New reference genomes of honey bee-associated bacteria Paenibacillus melissococcoides, Paenibacillus dendritiformis, and Paenibacillus thiaminolyticus.

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.

Prospects, challenges and perspectives in harnessing natural selection to solve the 'varroa problem' of honey bees.

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.

Genomic signatures underlying the oogenesis of the ectoparasitic mite Varroa destructor on its new host Apis mellifera.

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.

Influence of Honey bee Nutritive Jelly Type and Dilution on its Bactericidal Effect on Melissococcus plutonius, the Etiological Agent of European Foulbrood.

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.

Population genetic diversity and dynamics of the honey bee brood pathogen Melissococcus plutonius in a region with high prevalence.

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.

Lack of evidence for trans-generational immune priming against the honey bee pathogen Melissococcus plutonius.

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.

Decreased Mite Reproduction to Select Varroa destructor (Acari: Varroidae) Resistant Honey Bees (Hymenoptera: Apidae): Limitations and Potential Methodological Improvements.

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.

Compliance with recommended Varroa destructor treatment regimens improves the survival of honey bee colonies over winter.

The ectoparasitic mite Varroa destructor affects honey bee colony health and survival negatively, thus compelling beekeepers to treat their colonies every year. A broadly used mite control regimen is based on two organic molecules: formic and oxalic acids. To ensure optimal efficiency, several applications of these acids at pre-defined time points are recommended. These recommendations are mainly based on experiments conducted under controlled conditions. Studies evaluating the effectiveness under natural field conditions are lacking. We enrolled 30 beekeepers in a longitudinal study in three cantons in Switzerland and monitored the management and health of their colonies for two years. We assessed compliance with mite control recommendations and measured V. destructor infestation rates, indexes of colony productivity (brood size and honey harvest), and colony mortality in 300 colonies. We observed a 10-fold increased risk of colony death when beekeepers deviated slightly from the recommended treatment regimen compared to compliant beekeepers (odds ratio: 11.9, 95% CI: 2.6-55.2, p = 0.002). The risk of colony death increased 25-fold in apiaries with substantial deviations from the recommendations (odds ratio: 50.4, 95% CI: 9.7-262.5, p < 0.0001). The deviations led to increased levels of V. destructor infestation ahead of wintering, which was likely responsible for colony mortality. After communicating the apparent link between low compliance and poor colony survival at the end of the first year to the beekeepers, we observed better compliance and colony survival in the second year. Our results highlight the positive impact of compliance with the recommended V. destructor treatment regimen on the health of honeybee colonies and the need to better communicate the consequences of deviating from the recommendations to improve compliance. Compliance also occasionally decreased, which hints at concept implementation constraints that could be identified and possibly addressed in detail with the help of social sciences to further promote honey bee health.

Population genetics and host specificity of Varroa destructor mites infesting eastern and western honeybees.

In a globalized world, parasites are often brought in contact with new potential hosts. When parasites successfully shift host, severe diseases can emerge at a large cost to society. However, the evolutionary processes leading to successful shifts are rarely understood, hindering risk assessment, prevention, or mitigation of their effects. Here, we screened populations of Varroa destructor, an ectoparasitic mite of the honeybee genus Apis, to investigate their genetic structure and reproductive potential on new and original hosts. From the patterns identified, we deduce the factors that influenced the macro- and microevolutionary processes that led to the structure observed. Among the mite variants identified, we found two genetically similar populations that differed in their reproductive abilities and thus in their host specificity. These lineages could interbreed, which represents a threat due to the possible increased virulence of the parasite on its original host. However, interbreeding was unidirectional from the host-shifted to the nonshifted native mites and could thus lead to speciation of the former. The results improve our understanding of the processes affecting the population structure and evolution of this economically important mite genus and suggest that introgression between shifted and nonshifted lineages may endanger the original host. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10340-020-01322-7.

CSI Pollen: Diversity of Honey Bee Collected Pollen Studied by Citizen Scientists.

