Honeybee Cells Research Articles

Assessment of Mitochondrial Function in the AmE-711 Honey Bee Cell Line: Boscalid and Pyraclostrobin Effects.

There is a growing concern that chronic exposure to fungicides contributes to negative effects on honey bee development, life span, and behavior. Field and caged-bee studies have helped to characterize the adverse outcomes (AOs) of environmentally relevant exposures, but linking AOs to molecular/cellular mechanisms of toxicity would benefit from the use of readily controllable, simplified host platforms like cell lines. Our objective was to develop and optimize an in vitro-based mitochondrial toxicity assay suite using the honey bee as a model pollinator, and the electron transport chain (ETC) modulators boscalid and pyraclostrobin as model fungicides. We measured the effects of short (~30 min) and extended exposures (16-24 h) to boscalid and pyraclostrobin on AmE-711 honey bee cell viability and mitochondrial function. Short exposure to pyraclostrobin did not affect cell viability, but extended exposure reduced viability in a concentration-dependent manner (median lethal concentration = 4175 µg/L; ppb). Mitochondrial membrane potential (MMP) was affected by pyraclostrobin in both short (median effect concentration [EC50] = 515 µg/L) and extended exposure (EC50 = 982 µg/L) scenarios. Short exposure to 10 and 1000 µg/L pyraclostrobin resulted in a rapid decrease in the oxygen consumption rate (OCR), approximately 24% reduction by 10 µg/L relative to the baseline OCR, and 64% by 1000 µg/L. Extended exposure to 1000 µg/L pyraclostrobin reduced all respiratory parameters (e.g., spare capacity, coupling efficiency), whereas 1- and 10-µg/L treatments had no significant effects. The viability of AmE-711 cells, as well as the MMP and cellular respiration were unaffected by short and extended exposures to boscalid. The present study demonstrates that the AmE-711-based assessment of viability, MMP, and ETC functionality can provide a time- and cost-effective platform for mitochondrial toxicity screening relevant to bees. Environ Toxicol Chem 2024;43:976-987. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

The antiviral activities of Egyptian ethanolic propolis extract and honey bee venom against honey bees infected with multiple viruses in vitro

The demand for using natural compounds as control methods for honey bee viruses have increased in recent decades. In this study, a chemical profile of honey bee venom (HBV) has been determined by FTIR spectroscopy and Egyptian ethanolic propolis (EP) extract by GC-MS and HPLC. Also, the enzymatic activities of HBV were determined. HBV and EP were tested for antiviral activity. The cytotoxicity and cell viability were evaluated using the MTT assay. The results revealed the first identification of methyl gallate and phthalic acid using GC-MS and HPLC. HBV and EP showed antiviral activity against honey bee cell lines infected with deformed wing virus, black queen cell virus, Varroa destructor virus-1 and Kakugo virus. Also, cell proliferation was determined after incubating with HBV and EP, cells were produced with no evidence of cell death up to 1 g/L for HBV and 5 g/L for EP, despite cytotoxicity that was determined at higher doses of both treatments. The data demonstrated that EP and HBV exhibited a significant reduction of all identified viruses in the cultured cell. These findings suggest that HBV and EP in low concentrations could be used as potential supplements and antiviral drugs in honey bee apiaries.

Genotoxic Potential of Thymol on Honey Bee DNA in the Comet Assay

Thymol is a natural essential oil derived from the plant Thymus vulgaris L. It is known to be beneficial for human and animal health and has been used in beekeeping practice against Varroa mite for years. In this study, the genotoxic and antigenotoxic potential of thymol were evaluated on the honey bee (Apis mellifera L.) continuous cell line AmE-711 for the first time. Using the Comet assay, three increasing concentrations (10, 100, and 1000 µg/mL) of thymol were tested. Negative control (non-treated cells) and positive control (cells treated with 100 µM H2O2) were also included. The absence of thymol cytotoxicity was confirmed with the Trypan blue exclusion test. Thymol in the concentration of 10 µg/mL did not increase DNA damage in AmE-711 honey bee cells, while 100 and 1000 µg/mL concentrations showed genotoxic effects. For testing the antigenotoxic effect, all concentrations of thymol were mixed and incubated with H2O2. The antigenotoxic effect against was absent at all concentrations (10, 100, 1000 μg/mL) tested. Moreover, thymol enhanced the H2O2-induced DNA migration in the Comet assay. The obtained results indicate genotoxic effects of thymol on cultured honey bee cells suggesting its careful application in beekeeping practice to avoid possible negative effects on honey bees.

