Genetic variability of the honey bee mite, Varroa destructor, from a humid continental climatic region of Canada, and temperate and tropical climatic regions of Mexico.

Varroa destructor is a damaging mite of Western honey bees (Apis mellifera). Genetic variability of the mite in different regions of the world could be related to the movement of infested bees or other factors, such as climate. In this study, V. destructor samples were collected from tropical and temperate climate regions of Mexico, and a humid continental climate region of Canada. COX-1 AFLPs showed that all the mites were the Korean haplotype. Four microsatellites revealed nine haplogroups from the continental climate region of Canada, compared to three haplogroups from the tropical and temperate climate regions of Mexico. CytII-ATP sequences showed seven haplogroups from the humid continental climate region vs. two haplogroups from the temperate region and one haplogroup from the tropical region. CytB sequences revealed seven haplogroups from Canada vs. three from Mexico. A comparison of the cytB sequences of the samples from Canada and Mexico to those from a worldwide collection showed that one sequence, designated the cytB1 type, predominated, comprising 57% of the 86 sequences; it clustered with similar sequences that comprised 80% of the sequences, designated family A. CytB1 was predominant in Mexico, but not in Canada. The other 20% of sequences were in families B and C, and all those samples originated from East and Southeast Asia. The microsatellite, cytII-ATP, and cytB markers, all showed higher variability in mites collected in Canada than in Mexico, which could be related to the cooler climate or an earlier invasion and/or multiple mite invasions in Canada.

Effect of feeding chitosan or peptidoglycan on Nosema ceranae infection and gene expression related to stress and the innate immune response of honey bees (Apis mellifera).

Nosema ceranae is a microsporidian parasite that causes nosema disease, an infection of the honey bee (Apis mellifera) midgut. Two pathogen-associated molecular patterns (PAMPs), chitosan and peptidoglycan, and N. ceranae spores were fed to worker bees in sucrose syrup and compared to non-inoculated and N. ceranae-inoculated bees without PAMPs. Both chitosan and peptidoglycan significantly increased bee survivorship and reduced spore numbers due to N. ceranae infection. To determine if these results were related to changes in health status, expression of the immune-related genes, hymenoptaecin and defensin2, and the stress tolerance-related gene, blue cheese, was compared to that of control bees. Compared to the inoculated control, bees with the dose of chitosan that significantly reduced N. ceranae spore numbers showed lower expression of hymenoptaecin and defensin2 early after infection, higher expression mid-infection of defensin2 and lower expression of all three genes late in infection. In contrast, higher expression of defensin2 early in the infection and all three genes late in the infection was observed with peptidoglycan treatment. Changes late in the parasite multiplication stage when mature spores would be released from ruptured host cells are less likely to have contributed to reduced spore production. Based on these results, it is concluded that feeding bees chitosan or peptidoglycan can reduce N. ceranae infection, which is at least partially related to altering the health of the bee by inducing immune and stress-related gene expression.

Impact of Varroa destructor and deformed wing virus on emergence, cellular immunity, wing integrity and survivorship of Africanized honey bees in Mexico.

The ectoparasitic mite Varroa destructor is the primary health problem of honey bees (Apis mellifera) worldwide. Africanized honey bees in Brazil have demonstrated tolerance to the mite, but there is controversy about the degree of mite tolerance of Africanized bees in other countries. This study was conducted to quantify the effect of V. destructor parasitism on emergence, hemocyte concentration, wing integrity and longevity of Africanized honey bees in Mexico. Africanized bee brood were artificially infested with V. destructor mites and held in an incubator until emergence as adults and compared to non-infested controls. Deformed wing virus (DWV) presence was determined in the mites used to infest the bees. After emergence, the bees were maintained in an incubator to determine survivorship. The percentage of worker bees that emerged from parasitized cells (69%) was significantly lower than that of bees emerged from non-infested cells (96%). Newly-emerged parasitized bees had a significantly lower concentration of hemocytes in the hemolymph than non-parasitized bees. Additionally, the proportion of bees with deformed wings that emerged from V. destructor-parasitized cells was significantly higher (54%) than that of the control group (0%). The mean survival time of bees that emerged from infested and non-infested cells was 8.5 ±â€¯0.3 and 14.4 ±â€¯0.4 days, respectively, and the difference was significant. We conclude that V. destructor parasitism and DWV infections kill, cause deformities and inhibit cellular immunity in developing Africanized honey bees, and significantly reduce the lifespan of adult bees in Mexico. These results suggest that the tolerance of Africanized bees to V. destructor is related to adult bee mechanisms.

