Development of a 44K SNP assay focussing on the analysis of a varroa-specific defence behaviour in honey bees (Apis mellifera carnica)

Honey bees are exposed to a number of damaging pathogens and parasites. The most destructive among them, affecting mainly the brood, is Varroa destructor. A promising approach to prevent its spread is to breed for Varroa-tolerant honey bees. A trait that has been shown to provide significant resistance against the Varroa mite is hygienic behaviour, a behavioural response of honey bee workers to brood diseases in general. This study reports the development of a 44K SNP assay, specifically designed for the analysis of hygienic behaviour of individual worker bees (Apis mellifera carnica) directed against V. destructor. Initially, 70,000 SNPs chosen from a large set of SNPs published by the Honey Bee Genome Project were validated for their suitability in the analysis of the Varroa resistance trait 'uncapping of Varroa-infested brood'. This was achieved by genotyping of pooled DNA samples of trait bearers and two trait-negative controls using next-generation sequencing. Approximately 36,000 of these validated SNPs and another 8000 SNPs not validated in this study were selected for the construction of a SNP assay. This assay will be employed in following experiments to analyse individualized DNA samples in order to identify quantitative trait loci (QTL) involved in the control of the investigated trait and to evaluate and possibly confirm QTL found in other studies. However, this assay is not just suitable to study Varroa tolerance, it is as well applicable to analyse any other trait in honey bees. In addition, because of its high density, this assay provides access into genomic selection with respect to several traits considered in honey bee breeding. It will become publicly available via AROS Applied Biotechnology AS, Aarhus, Denmark, before the end of the year 2011.     

Tool for genomic selection and breeding to evolutionary adaptation: Development of a 100K single nucleotide polymorphism array for the honey bee

High‐throughput high‐density genotyping arrays continue to be a fast, accurate, and cost‐effective method for genotyping thousands of polymorphisms in high numbers of individuals. Here, we have developed a new high‐density SNP genotyping array (103,270 SNPs) for honey bees, one of the most ecologically and economically important pollinators worldwide. SNPs were detected by conducting whole‐genome resequencing of 61 honey bee drones (haploid males) from throughout Europe. Selection of SNPs for the chip was done in multiple steps using several criteria. The majority of SNPs were selected based on their location within known candidate regions or genes underlying a range of honey bee traits, including hygienic behavior against pathogens, foraging, and subspecies. Additionally, markers from a GWAS of hygienic behavior against the major honey bee parasite Varroa destructor were brought over. The chip also includes SNPs associated with each of three major breeding objectives—honey yield, gentleness, and Varroa resistance. We validated the chip and make recommendations for its use by determining error rates in repeat genotypings, examining the genotyping performance of different tissues, and by testing how well different sample types represent the queen's genotype. The latter is a key test because it is highly beneficial to be able to determine the queen's genotype by nonlethal means. The array is now publicly available and we suggest it will be a useful tool in genomic selection and honey bee breeding, as well as for GWAS of different traits, and for population genomic, adaptation, and conservation questions.     

Honey bee colony collapse disorder is possibly caused by a dietary pyrethrum deficiency

Industrialized farming relies on bee keepers transporting hives to the vicinity of large areas of mono-crops for crop pollination. Hives are typically moved multiple times per growing season to satisfy the pollination need. A phenomenon wherein colonies of honey bees collapse in large numbers has been threatening crops in North America. Honey bees are hosts to at least two pathogenic mites; Varroa destructor and Acarapis woodi (a tracheal mite). Pyrethrums are a group of flowering plants which include Chrysanthemum coccineum, Chrysanthemum cinerariifolium, Chrysanthemum marschallii, and related species. These plants produce potent insecticides, also named pyrethrums, which are powerful mite toxins. We believe that a honey bee dietary deficiency of pyrethrums and other micro-nutrients from pyrethrum producing plants allows parasitic mites to either kill the honey bees directly or reduce honey bee resistance to other pathogens. Intermittent feeding of honey bees on pyrethrum producing plants might reverse or prevent colony collapse disorder.     

