Abstract Although the roles of host-associated microbiomes in animal health are increasingly recognised, the factors influencing their variation remain understudied. The relatively simple microbiome of honey bees is a relevant system to address this gap. In particular, the relationship between variations in microbiome composition and the ectoparasite Varroa destructor , the main threat to honey bee health worldwide, is poorly established. In this study, we used metagenomic and statistical analyses of 1442 European honey bee colonies to investigate the relationships between the honey bee microbiome, temporality, location, V. destructor load, and behavioural response to its infestation by the host. While season, year, and location were identified as the main drivers of microbiome variation, V. destructor load emerged as a significant factor associated with microbiome variation. Notably, we identify several pathogens and opportunists that correlated positively with V. destructor load, while the core symbiont Bombilactobacillus correlated negatively. This is compatible with a shift in the microbiome toward dysbiosis, which may be driven by or promote V. destructor parasitism. By contrast, we found only limited evidence of an association between the microbiome and resistance behaviours of the host against this parasite. While the study cannot establish causal relationships, we present the largest metagenomic analysis of honey bee microbiomes to date, providing robust, generalisable evidence about the factors driving variation in the microbiome composition of this ecologically and economically important pollinator. These findings may serve as additional markers in selective breeding programs targeting V. destructor resistance, which could ultimately improve honey bee health.

Complex host-vector-virus interfaces can impose serious health challenges. Western honeybees have experienced high colony losses globally, mainly driven by the host-shifted, virus-vectoring ectoparasitic mites Varroa destructor and Tropilaelaps mercedesae. Host populations can survive mite infestations through natural selection, offering a long-term strategy for colony health. However, host-vector-virus coevolution requires local adaptations of this triad, which is poorly understood. We propose harnessing natural selection through a global approach focused on standardized monitoring of colony survival, mite infestation levels, and control of reproductives. Studying native and adapted mite hosts, host shifts, and comparing susceptible to surviving hosts will enhance understanding of this host-vector-virus system. This strategy promotes colony health in both managed and wild host populations and provides insights into other host-vector-virus interfaces.

Abstract Global food supply strongly depends on honeybee pollination services, which are threatened by insecticides and pests such as parasitic Varroa destructor mites. Chemical varroacides/acaricides are hampered by resistance development, necessitating the development of sustainable and environmentally friendly alternatives, with arthropod venom peptides being considered promising sources of acaricidal toxins. With only a few acaricidal venom peptides being reported, we performed a systematic topical screening of 50 arthropod venoms against V. destructor, with 78% of the venoms causing 100% mortality after 24 h. Deconvolution of the venoms from the Tasmanian cave spider Hickmania troglodytes and the Giant Japanese funnel-web spider Gigathele gigas led to identification of the varroacidal peptides Ht1a and Gg1a. Topical application of Ht1a and Gg1a reduced varroa mite but not honeybee survival, despite Ht1a inhibiting voltage-gated sodium channels from varroa and honeybee with equal potency. Ht1a and Gg1a were inactive against human skeletal muscle (hNaV1.4), cardiac (NaV1.5), neuronal NaV channel isoforms, and human voltage-gated calcium channel CaV2.2. At human α3β2/4 nicotinic acetylcholine receptors, Gg1a was inactive while 10 µM of Ht1a partially blocked nicotine-mediated Ca2+ influx. Our data reveal Ht1a and Gg1a as promising candidates for the development of novel varroa mite treatments of honeybee hives.

Honey bee colonies contain thousands of individuals living in close proximity in a thermally homeostatic nest, creating ideal conditions for the thriving of numerous pathogens. Among the bacterial pathogens, Paenibacillus larvae infects larvae via the nutritive jelly that adult workers feed them, causing the highly contagious American foulbrood disease. Further Paenibacillus species were anecdotally found in association with honey bees, including when affected by another disease, European foulbrood (EFB). However, their pathogenicity remains largely unknown. Our results indicate that Paenibacillus dendritiformis, Paenibacillus thiaminolyticus and newly described Paenibacillus melissococcoides are pathogenic towards honey bee brood and that their virulence correlates with their sporulation ability, which confers them resistance to the bactericidal properties of the nutritive jelly. Our survey occasionally but increasingly detected P. melissococcoides in confirmed and idiopathic cases of EFB but never in healthy colonies, suggesting that this bacterium is an emerging pathogen of honey bee brood. Overall, our results suggest that virulence traits allowing a pathogenic or opportunistically pathogenic habit towards honey bee brood are frequent in Paenibacillus spp., but that their degree of adaptation to this host varies. Our study clarifies the ecology of this ubiquitous genus, especially when infecting honey bees.