Evidence of presence and replication of honey bee viruses among wild bee pollinators in subtropical environments.

We determined the presence of six viruses in different bee species collected in subtropical environments. Deformed wing virus (DWV) and black queen cell virus (BQCV) were detected in >90% of honey bee samples and in 50-100% of four stingless bee, two bumble bee and one solitary bee species. Additionally, minus DWV and BQCV RNA strands were detected, indicating that the viruses replicate in several hosts. This is the first report of honey bee viruses replicating in six wild bee species in the tropics. If pathogenic to them, viral infections could result in negative impacts in agricultural and unmanaged ecosystems.

Varroa destructor parasitism reduces hemocyte concentrations and prophenol oxidase gene expression in bees from two populations.

Circulating hemocytes are responsible for defensive and healing mechanisms in the honey bee, Apis mellifera. Parasitism by the mite Varroa destructor and injection of V. destructor homogenate in buffer, but not buffer injection, showed similar reductions in total hemocyte concentrations in both Africanized and European adult honey bees. This indicated that compounds in V. destructor homogenate can have similar effects as V. destructor parasitism and that the response is not solely due to wounding. Samples from honey bees with different hemocyte concentrations were compared for the expression patterns of hemolectin (AmHml), prophenol oxidase (AmPpo), and class C scavenger receptor (AmSRC-C). Of the genes tested, only the expression of AmPpo correlated well with hemocyte counts for all the treatments, indicating that melanization is associated with those responses. Thus, the expression of AmPpo might be a suitable biomarker for hemocyte counts as part of cellular defenses against injection of buffer or mite compounds and V. destructor parasitism and perhaps other conditions involving healing and immunity.

Differential Gene Expression Associated with Honey Bee Grooming Behavior in Response to Varroa Mites.

Honey bee (Apis mellifera) grooming behavior is an important mechanism of resistance against the parasitic mite Varroa destructor. This research was conducted to study associations between grooming behavior and the expression of selected immune, neural, detoxification, developmental and health-related genes. Individual bees tested in a laboratory assay for various levels of grooming behavior in response to V. destructor were also analyzed for gene expression. Intense groomers (IG) were most efficient in that they needed significantly less time to start grooming and fewer grooming attempts to successfully remove mites from their bodies than did light groomers (LG). In addition, the relative abundance of the neurexin-1 mRNA, was significantly higher in IG than in LG, no groomers (NG) or control (bees without mite). The abundance of poly U binding factor kd 68 and cytochrome p450 mRNAs were significantly higher in IG than in control bees. The abundance of hymenoptaecin mRNA was significantly higher in IG than in NG, but it was not different from that of control bees. The abundance of vitellogenin mRNA was not changed by grooming activity. However, the abundance of blue cheese mRNA was significantly reduced in IG compared to LG or NG, but not to control bees. Efficient removal of mites by IG correlated with different gene expression patterns in bees. These results suggest that the level of grooming behavior may be related to the expression pattern of vital honey bee genes. Neurexin-1, in particular, might be useful as a bio-marker for behavioral traits in bees.

Continuous release of oregano oil effectively and safely controls Varroa destructor infestations in honey bee colonies in a northern climate.

The ectoparasitic mite Varroa destructor is responsible for the death of millions of honey bee (Apis mellifera) colonies worldwide. Testing potential miticide compounds with different delivery methods that effectively control V. destructor and have low toxicity for honey bees is crucial to manage this parasite in hives. We determined the varroacide efficacy of three natural compounds delivered to hives with three application methods over a 4-week period. Oxalic acid in a sucrose solution was applied impregnated in cardboard (T1). A mixture of oregano and clove oils in an ethanol-gelatin solution was applied impregnated in absorbent pads (T2). Oregano oil alone was delivered using electric vaporizers (T3) to test the hypothesis that continuous release of miticides increases the varroacidal efficacy of essential oils. The varroa mite control rates for treatments T1-T3 were 76.5 ± 7.11, 57.8 ± 12.79 and 97.4 ± 0.68%, respectively, and there were no differences for bee mortality between control and treatments 1 and 3. Additionally, most mites were killed in the first 2 weeks in T3 colonies compared to the last 2 weeks in colonies of the other treatments. These results demonstrate the importance of continuously releasing natural miticides to achieve safe and high rates of mite control in hives. They also show that oregano oil may be an effective miticide against V. destructor infestations in colonies.

