Influence of virus abundances in donor colonies and nurse hives on queens of Apis mellifera during the rearing process.

Background: Honeybees are one of the three most important animals for mankind. In order to be safe and increase number of bee colonies for pollination, the breeding of queens is necessary. For several decades, bees were selected on economic and behavioral aspects. With the appearance of the neozootic mite Varroa destructor beekeepers were forced to adapt their methods. Varroa destructor can act as a vector for many different bee pathogenic viruses and by this potentiates its devastating impact. Aim: Methods of rearing queens were not evaluated since the mites' appearance. Besides scientific approaches, viruses received too little attention in regard to the rearing process of honeybee queens. Herein, we present a detailed analysis of virus abundances [Aparavirus, acute bee paralysis virus (ABPV); Triatovirus, black queen cell virus (BQCV); Cripavirus, chronic bee paralysis virus (CBPV); and Iflaviruses, deformed wings virus (DWV), Sacbrood virus (SBV), VDV-1] in breeding hives, donating first instar larvae, hives that are nursing these larvae until the pupa stage, and on queens of Apis mellifera in a breeding apiary. Methods: Nurse and donor colonies of the queen-rearing process were sampled in the year 2020 and analyzed by RT qPCR. Virus quantifications were correlated with queen mortalities and seasonal effects. Results: Virus detections increased in reared queens, however, the elevated virus titers did not increase the mortality of the queens until their exclosure. Moreover, we observed a lower interrelation between virus abundance in queens and their original donor colonies, than between nurse hives and their nursed queens. Conclusion: The bee pathogenic viruses ABPV, BQCV, CBPV, DWV, SBV, and VDV-1 do not influence the mortality of bee queens during the rearing process. Whether respective virus loads result in sublethal or long-term effects remains to be elucidated.

Impact of a Microbial Pest Control Product Containing Bacillus thuringiensis on Brood Development and Gut Microbiota of Apis mellifera Worker Honey Bees.

To avoid potential adverse side effects of chemical plant protection products, microbial pest control products (MPCP) are commonly applied as biological alternatives. This study aimed to evaluate the biosafety of a MPCP with the active organism Bacillus thuringiensis ssp. aizawai (strain: ABTS-1857). An in-hive feeding experiment was performed under field-realistic conditions to examine the effect of B. thuringiensis (B. t.) on brood development and the bacterial abundance of the core gut microbiome (Bifidobacterium asteroids, Gilliamella apicola, the group of Lactobacillus and Snodgrasella alvi) in Apis mellifera worker bees. We detected a higher brood termination rate and a non-successful development into worker bees of treated colonies compared to those of the controls. For the gut microbiome, all tested core members showed a significantly lower normalized abundance in bees of the treated colonies than in those of the controls; thus, a general response of the gut microbiome may be assumed. Consequently, colony exposure to B. t. strain ABTS-1857 had a negative effect on brood development under field-realistic conditions and caused dysbiosis of the gut microbiome. Further studies with B. t.-based products, after field-realistic application in bee attractive crops, are needed to evaluate the potential risk of these MPCPs on honey bees.

Determination, distribution, and environmental fate of Bacillus thuringiensis spores in various honeybee matrices after field application as plant protection product.

The increasing use of Bacillus thuringiensis (Bt)-based plant protection products (PPPs) has recently raised some concerns regarding their environmental accumulation and possible chronic exposure of non-target species, including pollinators, to higher than expected doses. The exposure level of such microbial PPPs in bee's matrices under field conditions has not yet been described. Therefore, the current study aims at evaluating the realistic exposure level and comparing the distributions and persistence of Bt spores under field conditions. A field trial with spray application in oilseed rape (Brassica napus) as a representative bee-attractive crop was conducted. During the experimental period, different matrices, including honeybee-collected and -stored matrices as well as bee larvae and dead bees, were collected and analyzed using newly established methods. The concentration of Bt spores in the various matrices was quantified. The results show high levels of Bt spores in honey sac and pollen pellets with reduction over time but no reduction of Bt spores in the stored matrices within the colony, i.e., nectar and bee bread, over time. Our results show for the first time the exposure level of bees to Bt spores under realistic field conditions and are fundamentally important for assessing potential exposure and risks for pollinators.

Assessment of the impacts of microbial plant protection products containing Bacillus thuringiensis on the survival of adults and larvae of the honeybee (Apis mellifera).

This study was aimed at evaluating the effect of a microbial pest-controlling product (MPCP) with the active substance Bacillus thuringiensis ssp. aizawai (strain: ABTS-1857) on adults and larvae of honeybees. To determine the contamination levels of Bt spores in different matrices, a colony-feeding study under semi-field conditions was performed. Furthermore, two chronic adult trials and a chronic larval study were conducted under laboratory conditions to test the effects of different concentrations of the plant protection product (PPP) on the development and mortality. Possible modifications of the chronic oral toxicity test were assessed by additional pollen feeding. Our results showed that Bt spores were detected in all matrices over the entire test duration in different concentrations, decreasing over time. The survival of adult bees and larvae was negatively affected in laboratory conditions after a chronic exposure to the MPCP depending on the tested concentrations. Moreover, the earliest sign of bee mortality, resulting from exposure to ABTS-1857, was recorded only after 96 h at the highest tested concentration. Pollen feeding to adults significantly increased the survival of the treated bees. In conclusion, the PPP with the Bt strain ABTS-1857 showed an effect on the mortality of adults and larvae under laboratory conditions. Further studies with Bt-based PPPs under realistic field conditions are necessary to evaluate the potential risk of those MPCPs on honeybees.

