA polymerase but not so when it was based on the VP3 region master sequences. We propose that switching of DWV between the bee and the mite host is accompanied by changes in viral sequence, tissue tropism and virulence and that the RNA-dependent RNA polymerase is involved in determining host range and virulence.

American Foulbrood is a worldwide distributed, fatal disease of the brood of the Western honey bee (Apis mellifera). The causative agent of this fatal brood disease is the Gram-positive, spore-forming bacterium Paenibacillus larvae, which can be classified into four different genotypes (ERIC I-IV), with ERIC I and II being the ones isolated from contemporary AFB outbreaks. P. larvae is a peritrichously flagellated bacterium and, hence, we hypothesized that P. larvae is capable of coordinated and cooperative multicellular behaviors like swarming motility and biofilm formation. In order to analyze these behaviors of P. larvae, we firstly established appropriate functional assays. Using these assays we demonstrated that P. larvae ERIC II, but not P. larvae ERIC I, was capable of swarming. Swarming motility was hampered in a P. larvae ERIC II-mutant lacking production of paenilarvin, an iturin-like lipopeptide exclusively expressed by this genotype. Both genotypes were able to form free floating biofilm aggregates loosely attached to the walls of the culture wells. Visualizing the biofilms by Congo red and thioflavin S staining suggested structural differences between the biofilms formed. Biofilm formation was shown to be independent from paenilarvin production because the paenilarvin deficient mutant was comparably able to form a biofilm.

Recent large-scale colony losses among managed Western honey bees (Apis mellifera) have alarmed researchers and apiculturists alike. Here, the existing correlative evidence provided by monitoring studies is reviewed which (i) identified members of the deformed wing virus and acute bee paralysis virus clades as lethal pathogens for entire colonies, and (ii) identified novel viruses whose impact on honey bee health remains elusive. Also discussed in this review is related evidence obtained via controlled experimental infection assays and RNAi approaches underscoring the damage inflicted by some of these viruses on individuals and colonies. The relevance of the ectoparasitic mite Varroa destructor acting as mechanical and biological virus vector for the enhanced virulence of certain viruses or mite selected virus strains is carefully considered.

American foulbrood is one of the most devastating honey bee diseases. Although the causative agent, Paenibacillus larvae, only kills larval stages, diseased colonies will eventually die from the lack of progeny. Analysis of P. larvae virulence mechanisms revealed that P. larvae has evolved means to combat microbial competitors, expresses an enzyme able to degrade the peritrophic matrix (a chitin-containing protective layer lining the midgut epithelium), and uses toxins and an S-layer protein to attack and breach the midgut epithelium thereby killing the larva.

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.

The toxin Plx2A is an important virulence factor of Paenibacillus larvae, the etiological agent of American Foulbrood, the most destructive bacterial disease of honey bees. Biochemical and functional analyses as well as the crystal structure of Plx2A revealed that it belongs to the C3 mono-ADP-ribosylating toxin subgroup. RhoA was identified as the cellular target of Plx2A activity. The kinetic parameters (K

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3597-6) contains supplementary material, which is available to authorized users.

Bacterial diseases are quite common in animals, and in most cases one animal is host to many bacterial pathogens. However, only two bacteria are known to cause disease in honey bee and these two bacteria are only pathogenic to honey bee larvae. One of these bacterial brood pathogens is the bacterium Paenibacillus larvae (P. larvae), causing American Foulbrood (AFB). AFB is the most serious honey bee brood disease because AFB is not only able to kill infected larvae but may also lead to the death of entire colonies. AFB is highly contagious and can spread quite fast within an apiary and between apiaries. Therefore, AFB is a notifiable epizootic in many countries and mandatory control measures often include the culling of diseased colonies or even of all colonies of an affected apiary. So far, no satisfactory strategies which can prevent or cure the disease are available. To change this situation, a better understanding of the pathogenesis of AFB is urgently needed. In the following chapter, the current state of AFB research is presented. The two P. larvae genotypes ERIC I and ERIC II are introduced, and their relevance for both research and practice is explained. Several P. larvae factors necessary for pathogenesis and virulence are described together with the methods used for their discovery and evaluation. A model on the interaction between P. larvae and larvae during pathogenesis is presented integrating all the novel data. Special emphasis has been put into outlining the practical implications of the newly generated knowledge.