Nosema ceranae is an old resident of honey bee (Apis mellifera) colonies in Mexico, causing infection levels of one million spores per bee or higher during summer and fall.

This study was conducted to identify Nosema spp. and to determine their infection levels in honey bee (Apis mellifera) samples collected in Mexico in 1995-1996. Samples of historical surveys from different countries are of particular interest to support or challenge the hypothesis that the microsporidium Nosema ceranae is a new parasite of A. mellifera that has recently dispersed across the world. We demonstrate that N. ceranae has parasitized honey bees in Mexico since at least 1995 and that the infection levels of this parasite during summer and fall, exceed the threshold at which treatment of honey bee colonies is recommended.

Higher prevalence and levels of Nosema ceranae than Nosema apis infections in Canadian honey bee colonies.

This study was conducted to determine the prevalence and infection levels of the microsporidia fungi Nosema apis and/or Nosema ceranae in honey bee colonies of two Canadian provinces. Three surveys were conducted in the springs of 2008, 2010 and 2012 and PCR identification of Nosema species were performed in samples from 169 and 181 Ontario colonies and from 76 Alberta colonies that tested positive to Nosema spp. Infection levels of positive colonies were determined by microscopy and analyzed by Nosema spp. Results showed that N. ceranae was the dominant species in all three surveys (prevalence range of 41-91 vs. 4-34 % for N. apis), whereas mixed infections were less frequent than single infections (5-25 %). Infection levels of colonies parasitized by N. ceranae were three to five times higher than those of colonies parasitized by N. apis in the three surveys whereas mixed infections showed the highest spore counts. This is the first field study demonstrating significantly higher infection levels in colonies parasitized with either N. ceranae only or with both, N. ceranae and N. apis, than in colonies parasitized with N. apis only. Taken together, these results suggest that N. ceranae may be more virulent and better adapted than N. apis in cold climates such as Canadian environments.

Varroa destructor (Mesostigmata: Varroidae) Parasitism and Climate Differentially Influence the Prevalence, Levels, and Overt Infections of Deformed Wing Virus in Honey Bees (Hymenoptera: Apidae).

The prevalence and loads of deformed wing virus (DWV) between honey bee (Apis mellifera L.) colonies from a tropical and a temperate environment were compared. The interaction between these environments and the mite Varroa destructor in relation to DWV prevalence, levels, and overt infections, was also analyzed. V. destructor rates were determined, and samples of mites, adult bees, brood parasitized with varroa mites and brood not infested by mites were analyzed. DWV was detected in 100% of the mites and its prevalence and loads in honey bees were significantly higher in colonies from the temperate climate than in colonies from the tropical climate. Significant interactions were found between climate and type of sample, with the highest levels of DWV found in varroa-parasitized brood from temperate climate colonies. Additionally, overt infections were observed only in the temperate climate. Varroa parasitism and DWV loads in bees from colonies with overt infections were significantly higher than in bees from colonies with covert infections. These results suggest that interactions between climate, V. destructor, and possibly other factors, may play a significant role in the prevalence and levels of DWV in honey bee colonies, as well as in the development of overt infections. Several hypotheses are discussed to explain these results.

Differential responses of Africanized and European honey bees (Apis mellifera) to viral replication following mechanical transmission or Varroa destructor parasitism.

For the first time, adults and brood of Africanized and European honey bees (Apis mellifera) were compared for relative virus levels over 48 h following Varroa destructor parasitism or injection of V. destructor homogenate. Rates of increase of deformed wing virus (DWV) for Africanized versus European bees were temporarily lowered for 12h with parasitism and sustainably lowered over the entire experiment (48 h) with homogenate injection in adults. The rates were also temporarily lowered for 24h with parasitism but were not affected by homogenate injection in brood. Rates of increase of black queen cell virus (BQCV) for Africanized versus European bees were similar with parasitism but sustainably lowered over the entire experiment with homogenate injection in adults and were similar for parasitism and homogenate injection in brood. Analyses of sac brood bee virus and Israeli acute paralysis virus were limited as detection did not occur after both homogenate injection and parasitism treatment, or levels were not significantly higher than those following control buffer injection. Lower rates of replication of DWV and BQCV in Africanized bees shows that they may have greater viral resistance, at least early after treatment.