The resistance mechanism of the Asian honey bee,Apis cerana Fabr., to an ectoparasitic mite,Varroa jacobsoni Oudemans

A behavioral and physiological resistance mechanism of the Asian honey bee (Apis cerana) to an ectoparasitic mite, Varroa jacobsoni, which causes severe damage to the European honey bee (Apis mellifera) in the beekeeping industry worldwide, is reported here for the first time. Parasitism by the mite induced Asian worker bees to perform a series of cleaning behaviors that effectively removed the mites from the bodies of the adult host bees. The mites were subsequently killed and removed from the bee hives in a few seconds to a few minutes. The grooming behavior consists of self-cleaning, grooming dance, nestmate cleaning, and group cleaning. Worker bees can also rapidly and effectively remove the mites from the brood. The European bee showed cleaning behavior at low frequency and generally failed to remove the mites from both the adult bees and the brood.     

Bidirectional Transfer of RNAi between Honey Bee and Varroa destructor: Varroa Gene Silencing Reduces Varroa Population

The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.     

Correlation of proteome-wide changes with social immunity behaviors provides insight into resistance to the parasitic mite, Varroa destructor, in the honey bee (Apis mellifera)

Background

Disease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies.

Results

We sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae.

Conclusions

Our results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation.     

Twenty-five-year study of Nosema spp. in honey bees (Apis mellifera) in Serbia

A total of 7386 samples of adult honey bees from different areas of Serbia (fifteen regions and 79 municipalities) were selected for light microscopy analysis for Nosema species during 1992–2017. A selection of honey bee samples from colonies positive for microsporidian spores during 2009–2011, 2015 and 2017 were then subjected to molecular diagnosis by multiplex PCR using specific primers for a region of the 16S rRNA gene of Nosema species. The prevalence of microsporidian spore-positive bee colonies ranged between 14.4% in 2013 and 65.4% in 1992. PCR results show that Nosema ceranae is not the only Nosema species to infect honey bees in Serbia. Mixed N. apis/N. ceranae infections were detected in the two honey bee samples examined by mPCR during 2017. The beekeeping management of disease prevention, such as replacement of combs and queens and hygienic handling of colonies are useful in the prevention of Nosema infection.     

Genetic bases of tolerance to Varroa destructor in honey bees (Apis mellifera L.)

Currently, the Varroa destructor mite is the most serious parasite of honey bees (Apis mellifera) and has become a nearly cosmopolitan species. The mite not only causes damage by feeding on the haemolymph of honey bees, but it also transmits viruses, which have been implicated in colony collapse disorder. The major research goal has been to breed mite-tolerant honey bee lines in order to reduce the amount of pesticide used, because pesticides can promote the evolution of resistance in mites. In this review, we describe different behavioural traits and genes that may be part of the defence against the Varroa mite. Specifically, we review grooming behaviour, Varroa-sensitive hygiene and the suppression of mite reproduction. A large number of candidate genes have been identified by Quantitative Trait Loci studies, and through gene expression studies their function and effect have been elucidated. Results from the studies discussed can be used in apiary practice.     

Varroa destructor infestation in untreated honey bee (Hymenoptera: Apidae) colonies selected for hygienic behavior

Honey bee (Apis mellifera L.) colonies bred for hygienic behavior were tested in a large field trial to determine if they were able to resist the parasitic mite Varroa destructor better than unselected colonies of"Starline" stock. Colonies bred for hygienic behavior are able to detect, uncap, and remove experimentally infested brood from the nest, although the extent to which the behavior actually reduces the overall mite-load in untreated, naturally infested colonies needed further verification. The results indicate that hygienic colonies with queens mated naturally to unselected drones had significantly fewer mites on adult bees and within worker brood cells than Starline colonies for up to 1 yr without treatment in a commercial, migratory beekeeping operation. Hygienic colonies actively defended themselves against the mites when mite levels were relatively low. At high mite infestations (>15% of worker brood and of adult bees), the majority of hygienic colonies required treatment to prevent collapse. Overall, the hygienic colonies had similar adult populations and brood areas, produced as much honey, and had less brood disease than the Starline colonies. Thus, honey bees bred for hygienic behavior performed as well if not better than other commercial lines of bees and maintained lower mite loads for up to one year without treatment.     