A diverse supply of pollen is an important factor for honey bee health, but information about the pollen diversity available to colonies at the landscape scale is largely missing. In this COLOSS study, beekeeper citizen scientists sampled and analyzed the diversity of pollen collected by honey bee colonies. As a simple measure of diversity, beekeepers determined the number of colors found in pollen samples that were collected in a coordinated and standardized way. Altogether, 750 beekeepers from 28 different regions from 24 countries participated in the two-year study and collected and analyzed almost 18,000 pollen samples. Pollen samples contained approximately six different colors in total throughout the sampling period, of which four colors were abundant. We ran generalized linear mixed models to test for possible effects of diverse factors such as collection, i.e., whether a minimum amount of pollen was collected or not, and habitat type on the number of colors found in pollen samples. To identify habitat effects on pollen diversity, beekeepers' descriptions of the surrounding landscape and CORINE land cover classes were investigated in two different models, which both showed that both the total number and the rare number of colors in pollen samples were positively affected by 'urban' habitats or 'artificial surfaces', respectively. This citizen science study underlines the importance of the habitat for pollen diversity for bees and suggests higher diversity in urban areas.

Using Citizen Science to Scout Honey Bee Colonies That Naturally Survive Varroa destructor Infestations.

Citizen Science contributes significantly to the conservation of biodiversity, but its application to honey bee research has remained minimal. Even though certain European honey bee (Apis mellifera) populations are known to naturally survive Varroa destructor infestations, it is unclear how widespread or common such populations are. Such colonies are highly valuable for investigating the mechanisms enabling colony survival, as well as for tracking the conservation status of free-living honey bees. Here, we use targeted Citizen Science to identify potentially new cases of managed or free-living A. mellifera populations that survive V. destructor without mite control strategies. In 2018, a survey containing 20 questions was developed, translated into 13 languages, and promoted at beekeeping conferences and online. After three years, 305 reports were collected from 28 countries: 241 from managed colonies and 64 from free-living colonies. The collected data suggest that there could be twice as many naturally surviving colonies worldwide than are currently known. Further, online and personal promotion seem to be key for successful recruitment of participants. Although the survivor status of these colonies still needs to be confirmed, the volume of reports and responses already illustrate how effectively Citizen Science can contribute to bee research by massively increasing generated data, broadening opportunities for comparative research, and fostering collaboration between scientists, beekeepers, and citizens. The success of this survey spurred the development of a more advanced Citizen Science platform, Honey Bee Watch, that will enable a more accurate reporting, confirmation, and monitoring of surviving colonies, and strengthen the ties between science, stakeholders, and citizens to foster the protection of both free-living and managed honey bees.

Adaptive population structure shifts in invasive parasitic mites, Varroa destructor.

Comparative studies of genetic diversity and population structure can shed light on the ecological and evolutionary factors governing host-parasite interactions. Even though invasive parasites are considered of major biological importance, little is known about their adaptative potential when infesting the new hosts. Here, the genetic diversification of Varroa destructor, a novel parasite of Apis mellifera originating from Asia, was investigated using population genetics to determine how the genetic structure of the parasite changed in distinct European populations of its new host. To do so, mites infesting two categories of hosts in four European regions were compared: (a) adapted hosts surviving through means of natural selection, thereby expected to impose strong selective pressure on the mites, and (b) treated host populations, surviving mite infestations because acaricides are applied, therefore characterized by a relaxed selection imposed by the host on the mites. Significant genetic divergence was found across regions, partially reflecting the invasion pattern of V. destructor throughout Europe and indicating local adaptation of the mite to the host populations. Additionally, varying degrees of genotypic changes were found between mites from adapted and treated colonies. Altogether, these results indicate that V. destructor managed to overcome the genetic bottlenecks following its introduction in Europe and that host-mediated selection fostered changes in the genetic structure of this mite at diverse geographic scales. These findings highlight the potential of parasites to adapt to their local host populations and confirm that adaptations developed within coevolutionary dynamics are a major determinant of population genetic changes.