New meta-analysis tools reveal common transcriptional regulatory basis for multiple determinants of behavior.

A fundamental problem in meta-analysis is how to systematically combine information from multiple statistical tests to rigorously evaluate a single overarching hypothesis. This problem occurs in systems biology when attempting to map genomic attributes to complex phenotypes such as behavior. Behavior and other complex phenotypes are influenced by intrinsic and environmental determinants that act on the transcriptome, but little is known about how these determinants interact at the molecular level. We developed an informatic technique that identifies statistically significant meta-associations between gene expression patterns and transcription factor combinations. Deploying this technique for brain transcriptome profiles from ca. 400 individual bees, we show that diverse determinants of behavior rely on shared combinations of transcription factors. These relationships were revealed only when we considered complex and variable regulatory rules, suggesting that these shared transcription factors are used in distinct ways by different determinants. This regulatory code would have been missed by traditional gene coexpression or cis-regulatory analytic methods. We expect that our meta-analysis tools will be useful for a broad array of problems in systems biology and other fields.

Honey bee stressor networks are complex and dependent on crop and region.

Honey bees play a major role in crop pollination but have experienced declining health throughout most of the globe. Despite decades of research on key honey bee stressors (e.g., parasitic Varroa destructor mites and viruses), researchers cannot fully explain or predict colony mortality, potentially because it is caused by exposure to multiple interacting stressors in the field. Understanding which honey bee stressors co-occur and have the potential to interact is therefore of profound importance. Here, we used the emerging field of systems theory to characterize the stressor networks found in honey bee colonies after they were placed in fields containing economically valuable crops across Canada. Honey bee stressor networks were often highly complex, with hundreds of potential interactions between stressors. Their placement in crops for the pollination season generally exposed colonies to more complex stressor networks, with an average of 23 stressors and 307 interactions. We discovered that the most influential stressors in a network-those that substantively impacted network architecture-are not currently addressed by beekeepers. Finally, the stressor networks showed substantial divergence among crop systems from different regions, which is consistent with the knowledge that some crops (e.g., highbush blueberry) are traditionally riskier to honey bees than others. Our approach sheds light on the stressor networks that honey bees encounter in the field and underscores the importance of considering interactions among stressors. Clearly, addressing and managing these issues will require solutions that are tailored to specific crops and regions and their associated stressor networks.

Higher prevalence of sacbrood virus in Apis mellifera (Hymenoptera: Apidae) colonies after pollinating highbush blueberries.

Highbush blueberry pollination depends on managed honey bees (Apis mellifera) L. for adequate fruit sets; however, beekeepers have raised concerns about the poor health of colonies after pollinating this crop. Postulated causes include agrochemical exposure, nutritional deficits, and interactions with parasites and pathogens, particularly Melisococcus plutonius [(ex. White) Bailey and Collins, Lactobacillales: Enterococcaceae], the causal agent of European foulbrood disease, but other pathogens could be involved. To broadly investigate common honey bee pathogens in relation to blueberry pollination, we sampled adult honey bees from colonies at time points corresponding to before (t1), during (t2), at the end (t3), and after (t4) highbush blueberry pollination in British Columbia, Canada, across 2 years (2020 and 2021). Nine viruses, as well as M. plutonius, Vairimorpha ceranae, and V. apis [Tokarev et al., Microsporidia: Nosematidae; formerly Nosema ceranae (Fries et al.) and N. apis (Zander)], were detected by PCR and compared among colonies located near and far from blueberry fields. We found a significant interactive effect of time and blueberry proximity on the multivariate pathogen community, mainly due to differences at t4 (corresponding to ~6 wk after the beginning of the pollination period). Post hoc comparisons of pathogens in near and far groups at t4 showed that detections of sacbrood virus (SBV), which was significantly higher in the near group, not M. plutonius, was the primary driver. Further research is needed to determine if the association of SBV with highbush blueberry pollination is contributing to the health decline that beekeepers observe after pollinating this crop.

Viral Quantification in Bee Samples Using Synthetic DNA Sequences with Real-Time PCR (qPCR).