Bees are crucial for food security and biodiversity. However, managed bees are increasingly considered drivers of wild bee declines, leading to stakeholder conflicts and restrictive policies. We propose avenues to reconcile wild and managed bee proponents and point out knowledge gaps that hinder the development of evidence-based policies.

The invasion of the ectoparasitic mite Varroa destructor into European honey bee, Apis mellifera, populations has contributed to the collapse of most wild populations and economic losses in beekeeping operations. Understanding how some A. mellifera populations survive infestation by this parasite is of great fundamental and practical interest and has led to numerous studies of potential resistance mechanisms. One such mechanism is the uncapping and recapping of comb cells containing infested brood by nurse bees. Recapping has been observed in most surviving populations, but its link to V. destructor resistance remains unclear. Investigating the occurrence of recapping in the Eastern honey bee, Apis cerana, the original host of the parasite, could provide a better understanding of the evolution and function of this behaviour in the Apis genus. Here, we determined the frequency of recapping in two A. cerana populations in China and Thailand at different stages of brood development and compared them with a sympatric A. mellifera population in China. The species, which differ in their susceptibility to infestation, did not show significant differences in recapping frequency. A specific association between recapping and resistance to Varroa spp. in A. cerana is therefore not supported. We discuss possible functions and evolutionary scenarios for this behavior.

A novel, facultatively anaerobic, Gram-stain-positive, motile, endospore-forming bacterium of the genus Paenibacillus , designated strain 2.1 T , was isolated from a colony of Apis mellifera affected by European foulbrood disease in Switzerland. The rod-shaped cells of strain 2.1 T were 2.2–6.5 µm long and 0.7–1.1 µm wide. Colonies of strain 2.1 T were orange-pigmented under oxic growth conditions on solid basal medium at 35–37 °C. Strain 2.1 T showed catalase and cytochrome c oxidase activity. Its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and phospholipid. The only respiratory quinone was menaquinone 7, and the major cellular fatty acids were anteiso-C 15 : 0 , anteiso-C 17 : 0 , iso-C 15 : 0 , iso-C 17 : 0 and palmitic acid (C 16 : 0 ), which is consistent with other members of the genus Paenibacillus . The G+C content of the genomic DNA of strain 2.1 T was 53.3 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence similarity showed that strain 2.1 T was closely related to Paenibacillus dendritiformis LMG 21716 T (99.7 % similarity) and Paenibacillus thiaminolyticus DSM 7262 T (98.8 %). The whole-genome average nucleotide identity between strain 2.1 T and the type strains of P. dendritiformis and P. thiaminolyticus was 92 and 91 %, respectively, and thus lower than the 95 % threshold value for delineation of genomic prokaryotic species. Based on the results of phylogenetic, genomic, phenotypic and chemotaxonomic analyses we propose the name Paenibacillus melissococcoides sp. nov. for this novel Paenibacillus species. The type strain is 2.1 T (=CCOS 2000 T =DSM 113619 T =LMG 32539 T ).