Evaluation of genotoxic and genoprotective effects of Agaricus bisporus extract on AmE-711 honey bee cell line in the Comet assay

Mushroom extracts have been shown to contain bioactive compounds that confer health benefits to humans and animals, including honey bees. Honey bees are often challenged simultaneously with several abiotic (e.g., pesticides and poor nutrition) and biotic (e.g., parasites and pathogens) stressors. Mushroom extracts, for example, those made from Agaricus bisporus, may hold the potential to mitigate the negative effects of these stressors through actions on the honey bee’s immune system, metabolism, and other physiological processes. Exploring the health benefits of mushroom extracts for honey bees requires a basic understanding of their bioactive properties at the cellular level. The aim of the present study was to evaluate, for the first time, the genotoxic and antigenotoxic potential of A. bisporus on the honey bee cell line AmE-711 using the comet assay. Three increasing concentrations of A. bisporus water extract (100, 200, and 400 µg/mL) were tested. The cells in the negative control group were not treated either with A. bisporus extract or with H2O2. In the positive control group, DNA damage was induced with 100 µM H2O2. For antigenotoxic effect, tested extracts were mixed and incubated with H2O2. None of the tested concentrations exerted genotoxic potential, but all showed antigenotoxic effects against H2O2-induced DNA damage. The concentration of 200 μg/mL A. bisporus extract was the most effective in its action against DNA damage. To conclude, A. bisporus extract did not demonstrate genotoxic effects but showed promising antigenotoxic properties. AmE-711 cell line may serve as a cell culture system for genotoxicity investigations.

A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis.

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace’s insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

Investigating Virus-Host Interactions in Cultured Primary Honey Bee Cells.

Simple SummaryHoney bees are eusocial insects that live in colonies comprised of ~30,000 individuals. They are the primary pollinators of plants that produce fruits, nuts, and vegetables. High annual losses of honey bee colonies have made it challenging for beekeepers to provide enough colonies to meet the demand for pollination services. Multiple stressors, including viruses, contribute to colony deaths. To better understand viral pathogenesis and bee antiviral defense mechanisms, we obtained primary cells from honey bee larvae and pupae, maintained them in culture, and infected them with a panel of viruses. We determined that larval hemocytes, which are primary immune cells, support replication of a honey bee virus (sacbrood virus) and a model virus (Flock House virus). Similarly, we determined that mixed cell populations derived from honey bee pupae support replication of sacbrood virus, Flock House virus, and another honey bee virus (deformed wing virus). Evaluation of the host cellular level response to infections with each of these viruses revealed unique expression profiles of three immune genes. In summary, this study demonstrates the utility of honey bee primary cell cultures to investigate the impacts of virus infection on honey bees at the cellular level, which in turn affects individual bee and colony health.Honey bee (Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses (i.e., sacbrood virus (SBV) and deformed wing virus (DWV)) and an insect model virus, Flock House virus (FHV). Virus abundances were quantified over the course of infection. The production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer of deformed wing virus or Flock House virus from infected cell cultures. Initial characterization of the honey bee antiviral immune responses at the cellular level indicated that there were virus-specific responses, which included increased expression of bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells. Additional studies are required to further elucidate virus-specific honey bee antiviral defense mechanisms. The continued use of cultured primary honey bee cells for studies that involve multiple viruses will address this knowledge gap.