Pathogen spillover between honey bees and wild pollinators is a relatively new and exciting field of study. It is known that some viral diseases are a major threat to honey bee health and, thus, the diagnosis and quantification of honey bee viruses in wild pollinators have gained attention. Pathogen spillover from honey bees to wild bees and the consequences of viral replication to their health still need to be investigated. However, finding positive samples to produce standard curves and include positive controls in real-time PCR (qPCR) assays is challenging. Here we describe the use of synthetic DNA sequences of two variants of deformed wing virus (DWV-A and DWV-B), black queen cell virus (BQCV), sacbrood virus (SBV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV), and Israeli acute paralysis virus (IAPV), to construct standard curves for viral quantification, and for their use as positive controls in qPCR assays.

Breeding honey bees (Apis mellifera L.) for low and high Varroa destructor population growth: Gene expression of bees performing grooming behavior.

Introduction: Social organisms, including honey bees (Apis mellifera L.), have defense mechanisms to control the multiplication and transmission of parasites and pathogens within their colonies. Self-grooming, a mechanism of behavioral immunity, seems to contribute to restrain the population growth of the ectoparasitic mite Varroa destructor in honey bee colonies. Because V. destructor is the most damaging parasite of honey bees, breeding them for resistance against the mite is a high priority of the beekeeping industry. Methods: A bidirectional breeding program to select honey bee colonies with low and high V. destructor population growth (LVG and HVG, respectively) was conducted. Having high and low lines of bees allowed the study of genetic mechanisms underlying self-grooming behavior between the extreme genotypes. Worker bees were classified into two categories: 'light groomers' and 'intense groomers'. The brains of bees from the different categories (LVG-intense, LVG-light, HVG-intense, and HVG-light) were used for gene expression and viral quantification analyses. Differentially expressed genes (DEGs) associated with the LVG and HVG lines were identified. Results: Four odorant-binding proteins and a gustatory receptor were identified as differentially expressed genes. A functional enrichment analysis showed 19 enriched pathways from a list of 219 down-regulated DEGs in HVG bees, including the Kyoto Encyclopedia of Genes and Genomes (KEGG) term of oxidative phosphorylation. Additionally, bees from the LVG line showed lower levels of Apis rhabdovirus 1 and 2, Varroa destructor virus -1 (VDV-1/DWV-B), and Deformed wing virus-A (DWV-A) compared to bees of the HVG line. The difference in expression of odorant-binding protein genes and a gustatory receptor between bee lines suggests a possible link between them and the perception of irritants to trigger rapid self-grooming instances that require the activation of energy metabolic pathways. Discussion: These results provide new insights on the molecular mechanisms involved in honey bee grooming behavior. Differences in viral levels in the brains of LVG and HVG bees showed the importance of investigating the pathogenicity and potential impacts of neurotropic viruses on behavioral immunity. The results of this study advance the understanding of a trait used for selective breeding, self-grooming, and the potential of using genomic assisted selection to improve breeding programs.

The Fungus Nosema ceranae and a Sublethal Dose of the Neonicotinoid Insecticide Thiamethoxam Differentially Affected the Health and Immunity of Africanized Honey Bees.

Honey bees (Apis mellifera L.) are affected by different biotic and abiotic stressors, such as the fungus Nosema ceranae and neonicotinoid insecticides, that negatively impact their health. However, most studies so far conducted have focused on the effect of these stressors separately and in European honey bees. Therefore, this study was conducted to analyze the impact of both stressors, singly and in combination, on honey bees of African descent that have demonstrated resistance to parasites and pesticides. Africanized honey bees (AHBs, Apis mellifera scutellata Lepeletier) were inoculated with N. ceranae (1 × 105 spores/bee) and/or chronically exposed for 18 days to a sublethal dose of thiamethoxam (0.025 ng/bee) to evaluate their single and combined effects on food consumption, survivorship, N. ceranae infection, and immunity at the cellular and humoral levels. No significant effects by any of the stressors were found for food consumption. However, thiamethoxam was the main stressor associated to a significant decrease in AHB survivorship, whereas N. ceranae was the main stressor affecting their humoral immune response by upregulating the expression of the gene AmHym-1. Additionally, both stressors, separately and combined, significantly decreased the concentration of haemocytes in the haemolymph of the bees. These findings indicate that N. ceranae and thiamethoxam differentially affect the lifespan and immunity of AHBs and do not seem to have synergistic effects when AHBs are simultaneously exposed to both stressors.

Prevalence of Adult Honey Bee (Apis mellifera L.) Pests and Pathogens in the Five Beekeeping Regions of Mexico.