Honey bees play an essential role in modern agriculture as farm animals and crop pollinators, and they contribute to one third of our diet. Over the last few decades, managed honey bees have faced large-scale losses worldwide. Various causes include the spread of pathogens and parasites, habitat loss and loss of forage, pesticide use, and climate change. Many scientists investigated these issues worldwide separately and independently, often using different methodologies, and this approach might lead to conflicting and sometimes erroneous findings. To provide global and sustainable solutions, a group of bee scientists established COLOSS, a non-profit association for the prevention of honey bee COlony LOSSes. Its mission is to investigate the causes of declining bee health and find effective means to improve the well-being of bees. COLOSS comprises various Core Projects and Task Forces focusing on specific topics identified by the association to receive priority attention. Among the core projects, the COLOSS BEEBOOK is a unique venture aiming to provide a standardised methods manual for studying the honey bee. The project’s goal has been to create a comprehensive collection of established methods and techniques for honey bee research, with the aim of making studies conducted by different groups across the world more comparable. The resulting practical manual contains over 2,000 standardized methods across all fields of honey bee research. It is definitive, but evolving, research manual. There is a strong need for standardisation in the fast-growing field of edible insect farming and research. The COLOSS BEEBOOK stands as a testament to the achievements that can be made when researchers collaborate towards a common goal, and it can serve as an inspiration for the establishment of standardized methods for the mass rearing of edible insects.

Honey bees, Apis mellifera, of European origin are major pollinators of crops and wild flora. Their endemic and exported populations are threatened by a variety of abiotic and biotic factors. Among the latter, the ectoparasitic mite Varroa destructor is the most important single cause behind colony mortality. The selection of mite resistance in honey bee populations has been deemed a more sustainable solution to its control than varroacidal treatments. Because natural selection has led to the survival of some European and African honey bee populations to V. destructor infestations, harnessing its principles has recently been highlighted as a more efficient way to provide honey bee lineages that survive infestations when compared with conventional selection on resistance traits against the parasite. However, the challenges and drawbacks of harnessing natural selection to solve the varroa problem have only been minimally addressed. We argue that failing to consider these issues could lead to counterproductive results, such as increased mite virulence, loss of genetic diversity reducing host resilience, population collapses or poor acceptance by beekeepers. Therefore, it appears timely to evaluate the prospects for the success of such programmes and the qualities of the populations obtained. After reviewing the approaches proposed in the literature and their outcomes, we consider their advantages and drawbacks and propose perspectives to overcome their limitations. In these considerations, we not only reflect on the theoretical aspects of host-parasite relationships but also on the currently largely neglected practical constraints, that is, the requirements for productive beekeeping, conservation or rewilding objectives. To optimize natural selection-based programmes towards these objectives, we suggest designs based on a combination of nature-driven phenotypic differentiation and human-directed selection of traits. Such a dual strategy aims at allowing field-realistic evolutionary approaches towards the survival of V. destructor infestations and the improvement of honey bee health.

Melissococcus plutonius is a pathogenic bacterium affecting immature stages of the western honey bee ( Apis mellifera ) and leads to European foulbrood (EFB) disease. Despite EFB outbreaks increasing in frequency in several countries in recent decades, there is little knowledge on the epidemiology of M. plutonius or on the defence mechanisms of honey bees against this pathogen. Mating of honey bee queens with multiple males (polyandry) can be such a mechanism, as it has been shown to be beneficial to colony health and fitness. It is hypothesized that a high level of polyandry was selected for in response to pathogen pressure to maximize the probability that at least some patrilines among nestmates in a colony possess a high degree of resistance to specific pathogens, ultimately protecting colonies against infections. We show that M. plutonius infection provokes differential mortality among patrilines of immature honey bee workers. Such differences indicate a genetic origin of resistance against this pathogen—supporting the polyandry hypothesis—and open up avenues to improve control of EFB disease via selective breeding.

The homogenization of agricultural landscapes has led to a decrease in pollinator diversity and abundance. In response to this decline, farmers have implemented agroecological measures, which, in meadows, aim at providing more floral resources. These measures are the availability of unmown floral strips, delayed mowing, and discouraging the use of the conditioner, a device known to harm insects. The aim of our study was to investigate the cascade of effects of these agroecological measures on honey bee colony development and winter survival. We (1) determined the effect of these measures on colony size during the nectar and pollen collecting season in spring and summer, (2) evaluated the effect of spring and summer colony sizes on autumn size, and (3) described the effect of colony size in autumn on winter mortality. In this study, 300 honey bee colonies were monitored over three years in three cantons of Switzerland. Colony size was defined by the numbers of brood cells and of adult workers. Honey bee colony size in summer and autumn was improved by agroecological measures on meadows and likely contributed to the increased overwintering success. This study is a first step toward the targeted identification of viable agroecological measures on temporary meadows that can be implemented to promote honey bee colonies health in the agricultural landscape.