Evaluation of the Distribution and Impacts of Parasites,Pathogens, and Pesticides on Honey Bee (Apis mellifera) Populations in East Africa

In East Africa, honey bees (Apis mellifera) provide critical pollination services and income for small-holder farmers and rural families. While honey bee populations in North America and Europe are in decline, little is known about the status of honey bee populations in Africa. We initiated a nationwide survey encompassing 24 locations across Kenya in 2010 to evaluate the numbers and sizes of honey bee colonies, assess the presence of parasites (Varroa mites and Nosema microsporidia) and viruses, identify and quantify pesticide contaminants in hives, and assay for levels of hygienic behavior. Varroa mites were present throughout Kenya, except in the remote north. Levels of Varroa were positively correlated with elevation, suggesting that environmental factors may play a role in honey bee host-parasite interactions. Levels of Varroa were negatively correlated with levels of hygienic behavior: however, while Varroa infestation dramatically reduces honey bee colony survival in the US and Europe, in Kenya Varroa presence alone does not appear to impact colony size. Nosema apis was found at three sites along the coast and one interior site. Only a small number of pesticides at low concentrations were found. Of the seven common US/European honey bee viruses, only three were identified but, like Varroa, were absent from northern Kenya. The number of viruses present was positively correlated with Varroa levels, but was not correlated with colony size or hygienic behavior. Our results suggest that Varroa, the three viruses, and Nosema have been relatively recently introduced into Kenya, but these factors do not yet appear to be impacting Kenyan bee populations. Thus chemical control for Varroa and Nosema are not necessary for Kenyan bees at this time. This study provides baseline data for future analyses of the possible mechanisms underlying resistance to and the long-term impacts of these factors on African bee populations.     

PCR-based detection of a tracheal mite of the honey bee Acarapis woodi

The effects of the tracheal mite Acarapis woodi on the health of honey bees have been neglected since the prevalence of Varroa mites to Apis mellifera colonies. However, tracheal mite infestation of honey bee colonies still occurs worldwide and could impose negative impact on apiculture. The detection of A. woodi requires the dissection of honey bees followed by microscopic observation of the tracheal sacs. We thus developed PCR methods to detect A. woodi. These methods facilitate rapid and sensitive detection of A. woodi in many honey bee samples for epidemiologic surveys.     

Population growth of Varroa destructor (Acari: Varroidae) in commercial honey bee colonies treated with beta plant acids

Varroa (Varroa destuctor Anderson and Trueman) populations in honey bee (Apis mellifera L.) colonies might be kept at low levels by well-timed miticide applications. HopGuard^® (HG) that contains beta plant acids as the active ingredient was used to reduce mite populations. Schedules for applications of the miticide that could maintain low mite levels were tested in hives started from either package bees or splits of larger colonies. The schedules were developed based on defined parameters for efficacy of the miticide and predictions of varroa population growth generated from a mathematical model of honey bee colony–varroa population dynamics. Colonies started from package bees and treated with HG in the package only or with subsequent HG treatments in the summer had 1.2–2.1 mites per 100 bees in August. Untreated controls averaged significantly more mites than treated colonies (3.3 mites per 100 bees). By October, mite populations ranged from 6.3 to 15.0 mites per 100 bees with the lowest mite numbers in colonies treated with HG in August. HG applications in colonies started from splits in April reduced mite populations to 0.12 mites per 100 bees. In September, the treated colonies had significantly fewer mites than the untreated controls. Subsequent HG applications in September that lasted for 3 weeks reduced mite populations to levels in November that were significantly lower than in colonies that were untreated or had an HG treatment that lasted for 1 week. The model accurately predicted colony population growth and varroa levels until the fall when varroa populations measured in colonies established from package bees or splits were much greater than predicted. Possible explanations for the differences between actual and predicted mite populations are discussed.     

Deformed wing virus associated with <Emphasis Type="Italic">Tropilaelaps mercedesae</Emphasis> infesting European honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelaps mercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.     

Host brood traits,independent of adult behaviours,reduce Varroa destructor mite reproduction in resistant honeybee populations