Characterizing the Honey Bee Interactome using Mass Spectrometry‐Based Proteomics

Western honey bees (Apis mellifera), as a superorganism, have an essential ecological and economic role as pollinators. However, various understudied pathogens infecting honey bees pose a significant threat to their health. One such pathogen is Nosema ceranae, a microsporidian parasite which rapidly proliferates in the honey bee ventriculus (midgut) cells, weakening their immune systems by competing for nutrients. Upon completing their lifecycle in the midgut cells, Nosema spores rupture host cells and spread into the rest of the gut and subsequently to other bees in the colony. The resulting symptoms of the diseased colony have been attributed to colony death. We hypothesize that host-pathogen interactions underpin Nosema infection in honey bees, and therefore understanding pathological protein interactions will suggest tools for combating Nosema. To this end, we used an optimized version of the mass spectrometry-based co-fractionation experiment described in Kristensen et al. (2012) This method uses size exclusion chromatography (SEC) to separate protein complexes into fractionated samples. After measuring protein amounts in each fraction through mass spectrometry, a machine learning pipeline detects proteins with similar separation profiles as protein interactions (Stacey et al., 2017). We can then assess the effects of Nosema infection. Using an in vivo approach, we first constructed a honey bee interactome from uninfected midgut cells. This is the first honey bee interactome to date. Validation experiments using co-immunoprecipitation coupled with mass spectrometry (IP-MS) demonstrate that this interactome is high quality and will be a valuable tool to future researchers. Using this interactome as a baseline, we can then compare interactomes derived from infected honey bee cells to identify the protein-based mechanisms of Nosema infection. Preliminary results from this highlight interactome changes in various processes such as peroxiredoxin activity and carbohydrate binding. This work presents the first honey bee interactome map. In addition, we are gaining novel insight into how protein interactions change upon infection.

Effects of Prebiotics and Probiotics on Honey Bees (Apis mellifera) Infected with the Microsporidian Parasite Nosema ceranae.

Nosema ceranae is a microsporidian fungus that parasitizes the midgut epithelial cells of honey bees, Apis mellifera. Due to the role that midgut microorganisms play in bee health and immunity, food supplementation with prebiotics and probiotics may assist in the control of N. ceranae. The dietary fiber prebiotics acacia gum, inulin, and fructooligosaccharides, as well as the commercial probiotics Vetafarm Probotic, Protexin Concentrate single-strain (Enterococcus faecium), and Protexin Concentrate multi-strain (Lactobacillus acidophilus, L. plantarum, L. rhamnosus, L. delbrueckii, Bifidobacterium bifidum, Streptococcus salivarius, and E. faecium) were tested for their effect on N. ceranae spore loads and honey bee survivorship. Bees kept in cages were inoculated with N. ceranae spores and single-dose treatments were administered in sugar syrup. Acacia gum caused the greatest reduction in N. ceranae spore numbers (67%) but also significantly increased bee mortality (62.2%). However, Protexin Concentrate single-strain gave similarly reduced spore numbers (59%) without affecting the mortality. In a second experiment, multiple doses of the probiotics revealed significantly reduced spore numbers with 2.50 mg/mL Vetafarm Probotic, and 0.25, 1.25, and 2.50 mg/mL Protexin Concentrate single-strain. Mortality was also significantly reduced with 1.25 mg/mL Protexin Concentrate single-strain. N. ceranae-inoculated bees fed 3.75 mg/mL Vetafarm Probotic had higher survival than N. ceranae-inoculated bees, which was similar to that of non-inoculated bees, while N. ceranae-inoculated bees fed 2.50 mg/mL Protexin Concentrate single-strain, had significantly higher survival than both N. ceranae-inoculated and non-inoculated bees. Protexin Concentrate single-strain is promising as it can reduce N. ceranae proliferation and increase bee survivorship of infected bees, even compared to healthy, non-infected bees.

DWV Infection in vitro Using Honey Bee Pupal Tissue.

The deformed wing virus (DWV) has been best characterized among honey bee viruses; however, very little is known regarding the mechanisms of viral infection and replication due to the lack of immortalized honey bee cell lines. To solve this problem, we established an in vitro system using honey bee pupal tissue to reconstruct DWV binding and entry into the host cell, followed by translation of the RNA genome and polyprotein processing using RNA-dependent RNA polymerase (RdRP) as a marker. Using this system, the P-domain of the virion subunit VP1 was found to be essential for DWV infection, but not for binding and entry into the cell. DWV efficiently infected the head tissue derived from early but not late pupa, suggesting that undifferentiated cells are targeted for viral infection. Furthermore, we found that inhibitors of mammalian picornavirus 3C-protease, rupintrivir and quercetin suppressed RdRP synthesis, indicating that this in vitro system is also useful for screening a compound to control viral infection. Our in vitro system may help to understand the mechanism of DWV infection in host cells.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis Mellifera).