Host-Parasite Co-Evolution in Real-Time: Changes in Honey Bee Resistance Mechanisms and Mite Reproductive Strategies.

Co-evolution is a major driving force shaping the outcome of host-parasite interactions over time. After host shifts, the lack of co-evolution can have a drastic impact on novel host populations. Nevertheless, it is known that Western honey bee (Apismellifera) populations can cope with host-shifted ectoparasitic mites (Varroa destructor) by means of natural selection. However, adaptive phenotypic traits of the parasites and temporal variations in host resistance behavior are poorly understood. Here, we show that mites made adaptive shifts in reproductive strategy when associated with resistant hosts and that host resistance traits can change over time. In a fully-crossed field experiment, worker brood cells of local adapted and non-adapted (control) A.mellifera host colonies were infested with mites originating from both types of host colonies. Then, mite reproduction as well as recapping of cells and removal of infested brood (i.e., Varroa Sensitive Hygiene, VSH) by host workers were investigated and compared to data from the same groups of host colonies three years earlier. The data suggest adaptive shifts in mite reproductive strategies, because mites from adapted hosts have higher probabilities of reproduction, but lower fecundity, when infesting their associated hosts than mites in treated colonies. The results confirm that adapted hosts can reduce mite reproductive success. However, neither recapping of cells nor VSH were significantly expressed, even though the latter was significantly expressed in this adapted population three years earlier. This suggests temporal variation in the expression of adaptive host traits. It also appears as if mechanisms not investigated here were responsible for the reduced mite reproduction in the adapted hosts. In conclusion, a holistic view including mite adaptations and studies of the same parasite/host populations over time appears overdue to finally understand the mechanisms enabling survival of V.destructor-infested honey bee host colonies.

Advances and perspectives in selecting resistance traits against the parasitic mite Varroa destructor in honey bees.

BACKGROUND: In spite of the implementation of control strategies in honey bee (Apis mellifera) keeping, the invasive parasitic mite Varroa destructor remains one of the main causes of colony losses in numerous countries. Therefore, this parasite represents a serious threat to beekeeping and agro-ecosystems that benefit from the pollination services provided by honey bees. To maintain their stocks, beekeepers have to treat their colonies with acaricides every year. Selecting lineages that are resistant to infestations is deemed to be a more sustainable approach. REVIEW: Over the last three decades, numerous selection programs have been initiated to improve the host-parasite relationship and to support honey bee survival in the presence of the parasite without the need for acaricide treatments. Although resistance traits have been included in the selection strategy of honey bees, it has not been possible to globally solve the V. destructor problem. In this study, we review the literature on the reasons that have potentially limited the success of such selection programs. We compile the available information to assess the relevance of selected traits and the potential environmental effects that distort trait expression and colony survival. Limitations to the implementation of these traits in the field are also discussed. CONCLUSIONS: Improving our knowledge of the mechanisms underlying resistance to V. destructor to increase trait relevance, optimizing selection programs to reduce environmental effects, and communicating selection outcomes are all crucial to efforts aiming at establishing a balanced relationship between the invasive parasite and its new host.

Putative determinants of virulence in Melissococcus plutonius, the bacterial agent causing European foulbrood in honey bees.

MELISSOCOCCUS PLUTONIUS: is a bacterial pathogen that causes epidemic outbreaks of European foulbrood (EFB) in honey bee populations. The pathogenicity of a bacterium depends on its virulence, and understanding the mechanisms influencing virulence may allow for improved disease control and containment. Using a standardized in vitro assay, we demonstrate that virulence varies greatly among sixteen M. plutonius isolates from five European countries. Additionally, we explore the causes of this variation. In this study, virulence was independent of the multilocus sequence type of the tested pathogen, and was not affected by experimental co-infection with Paenibacillus alvei, a bacterium often associated with EFB outbreaks. Virulence in vitro was correlated with the growth dynamics of M. plutonius isolates in artificial medium, and with the presence of a plasmid carrying a gene coding for the putative toxin melissotoxin A. Our results suggest that some M. plutonius strains showed an increased virulence due to the acquisition of a toxin-carrying mobile genetic element. We discuss whether strains with increased virulence play a role in recent EFB outbreaks.