INTRODUCTION:Host shift of parasites may have devastating effects on the novel hosts. One remarkable example is that of the ectoparasitic mite Varroa destructor, which has shifted its host from Eastern honey bees (Apis cerana) to Western honey bees (Apis mellifera) and posed a global threat to apiculture.OBJECTIVES:To identify the genetic factors underlying the reproduction of host-shifted V. destructor on the new host.METHODS:Genome sequencing was conducted to construct the phylogeny of the host-shifted and non-shifted mites and to screen for genomic signatures that differentiated them. Artificial infestation experiment was conducted to compare the reproductive difference between the mites, and transcriptome sequencing was conducted to find differentially expressed genes (DEGs) during the reproduction process.RESULTS:The host-shifted and non-shifted V. destructor mites constituted two genetically distinct lineages, with 15,362 high-FST SNPs identified between them. Oogenesis was upregulated in host-shifted mites on the new host A. mellifera relative to non-shifted mites. The transcriptomes of the host-shifted and non-shifted mites differed significantly as early as 1h post-infestation. The DEGs were associated with nine genes carrying nonsynonymous high-FST SNPs, including mGluR2-like, Lamb2-like and Vitellogenin 6-like, which were also differentially expressed, and eIF4G, CG5800, Dap160 and Sas10, which were located in the center of the networks regulating the DEGs based on protein-protein interaction analysis.CONCLUSIONS:The annotated functions of these genes were all associated with oogenesis. These genes appear to be the key genetic determinants of the oogenesis of host-shifted mites on the new host. Further study of these candidate genes will help elucidate the key mechanism underlying the success of host shifts of V. destructor.

European foulbrood (EFB) is a honey bee brood disease caused by the bacterium Melissococcus plutonius. Large-scale EFB outbreaks have been reported in several countries in recent decades, which entail costly sanitation measures of affected apiaries to restrict the spread of this contagious pathogen. To mitigate its impact, a better understanding of the population dynamics of the etiological agent is required. We here used multi-locus sequence typing (MLST) to infer the genetic diversity and geographical distribution of 160 M. plutonius isolates collected from EFB symptomatic honey bee colonies seven years apart. Isolates belonged to three clonal complexes (CCs) known worldwide and to 12 sequence types (STs), of which five were novel. Phylogenetic and clustering analyses showed that some of these novel sequence types have likely evolved locally during a period of outbreak, but most disappeared again. We further screened the isolates for melissotoxin A (mtxA), a putative virulence gene. The prevalence of STs in which mtxA was frequent increased over time, suggesting that this gene promotes spread. Despite the increased frequency of this gene in the population, the total number of cases decreased, which could be due to stricter control measures implemented before the second sampling period. Our results provide a better understanding of M. plutonius population dynamics and help identify knowledge gaps that limit efficient control of this emerging disease.

We sequenced the genomes of recently discovered Paenibacillus melissococcoides (CCOS 2000) and of the type strains of closely related P. thiaminolyticus (DSM 7262) and P. dendritiformis (LMG 21716). The three genomes set the basis to unambiguous diagnostic of these honey bee associated Paenibacillus bacteria.

The invasive parasitic mite, Varroa destructor (Anderson and Trueman), is the major biotic threat to the survival of European honey bees, Apis mellifera L. To improve colony survival against V. destructor, the selection of resistant lineages against this parasite is considered a sustainable solution. Among selected traits, mite fertility and fecundity, often referred to as suppressed mite reproduction are increasingly used in breeding programmes. However, the current literature leaves some gaps in the assessment of the effectiveness of selecting these traits toward achieving resistance. In the population studied here, we show a low repeatability and reproducibility of mite fertility and fecundity phenotypes, as well as a low correlation of these traits with infestation rates of colonies. Phenotyping reliability could neither be improved by increasing the number of worker brood cells screened, nor by screening drone brood, which is highly attractive for the parasite and available early in the season, theoretically allowing a reduction of generation time and thus an acceleration of genetic progress in selected lineages. Our results provide an evaluation of the potential and limitations of selecting on decreased mite reproduction traits to obtain V. destructor-resistant honeybee colonies. To allow for a more precise implementation of such selection and output reporting, we propose a refined nomenclature by introducing the terms of decreased mite reproduction and reduced mite reproduction, depending on the extent of mite reproduction targeted. We also highlight the importance of ensuring accurate phenotyping ahead of initiating long-lasting selection programmes.