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Aberrant cocoons found on honey bee comb cells are found to be Osmia cornifrons (Radoszkowski) (Hymenoptera: Megachillidae)

Potential biological threats to honey bees must be identified quickly, before making disruptive and costly decisions. Here we describe numerous Osmia cornifrons (Hymenoptera: Megachillidae) cocoons in honey bee cells from one bee hive in Ohio. The developing Osmia cells presented themselves as a mystery at first, catching the attention of regulatory agencies. Along with identifying this species as a presumably benign resident in honey bee colonies, our observations suggest Osmia may use stored honey bee resources to provision offspring. Conceivably, resident honey bees might even act as surrogates because Osmia cocoons were attached to one another with honey bee wax, it seems likely that honey bee hosts were present during Osmia development. Along with resolving a potentially new biotic threat to honey bees, this diagnosis suggests a method for mass production of Osmia pollinators using an array of single-cell foundation.

Cell Lines for Honey Bee Virus Research.

With ongoing colony losses driven in part by the Varroa mite and the associated exacerbation of the virus load, there is an urgent need to protect honey bees (Apis mellifera) from fatal levels of virus infection and from the non-target effects of insecticides used in agricultural settings. A continuously replicating cell line derived from the honey bee would provide a valuable tool for the study of molecular mechanisms of virus–host interaction, for the screening of antiviral agents for potential use within the hive, and for the assessment of the risk of current and candidate insecticides to the honey bee. However, the establishment of a continuously replicating honey bee cell line has proved challenging. Here, we provide an overview of attempts to establish primary and continuously replicating hymenopteran cell lines, methods (including recent results) of establishing honey bee cell lines, challenges associated with the presence of latent viruses (especially Deformed wing virus) in established cell lines and methods to establish virus-free cell lines. We also describe the potential use of honey bee cell lines in conjunction with infectious clones of honey bee viruses for examination of fundamental virology.

Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa destructor in a non-propagative manner

Honey bees, the primary managed insect pollinator, suffer considerable losses due to Deformed wing virus (DWV), an RNA virus vectored by the mite Varroa destructor. Mite vectoring has resulted in the emergence of virulent DWV variants. The basis for such changes in DWV is poorly understood. Most importantly, it remains unclear whether replication of DWV occurs in the mite. In this study, we exposed Varroa mites to DWV type A via feeding on artificially infected honey bees. A significant, 357-fold increase in DWV load was observed in these mites after 2 days. However, after 8 additional days of passage on honey bee pupae with low viral loads, the DWV load dropped by 29-fold. This decrease significantly reduced the mites’ ability to transmit DWV to honey bees. Notably, negative-strand DWV RNA, which could indicate viral replication, was detected only in mites collected from pupae with high DWV levels but not in the passaged mites. We also found that Varroa mites contain honey bee mRNAs, consistent with the acquisition of honey bee cells which would additionally contain DWV replication complexes with negative-strand DWV RNA. We propose that transmission of DWV type A by Varroa mites occurs in a non-propagative manner.

Impact of Nosema ceranae and Nosema apis on individual worker bees of the two host species (Apis cerana and Apis mellifera) and regulation of host immune response

Nosema apis and Nosema ceranae are obligate intracellular microsporidian parasites infecting midgut epithelial cells of host adult honey bees, originally Apis mellifera and Apis cerana respectively. Each microsporidia cross-infects the other host and both microsporidia nowadays have a worldwide distribution. In this study, cross-infection experiments using both N. apis and N. ceranae in both A. mellifera and A. cerana were carried out to compare pathogen proliferation and impact on hosts, including host immune response. Infection by N. ceranae led to higher spore loads than by N. apis in both host species, and there was greater proliferation of microsporidia in A. mellifera compared to A. cerana. Both N. apis and N. ceranae were pathogenic in both host Apis species. N. ceranae induced subtly, though not significantly, higher mortality than N. apis in both host species, yet survival of A. cerana was no different to that of A. mellifera in response to N. apis or N. ceranae. Infections of both host species with N. apis and N. ceranae caused significant up-regulation of AMP genes and cellular mediated immune genes but did not greatly alter apoptosis-related gene expression. In this study, A. cerana enlisted a higher immune response and displayed lower loads of N. apis and N. ceranae spores than A. mellifera, suggesting it may be better able to defend itself against microsporidia infection. We caution against over-interpretation of our results, though, because differences between host and parasite species in survival were insignificant and because size differences between microsporidia species and between host Apis species may alternatively explain the differential proliferation of N. ceranae in A. mellifera.