Reproduction of ectoparasitic mites in a coevolved system: Varroa spp.-Eastern honey bees, Apis cerana.

Parasite host shifts can impose a high selective pressure on novel hosts. Even though the coevolved systems can reveal fundamental aspects of host-parasite interactions, research often focuses on the new host-parasite relationships. This holds true for two ectoparasitic mite species, Varroa destructor and Varroa jacobsonii, which have shifted hosts from Eastern honey bees, Apis cerana, to Western honey bees, Apis mellifera, generating colony losses of these pollinators globally. Here, we study infestation rates and reproduction of V. destructor and V. jacobsonii haplotypes in 185 A. cerana colonies of six populations in China and Thailand to investigate how coevolution shaped these features. Reproductive success was mostly similar and low, indicating constraints imposed by hosts and/or mite physiology. Infestation rates varied between mite haplotypes, suggesting distinct local co-evolutionary scenarios. The differences in infestation rates and reproductive output between haplotypes did not correlate with the virulence of the respective host-shifted lineages suggesting distinct selection scenarios in novel and original host. The occasional worker brood infestation was significantly lower than that of drone brood, except for the V. destructor haplotype (Korea) from which the invasive lineage derived. Whether mites infesting and reproducing in atypical intraspecific hosts (i.e., workers and queens) actually predisposes for and may govern the impact of host shifts on novel hosts should be determined by identifying the underlying mechanisms. In general, the apparent gaps in our knowledge of this coevolved system need to be further addressed to foster the adequate protection of wild and managed honey bees from these mites globally.

Population genetics of ectoparasitic mites Varroa spp. in Eastern and Western honey bees.

Host shifts of parasites are often causing devastating effects in the new hosts. The Varroa genus is known for a lineage of Varroa destructor that shifted to the Western honey bee, Apis mellifera, with disastrous effects on wild populations and the beekeeping industry. Despite this, the biology of Varroa spp. remains poorly understood in its native distribution range, where it naturally parasitizes the Eastern honey bee, Apis cerana. Here, we combined mitochondrial and nuclear DNA analyses with the assessment of mite reproduction to determine the population structure and host specificity of V. destructor and Varroa jacobsonii in Thailand, where both hosts and several Varroa species and haplotypes are sympatric. Our data confirm previously described mite haplogroups, and show three novel haplotypes. Multiple infestations of single host colonies by both mite species and introgression of alleles between V. destructor and V. jacobsonii suggest that hybridization occurs between the two species. Our results indicate that host specificity and population genetic structure in the genus Varroa is more labile than previously thought. The ability of the host shifted V. destructor haplotype to spillback to A. cerana and to hybridize with V. jacobsonii could threaten honey bee populations of Asia and beyond.

Neonicotinoids and ectoparasitic mites synergistically impact honeybees.

The Western honeybee, Apis mellifera, is the most important managed pollinator globally and has recently experienced unsustainably high colony losses. Synergistic interactions among stressors are believed to be primarily responsible. However, despite clear evidence of strong effect on honeybee longevity of widely-employed neonicotinoid insecticides and of the ubiquitous ectoparasitic mite Varroa destructor, no data exist to show synergistic effects between these two stressors. Even though neonicotinoids had no significant impact by themselves, we here show for the first time a synergistic time-lag interaction between mites and neonicotinoids that resulted in significantly reduced survival of long-lived winter honeybees. Even though these mites are potent vectors of viruses, the virus-insecticide interaction had no significant impact. The data suggest a previously overlooked mechanism possibly explaining recent unsustainably high losses of managed A. mellifera honeybee colonies in many regions of the world. Future mitigation efforts should concentrate on developing sustainable agro-ecosystem management schemes that incorporate reduced use of neonicotinoids and sustainable solutions for V. destructor mites.