American foulbrood is the most destructive brood disease of honeybees (Apis mellifera) globally. The absence of a repeatable, universal typing scheme for the causative bacterium Paenibacillus larvae has restricted our understanding of disease epidemiology. We have created the first multilocus sequence typing scheme (MLST) for P. larvae, which largely confirms the previous enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR)-based typing scheme's divisions while providing added resolution and improved repeatability. We have used the new scheme to determine the distribution and biogeography of 294 samples of P. larvae from across six continents. We found that of the two most epidemiologically important ERIC types, ERIC I was more diverse than ERIC II. Analysis of the fixation index (FST ) by distance suggested a significant relationship between genetic and geographic distance, suggesting that population structure exists in populations of P. larvae. Interestingly, this effect was only observed within the native range of the host and was absent in areas where international trade has moved honeybees and their disease. Correspondence analysis demonstrated similar sequence type (ST) distributions between native and non-native countries and that ERIC I and II STs mainly have differing distributions. The new typing scheme facilitates epidemiological study of this costly disease of a key pollinator.

Das sporenbildende Bakterium Paenibacillus larvae ist der Erreger der Amerikanischen Faulbrut, einer weltweit auftretenden, hochpathogenen Bienenkrankheit. Wir konnten ein komplexes Hybrid-Gencluster aus nichtribosomalen Peptidsynthetasen (NRPS) und Polyketidsynthasen (PKS) im Genom von P. larvae aufklaren. Hier prasentieren wir die Isolierung und die Strukturaufklarung der antibakteriellen und antifungalen Produkte dieses Genclusters, die Paenilamicine genannt wurden. Die einzigartigen Strukturen der Paenilamicine geben tiefe Einblicke in die zugrunde liegende, komplexe Biosynthese-Maschinerie. Bienenlarven-Koinfektionstests zeigten, dass die Paenilamicine von P. larvae fur den Kampf um seine okologische Nische benotigt werden und nicht direkt fur das Sterben der Bienenlarven verantwortlich sind. Ihre antibakterielle und antimykotische Aktivitat machen die Paenilamicine zu vielversprechenden Kandidaten fur die Wirkstoffentwicklung.

Infection with Paenibacillus larvae, the etiological agent of American foulbrood, is lethal for honey bee larvae and may lead to loss of the entire colony. Of the four known ERIC-genotypes of P. larvae, ERIC I and II are most frequently observed and differ significantly in virulence. The course of the disease on the larval level is more accelerated after infection with genotype II strains allowing nurse bees to remove diseased larvae more efficiently before capping. For this reason the lead clinical symptom, conversion of capped larvae into 'ropy mass', is less frequently found than after infection with ERIC I strains bearing the risk of false negative diagnosis. In this study, the potential of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the discrimination of P. larvae genotypes ERIC I and II was explored on the basis of a comprehensive set of isolates. Using commercial software and a reference database constructed from field and type strains, ERIC I and II genotypes of all field isolates could be unambiguously identified on basis of mass spectra. Statistical analysis showed that the genotype is the main determinant for the spectral phenotype and MS-based ERIC-type determination is robust against sample selection. Furthermore, analysis of samples from Canada and New Zealand showed that distribution of ERIC II is not restricted to Europe as previously assumed. We suggest adding ERIC I and II genotype isolates as type-specific reference spectra for use in routine diagnostics.

American foulbrood (AFB) caused by the bee pathogenic bacterium Paenibacillus larvae is the most devastating bacterial disease of honey bees worldwide. From AFB-dead larvae, pure cultures of P. larvae can normally be cultivated indicating that P. larvae is able to defend its niche against all other bacteria present. Recently, comparative genome analysis within the species P. larvae suggested the presence of gene clusters coding for multi-enzyme complexes, such as non-ribosomal peptide synthetases (NRPSs). The products of these enzyme complexes are known to have a wide range of biological activities including antibacterial activities. We here present our results on antibacterial activity exhibited by vegetative P. larvae and the identification and analysis of a novel antibacterially active P. larvae tripeptide (called sevadicin; Sev) produced by a NRPS encoded by a gene cluster found in the genome of P. larvae. Identification of Sev was ultimately achieved by comparing the secretome of wild-type P. larvae with knockout mutants of P. larvae lacking production of Sev. Subsequent mass spectrometric studies, enantiomer analytics and chemical synthesis revealed the sequence and configuration of the tripeptide, D-Phe-D-ALa-Trp, which was shown to have antibacterial activity. The relevance of our findings is discussed in respect to host-pathogen interactions.