Rapid identification and genotyping of the honeybee pathogen Paenibacillus larvae by combining culturing and multiplex quantitative PCR.

Background: American Foulbrood (AFB) is a devastating disease of honey bee (Apis mellifera) larvae caused by the spore-forming, Gram-positive bacterium Paenibacillus larvae. In most countries, the law requires mandatory reporting of AFB to the veterinary authority. Aim and Methods: To speed up detection and genotyping of P. larvae spores, we compared different culturing protocols on Columbia sheep blood agar and developed a new multiplex quantitative polymerase chain reaction to distinguish between the two relevant P. larvae genotypes enterobacterial repetitive intergenic consensus (ERIC) I and ERIC II. Results and Conclusion: As confirmed by P. larvae reference strains and field isolates, the new identification and genotyping protocol halves the time of current workflows, lessens labor-intension, allows a higher throughput of samples for monitoring, and permits a faster intervention to prevent the spread of AFB.

Discovery of Paenibacillus larvae ERIC V: Phenotypic and genomic comparison to genotypes ERIC I-IV reveal different inventories of virulence factors which correlate with epidemiological prevalences of American Foulbrood.

Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a highly contagious brood disease of honey bees (Apis mellifera). AFB requires mandatory reporting to the veterinary authority in many countries and until now four genotypes, P. larvae ERIC I-IV, have been identified. We isolated a new genotype, ERIC V, from a Spanish honey sample. After a detailed phenotypic comparison with the reference strains of the ERIC I-IV genotypes, including spore morphology, non-ribosomal peptide (NRP) profiling, and in vivo infections of A. mellifera larvae, we established a genomic DNA Macrorestriction Fragment Pattern Analysis (MRFPA) scheme for future epidemiologic discrimination. Whole genome comparison of the reference strains and the new ERIC V genotype (DSM 106052) revealed that the respective virulence gene inventories of the five genotypes corresponded with the time needed to kill 100 % of the infected bee larvae (LT100) in in vivo infection assays. The rarely isolated P. larvae genotypes ERIC II I-V with a fast-killing phenotype (LT100 3 days) harbor genes with high homology to virulence factors of other insect pathogens. These virulence genes are absent in the epidemiologically prevalent genotypes ERIC I (LT100 12 days) and ERIC II (LT100 7 days), which exhibit slower killing phenotypes. Since killing-retardation is known to reduce the success of hygienic cleaning by nurse bees, the identified absence of virulence factors might explain the epidemiological prevalences of ERIC genotypes. The discovery of the P. larvae ERIC V isolate suggests that more unknown ERIC genotypes exist in bee colonies. Since inactivation or loss of a few genes can transform a fast-killing phenotype into a more dangerous slow-killing phenotype, these rarely isolated genotypes may represent a hidden reservoir for future AFB outbreaks.

Paenibacillus larvae-Directed Bacteriophage HB10c2 and Its Application in American Foulbrood-Affected Honey Bee Larvae.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious honey bee brood bacterial disease. We isolated and characterized P. larvae-directed bacteriophages and developed criteria for safe phage therapy. Whole-genome analysis of a highly lytic virus of the family Siphoviridae (HB10c2) provided a detailed safety profile and uncovered its lysogenic nature and a putative beta-lactamase-like protein. To rate its antagonistic activity against the pathogens targeted and to specify potentially harmful effects on the bee population and the environment, P. larvae genotypes ERIC I to IV, representatives of the bee gut microbiota, and a broad panel of members of the order Bacillales were analyzed for phage HB10c2-induced lysis. Breeding assays with infected bee larvae revealed that the in vitro phage activity observed was not predictive of the real-life scenario and therapeutic efficacy. On the basis of the disclosed P. larvae-bacteriophage coevolution, we discuss the future prospects of AFB phage therapy.

Paenilarvins: Iturin family lipopeptides from the honey bee pathogen Paenibacillus larvae.

The bacterium Paenibacillus larvae has been extensively studied as it is an appalling honey bee pathogen. In the present work, we screened crude extracts derived from fermentations of P. larvae genotypes ERIC I and II for antimicrobial activity, following the detection of four putative secondary metabolite gene clusters that show high sequence homology to known biosynthetic gene clusters for the biosynthesis of antibiotics. Low molecular weight metabolites produced by P. larvae have recently been shown to have toxic effects on honey bee larvae. Moreover, a novel tripeptide, sevadicin, was recently characterized from laboratory cultures of P. larvae. In this study, paenilarvins, which are iturinic lipopeptides exhibiting strong antifungal activities, were obtained by bioassay-guided fractionation from cultures of P. larvae, genotype ERIC II. Their molecular structures were determined by extensive 2D NMR spectroscopy, high resolution mass spectrometry, and other methods. Paenilarvins are the first antifungal secondary metabolites to be identified from P. larvae. In preliminary experiments, these lipopeptides also affected honey bee larvae and might thus play a role in P. larvae survival and pathogenesis. However, further studies are needed to investigate their function.