Mexico is a major honey producer, but not much information exists about the health status of honey bees (Apis mellifera L.) in the country. This study was conducted to determine the sanitary status of adult honey bees in Mexico's five beekeeping regions. Samples from 369 apiaries were diagnosed to identify pathogens such as Varroa destructor, which was quantified, Acarapis woodi, Nosema spp., and five viruses. Colonies were also inspected for the presence of the small hive beetle (SHB), Aethina tumida. Varroa destructor was found in 83.5% of the apiaries, with the Pacific Coast region having the highest prevalence (>95%) and rates (4.5% ± 0.6). Acarapis woodi was detected in only one apiary from the Pacific Coast, whereas Nosema spp. were prevalent in 48.5% of the apiaries, with the highest and lowest frequencies in the Yucatan Peninsula and North regions (64.6% and 10.2%, respectively). For viruses, deformed wing virus (DWV) was detected in 26.1% of the apiaries, with the highest frequency in the Pacific Coast region (44.7%). Israeli acute paralysis virus (IAPV) was diagnosed in 3.2% of the samples and sacbrood bee virus (SBV) in 23.3% of them, with the highest frequency in the High Plateau region (36.4%). Chronic bee paralysis and Kashmir bee viruses were not detected. SHB prevalence was 25.2% nationwide, with the highest frequency in the Yucatan Peninsula (39.2%). This study shows that the most common parasites of adult honey bees in Mexico are V. destructor and Nosema spp., and that the most prevalent virus is DWV, whereas SHB is highly prevalent in the Yucatan Peninsula. This information could be useful to design disease control strategies for honey bee colonies in different regions of Mexico.

Environmental metagenetics unveil novel plant-pollinator interactions.

Honey bees are efficient pollinators of flowering plants, aiding in the plant reproductive cycle and acting as vehicles for evolutionary processes. Their role as agents of selection and drivers of gene flow is instrumental to the structure of plant populations, but historically, our understanding of their influence has been limited to predominantly insect-dispersed flowering species. Recent metagenetic work has provided evidence that honey bees also forage on pollen from anemophilous species, suggesting that their role as vectors for transmission of plant genetic material is not confined to groups designated as entomophilous, and leading us to ask: could honey bees act as dispersal agents for non-flowering plant taxa? Using an extensive pollen metabarcoding dataset from Canada, we discovered that honey bees may serve as dispersal agents for an array of sporophytes (Anchistea, Claytosmunda, Dryopteris, Osmunda, Osmundastrum, Equisetum) and bryophytes (Funaria, Orthotrichum, Sphagnum, Ulota). Our findings also suggest that honey bees may occasionally act as vectors for the dispersal of aquatic phototrophs, specifically Coccomyxa and Protosiphon, species of green algae. Our work has shed light on the broad resource-access patterns that guide plant-pollinator interactions and suggests that bees could act as vectors of gene flow, and potentially even agents of selection, across Plantae.

Entomopathogenic fungi as potential biocontrol agents of the ecto-parasitic mite, Varroa destructor, and their effect on the immune response of honey bees (Apis mellifera L.).

Three isolates of each of the entomopathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Clonostachys rosea, were assessed for their pathogenicity to the honey bee parasitic mite, Varroa destructor. The fungi were applied to varroa mites by immersing them in a spore solution, and then the inoculated mites were placed on honey bee brood inside capped cells. At 7 days post inoculation (dpi), the three fungi caused significant varroa mortality compared to non-inoculated mites. In brood treated only with varroa mites, expression of the honey bee genes, hymenoptaecin and poly U binding factor 68 Kd (pUf68), decreased over time, while expression of blue cheese (BlCh) and single minded (SiMd) was not affected. In brood inoculated directly only with M. anisopliae or B. bassiana, the emerged adults showed reduced weight indicating infection by the fungi, which was confirmed by observation of hyphae in the brood. Fungal infection of the brood resulted in increased expression of hymenoptaecin, pUf68 and BlCh, but not SiMd. In brood treated with varroa mites that had been inoculated with the fungi, expression of hymenoptaecin, pUf68 and BlCh, but not SiMd, was even more up-regulated. While varroa mites can suppress gene expression in honey bee brood, varroa mites infected with entomopathogenic fungi induced their expression. This may be due to a low level of fungal infection of the bee, which negated the immunosuppression by the mites. Therefore, entomopathogenic fungi could reduce varroa mite damage to honey bee brood by both infecting the parasite and preventing varroa-associated suppression of honey bee immunity.