The ectoparasitic mite Varroa destructor is an invasive species of Western honey bees (Apis mellifera) and the largest pathogenic threat to their health world-wide. Its successful invasion and expansion is related to its ability to exploit the worker brood for reproduction, which results in an exponential population growth rate in the new host. With invasion of the mite, wild honeybee populations have been nearly eradicated from Europe and North America, and the survival of managed honeybee populations relies on mite population control treatments. However, there are a few documented honeybee populations surviving extended periods without control treatments due to adapted host traits that directly impact Varroa mite fitness. The aim of this study was to investigate if Varroa mite reproductive success was affected by traits of adult bee behaviours or by traits of the worker brood, in three mite-resistant honey bee populations from Sweden, France and Norway. The mite’s reproductive success was measured and compared in broods that were either exposed to, or excluded from, adult bee access. Mite-resistant bee populations were also compared with a local mite-susceptible population, as a control group. Our results show that mite reproductive success rates and mite fecundity in the three mite-resistant populations were significantly different from the control population, with the French and Swedish populations having significantly lower reproductive rates than the Norwegian population. When comparing mite reproduction in exposed or excluded brood treatments, no differences were observed, regardless of population. This result clearly demonstrates that Varroa mite reproductive success can be suppressed by traits of the brood, independent of adult worker bees.     

Selection for barriers between honey bees and a devastating parasite

Rivaling pesticides and a dearth of flowers, the parasitic mite Varroa destructor presents a tremendous threat to western honey bees, Apis mellifera. A longstanding, but minor, pest for the Asian honey bee Apis cerana, these obligate bee parasites feast on developing and adult A. mellifera across several continents. Varroa reproduction is limited to a short window when developing bee pupae are concealed in wax cells. Mated females target developing bees just before pupation and then have about one day to initiate reproduction, eventually laying one male and up to several female offspring. Female mites often fail to reproduce at all, instead waiting in cells until their bee host finishes development and then hitching dangerous rides on a succession of adult bees for up to several weeks, before scouting for a new host pupa. In this issue of Molecular Ecology, Conlon et al. (2019) have explored mite reproductive success via a clever and thought‐provoking association study. In so doing, they have identified a protein whose actions could be integral to the dance between bees and their mite parasites.     

Honey bee colonies from different races show variation in defenses against the varroa mite in a ‘common garden’

Honey bee [Apis mellifera L. (Hymenoptera: Apidae)] genetic diversity may be the key to responding to novel health challenges faced by this important pollinator. In this study, we first compared colonies of four honey bee races, A. m. anatoliaca, A. m. carnica, A. m. caucasica, and A. m. syriaca from Turkey, with respect to honey storage, bee population size, and defenses against varroa. The mite Varroa destructor Anderson & Trueman (Acari: Varroidae) is an important pest of honey bee colonies. There are genetic correlates with two main defenses of bees against this parasite: hygienic behavior, or removing infested brood, and grooming, which involves shaking and swiping off mites and biting them. In the second part of this study, we examined the relationship of these two types of defenses, hygiene and grooming, and their correlation with infestation rates in 32 genetically diverse colonies in a ‘common garden’ apiary. Mite biting was found to be negatively correlated with mite infestation levels.     

The prevalence of pathogens in honey bee (Apis mellifera) colonies infested with the parasitic mite Varroa jacobsoni

The prevalence of nine honey bee viruses in samples of dead adult bees from Apis mellifera colonies in the Netherlands and Germany infested with the parasitic mite Varroa jacobsoni was compared with virus incidence in uninfested colonies in Britain. In colonies with low mite populations the viruses present and their incidence during the year were similar to the results obtained from British colonies. However, in marked contrast with findings in Britain, acute paralysis virus (APV) was the primary cause of adult bee mortality in German honey bee colonies severely infested with V. jacobsoni. Dead brood from unsealed and sealed infested cells from German colonies with high mite populations also contained much APV. The evidence suggests that V. jacobsoni activates APV replication in adult bees by its feeding behaviour and transmits virus from adult honey bees to pupae. In addition, adult bees, in which APV is multiplying, transmit the virus to unsealed brood in the larval food.     

Intensively Cultivated Landscape and Varroa Mite Infestation Are Associated with Reduced Honey Bee Nutritional State