The hexagonal shape of the honeycomb cells depends on the construction behavior of bees.

The hexagonal shape of the honey bee cells has attracted the attention of humans for centuries. It is now accepted that bees build cylindrical cells that later transform into hexagonal prisms through a process that it is still debated. The early explanations involving the geometers’ skills of bees have been abandoned in favor of new hypotheses involving the action of physical forces, but recent data suggest that mechanical shaping by bees plays a role. However, the observed geometry can arise only if isodiametric cells are previously arranged in a way that each one is surrounded by six other similar cells; here I suggest that this is a consequence of the building program adopted by bees and propose a possible behavioral rule ultimately accounting for the hexagonal shape of bee cells.

Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees.

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection.

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Proteome profile and lentiviral transduction of cultured honey bee (Apis mellifera L.) cells

Honey bees (Apis mellifera L.) play a vital role in agriculture as pollinators, and serve as model organisms of social behaviour and immunity. The lack of both immortalized cell lines and methods to introduce recombinant DNA reliably into primary cells hinders cellular and molecular studies in this organism. We hereby demonstrate the expression of a GFP gene delivered by lentivirus transduction to cultured embryonic cells. The success of this approach indicates that viral transduction could be used to deliver constitutively active oncogenes in order to immortalize honey bee cells. We were able to revive cells successfully after several months of cryogenic storage and we show how the proteome varies between freshly collected and cultured embryonic cells.

Comparative virulence of Nosema ceranae and Nosema apis in individual European honey bees

Nosema apis and Nosema ceranae are intracellular microsporidian parasites infecting the midgut epithelial cells of adult honey bees. N. ceranae was considered to be restricted to the Asian honey bee, Apis cerana, but is nowadays a parasite found also in the European honey bee ( Apis mellifera) across most of the world. Recent surveys and experimental work suggest that N. ceranae is a serious threat to the global beekeeping industry. It has been suggested that N. ceranae induces significantly higher mortality in honey bees than N. apis, but little is known about their comparative virulence. In this study, we used in vivo infection experiments to study the two parasites’ different virulence (i.e. multiplication rate and infectivity). A qPCR was developed to elucidate within host competition between the two parasites using mixed infections. The outcome of the experiments indicates minor differences in infectious dose and multiplication rate between the two species. Moreover, the mortality caused by N. ceranae was not significantly higher than for N. apis and N. ceranae appeared to have no competitive advantage within host.

A scientific note on the fungusBeauveria bassianainfectingVarroa destructorin worker brood cells in honey bee hives

We report on the isolation of the mitosporic fungus Beauveria bassiana from varroa mites, Varroa destructor, in capped worker brood cells of honey bees, Apis mellifera. To our knowledge, this is the first record of B. bassiana from mites collected from bee brood. The brood chamber is the area of the hive where bees maintain high constant temperatures of 33–36 ◦C (Le Conte et al., 1990). An entomopathogenic fungus may be less efficacious because of poor germination at the high temperatures inside brood cells. Natural enemies of the varroa mite are few and, until recently, included no records of fungal pathogens (Chandler et al., 2001). However, a number of studies have shown that varroa mites are highly susceptible to infection by a range of entomopathogenic fungi, including B. bassiana (James, 2009). Many entomopathogenic fungi have a ubiquitous distribution and a wide host range, thus one might expect foraging bees to frequently carry fungus conidia into the bee hive from their environment. The lack of observed natural infections of varroa mites by these fungi could be due to a combination of hygienic behaviour of worker bees and the harsh environmental conditions in bee colonies. Nevertheless, Meikle et al. (2006) isolated B. bassiana from approximately 0.2% of varroa mites collected from a number of apiaries in southern France and documented that natural infections could indeed be found. B. bassiana has also been reported from varroa mites in southern Spain (Garcia-Fernandez et al., 2008).