Nosema apis and Nosema ceranae are two microsporidian pathogens of the European honey bee, Apis mellifera. There is evidence that N. ceranae is more virulent than N. apis subject to environmental factors like climate. This makes N. ceranae one of the suspects in the increasing colony losses recently observed in many regions of the world. Correct differentiation between N. apis and N. ceranae is important and best accomplished by molecular methods. So far only protocols based on species-specific sequence differences in the 16S rRNA gene are available. However, recent studies indicated that these methods may lead to confusing results due to polymorphisms in and recombination between the multi-copy 16S rRNA genes. To solve this problem and to provide a reliable molecular tool for the differentiation between the two bee pathogenic microsporidia we here present and evaluate a duplex-PCR protocol based on species-specific sequence differences in the highly conserved gene coding for the DNA-dependent RNA polymerase II largest subunit. A total of 102 honey bee samples were analyzed by the novel PCR protocol and the results were compared with the results of the originally published PCR-RFLP analysis and two recently published differentiation protocols, based on 16S rRNA sequence differences. Although the novel PCR protocol proved to be as reliable as the 16S rRNA gene based PCR-RFLP it was superior to simple 16S rRNA based PCR protocols which tended to overestimate the rate of N. ceranae infections. Therefore, we propose that species-specific sequence differences of highly conserved protein coding genes should become the preferred molecular tool for differentiation of Nosema spp.

SummaryMethods are described for working with Nosema apis and Nosema ceranae in the field and in the laboratory. For fieldwork, different sampling methods are described to determine colony level infections at a given point in time, but also for following the temporal infection dynamics. Suggestions are made for how to standardise field trials for evaluating treatments and disease impact. The laboratory methods described include different means for determining colony level and individual bee infection levels and methods for species determination, including light microscopy, electron microscopy, and molecular methods (PCR). Suggestions are made for how to standardise cage trials, and different inoculation methods for infecting bees are described, including control methods for spore viability. A cell culture system for in vitro rearing of Nosema spp. is described. Finally, how to conduct different types of experiments are described, including infectious dose, dose effects, course of infection and longevity t...

SummaryCell culture techniques are indispensable in most, if not all life science disciplines to date. Wherever appropriate cell culture models are lacking, scientific development is hampered. Unfortunately this has been and still is the case in honey bee research, because permanent honey bee cell lines have not so far been established. To overcome this hurdle, protocols for the cultivation of primary honey bee cells and of non-permanent honey bee cell lines have been developed. In addition, heterologous cell culture models for honey bee pathogens based on non-Apis insect cell lines have recently been developed. To further advance this progress and to encourage bee scientists to enter the field of cell biology based research, here we present protocols for the cultivation of honey bee primary cells and non-permanent cell lines, as well as hints for the cultivation of permanent insect cell lines suitable for honey bee research.

Summary 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.

Managed insect pollinators are indispensable in modern agriculture. They are used worldwide not only in the open field but also in greenhouses to enhance fruit set, seed production, and crop yield. Managed honey bee (Apis mellifera, Apis cerana) colonies provide the majority of commercial pollination although other members of the superfamily Apoidea are also exploited and commercialized as managed pollinators. In the recent past, it became more and more evident that viral diseases play a key role in devastating honey bee colony losses and it was also recognized that many viruses originally thought to be honey bee specific can also be detected in other pollinating insects. However, while research on viruses infecting honey bees started more than 50years ago and the knowledge on these viruses is growing ever since, little is known on virus diseases of other pollinating bee species. Recent virus surveys suggested that many of the viruses thought to be honey bee specific are actually circulating in the pollinator community and that pollinator management and commercialization of pollinators provide ample opportunity for viral diseases to spread. However, the direction of disease transmission is not always clear and the impact of these viral diseases on the different hosts remains elusive in many cases. With our review we want to provide an up-to-date overview on the viruses detected in different commercialized pollinators in order to encourage research in the field of pollinator virology that goes beyond molecular detection of viruses. A deeper understanding of this field of virology is urgently needed to be able to evaluate the impact of viruses on pollinator health and the role of different pollinators in spreading viral diseases and to be able to decide on appropriate measures to prevent virus-driven pollinator decline.

The gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood of honey bees, a notifiable disease in many countries. Hence, P. larvae can be considered as an entomopathogen of considerable relevance in veterinary medicine. P. larvae is a highly specialized pathogen with only one established host, the honey bee larva. No other natural environment supporting germination and proliferation of P. larvae is known. Over the last decade, tremendous progress in the understanding of P. larvae and its interactions with honey bee larvae at a molecular level has been made. In this review, we will present the recent highlights and developments in P. larvae research and discuss the impact of some of the findings in a broader context to demonstrate what we can learn from studying "exotic" pathogens.