The invasive parasitic mite, Varroa destructor (Anderson and Trueman), is the major biotic threat to the survival of European honey bees, Apis mellifera L. To improve colony survival against V. destructor, the selection of resistant lineages against this parasite is considered a sustainable solution. Among selected traits, mite fertility and fecundity, often referred to as suppressed mite reproduction are increasingly used in breeding programmes. However, the current literature leaves some gaps in the assessment of the effectiveness of selecting these traits toward achieving resistance. In the population studied here, we show a low repeatability and reproducibility of mite fertility and fecundity phenotypes, as well as a low correlation of these traits with infestation rates of colonies. Phenotyping reliability could neither be improved by increasing the number of worker brood cells screened, nor by screening drone brood, which is highly attractive for the parasite and available early in the season, theoretically allowing a reduction of generation time and thus an acceleration of genetic progress in selected lineages. Our results provide an evaluation of the potential and limitations of selecting on decreased mite reproduction traits to obtain V. destructor-resistant honeybee colonies. To allow for a more precise implementation of such selection and output reporting, we propose a refined nomenclature by introducing the terms of decreased mite reproduction and reduced mite reproduction, depending on the extent of mite reproduction targeted. We also highlight the importance of ensuring accurate phenotyping ahead of initiating long-lasting selection programmes.

Trans-generational immune priming involves the transfer of immunological experience, acquired by the parents after exposure to pathogens, to protect their progeny against infections by these pathogens. Such natural mechanisms could be exploited to prevent disease expression in economically important insects, such as the honey bee. This mechanism occurs when honey bee queens are exposed to the pathogenic bacterium Paenibacillus larvae. Here, we tested whether natural or experimental exposure to Melissococcus plutonius-another bacterium triggering a disease in honey bee larvae-reduced the susceptibility of the queen's progeny to infection by this pathogen. Because the immunological response upon pathogen exposure can lead to fitness costs, we also determined whether experimental exposure of the queens affected them or their colony negatively. Neither natural nor experimental exposure induced protection in the honey bee larvae against the deleterious effects of M. plutonius. Our results provided no evidence for the occurrence of trans-generational immune priming upon exposure of the queen to M. plutonius. Whether this lack was due to confounding genetic resistance, to unsuitable exposure procedure or to the absence of trans-generational immune priming against this pathogen in honey bees remains to be determined.

To defend themselves against pathogenic microorganisms, honey bees resort to social immunity mechanisms, such as the secretion of antibiotic compounds in the jelly they feed to their larvae. Whereas the bactericidal activity of jelly fed to queen larvae is well studied, little is known about the bioactivity of compositionally different jelly fed to worker larvae. However, the numerous worker larvae are likely to drive the spread of the microorganism and influence its virulence and pathogenesis. Diluted jelly or extracts are mostly used for jelly bioactivity tests, which may bias the evaluation of the pathogen's resistance and virulence. Here, we compared the bactericidal effect of pure and diluted jellies destined for queen and worker larvae on Melissococcus plutonius, the etiological agent of the European foulbrood (EFB) disease of honey bees, and on a secondary invader bacteria, Enterococcus faecalis. We tested three strains of M. plutonius with varying virulence to investigate the association between resistance to antibacterial compounds and virulence. The resistance of the bacteria varied but was not strictly correlated with their virulence and was lower in pure than in diluted jelly. Resistance differed according to whether the jelly was destined for queen or worker larvae, with some strains being more resistant to queen jelly and others to worker jelly. Our results provide a biologically realistic assessment of host defenses via nutritive jelly and contribute to a better understanding of the ecology of M. plutonius and of secondary invaders bacteria in the honey bee colony environment, thus shedding light on the selective forces affecting their virulence and on their role in EFB pathogenesis.