As key pollinators, honey bees are crucial to many natural and agricultural ecosystems. An important factor in the health of honey bees is the availability of diverse floral resources. However, in many parts of the world, high-intensity agriculture could result in a reduction in honey bee forage. Previous studies have investigated how the landscape surrounding honey bee hives affects some aspects of honey bee health, but to our knowledge there have been no investigations of the effects of intensively cultivated landscapes on indicators of individual bee health such as nutritional physiology and pathogen loads. Furthermore, agricultural landscapes in different regions vary greatly in forage and land management, indicating a need for additional information on the relationship between honey bee health and landscape cultivation. Here, we add to this growing body of information by investigating differences in nutritional physiology between honey bees kept in areas of comparatively low and high cultivation in an area generally high agricultural intensity in the Midwestern United States. We focused on bees collected directly before winter, because overwintering stress poses one of the most serious problems for honey bees in temperate climates. We found that honey bees kept in areas of lower cultivation exhibited higher lipid levels than those kept in areas of high cultivation, but this effect was observed only in colonies that were free of Varroa mites. Furthermore, we found that the presence of mites was associated with lower lipid levels and higher titers of deformed wing virus (DWV), as well as a non-significant trend towards higher overwinter losses. Overall, these results show that mite infestation interacts with landscape, obscuring the effects of landscape alone and suggesting that the benefits of improved foraging landscape could be lost without adequate control of mite infestations.     

Reproductive biology of <Emphasis Type="Italic">Varroa destructor</Emphasis> in Africanized honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Since its first contact with Apis mellifera, the population dynamics of the parasitic mite Varroa destructor varies from one region to another. In many regions of the world, apiculture has come to depend on the use of acaricides, because of the extensive damage caused by varroa to bee colonies. At present, the mite is considered to contribute to the recent decline of honey bee colonies in North America and Europe. Because in tropical climates worker brood rearing and varroa reproduction occurs all year round, it could be expected that here the impact of the parasite will be even more devastating. Yet, this has not been the case in tropical areas of South America. In Brazil, varroa was introduced more than 30 years ago and got established at low levels of infestation, without causing apparent damage to apiculture with Africanized honey bees (AHB). The tolerance of AHB to varroa is apparently attributable, at least in part, to resistance in the bees. The low fertility of this parasite in Africanized worker brood and the grooming and hygienic behavior of the bees are referred as important factors in keeping mite infestation low in the colonies. It has also been suggested that the type of mite influences the level of tolerance in a honey bee population. The Korea haplotype is predominant in unbalanced host-parasite systems, as exist in Europe, whereas in stable systems, as in Brazil, the Japan haplotype used to predominate. However, the patterns of varroa genetic variation have changed in Brazil. All recently sampled mites were of the Korea haplotype, regardless whether the mites had reproduced or not. The fertile mites on AHB in Brazil significantly increased from 56% in the 1980s to 86% in recent years. Nevertheless, despite the increased fertility, no increase in mite infestation rates in the colonies has been detected so far. A comprehensive literature review of varroa reproduction data, focusing on fertility and production of viable female mites, was conducted to provide insight into the Africanized bee host-parasite relationship.     

The Honey Bee Pathosphere of Mongolia: European Viruses in Central Asia

Parasites and pathogens are apparent key factors for the detrimental health of managed European honey bee subspecies, Apis mellifera. Apicultural trade is arguably the main factor for the almost global distribution of most honey bee diseases, thereby increasing chances for multiple infestations/infections of regions, apiaries, colonies and even individual bees. This imposes difficulties to evaluate the effects of pathogens in isolation, thereby creating demand to survey remote areas. Here, we conducted the first comprehensive survey for 14 honey bee pathogens in Mongolia (N = 3 regions, N = 9 locations, N = 151 colonies), where honey bee colonies depend on humans to overwinter. In Mongolia, honey bees, Apis spp., are not native and colonies of European A. mellifera subspecies have been introduced ~60 years ago. Despite the high detection power and large sample size across Mongolian regions with beekeeping, the mite Acarapis woodi, the bacteria Melissococcus plutonius and Paenibacillus larvae, the microsporidian Nosema apis, Acute bee paralysis virus, Kashmir bee virus, Israeli acute paralysis virus and Lake Sinai virus strain 2 were not detected, suggesting that they are either very rare or absent. The mite Varroa destructor, Nosema ceranae and four viruses (Sacbrood virus, Black queen cell virus, Deformed wing virus (DWV) and Chronic bee paralysis virus) were found with different prevalence. Despite the positive correlation between the prevalence of V. destructor mites and DWV, some areas had only mites, but not DWV, which is most likely due to the exceptional isolation of apiaries (up to 600 km). Phylogenetic analyses of the detected viruses reveal their clustering and European origin, thereby supporting the role of trade for pathogen spread and the isolation of Mongolia from South-Asian countries. In conclusion, this survey reveals the distinctive honey bee pathosphere of Mongolia, which offers opportunities for exciting future research.