Managed honey bee colonies (Apis mellifera) in the US continue to experience high overwinter loss rates driven by parasites, pathogens, poor nutrition, and pesticides. To mitigate these losses, inspection and monitoring are critical for identifying traits of colonies in decline and potential causal factors. In this study, we apply molecular methods to associate potential causative agents with colonies in various stages of decline. Initially, we investigated in-hive bee metagenomic RNA isolated from 15 colonies across seven managed operations in California whose adult bee and brood populations were classified as Strong, Medium, or Weak in strength. We discovered that Weak colonies harbored 2.2- and 3.6- fold more viral species than Medium and Strong colonies, respectively, as well as larger viral read pools despite similar library sizes. They also displayed higher nucleotide variation in Varroa-vectored viruses, indicating associations with high mite populations. When investigating differences in host gene expression, we discovered an upregulation of immune-related pathways in Weak colonies relative to Strong. Specifically, Weak colonies upregulated genes related to wound healing, phagocytosis, oxidative stress resistance, apoptosis, and RNA interference. Most antimicrobial peptides were upregulated in Weak colonies, although defensin1 was significantly higher in Strong colonies, along with several detoxification enzymes and the royal jelly peptide apisimin. Weak colonies also showed an upregulation of transcripts tied to abnormal protein digestion. The low levels of viral replication and fewer species of mite-vectored viruses in Strong colonies may be due to successful Varroa management. Strong colonies also displayed upregulated levels of nine different ubiquinone transcripts, arguably reflecting increasing longevity or a younger in-hive population compared to Weak colonies. Overall, these results provide a detailed account of viral metagenomics and associated host responses, providing new insights into the mechanisms underlying honey bee colony decline under comparable management conditions.

In 2025, a swarm of giant honey bees, Apis dorsata dorsata, was detected on board a cargo vessel prior to arrival at the port of Elizabeth, New Jersey, USA. Apis dorsata is a quarantine species not native to the United States, and further inspection of the bees uncovered 28 mites that were identified as Tropilaelaps mercedesae by morphological and DNA barcoding analysis. Tropilaelaps mercedesae is an ectoparasite of honey bees that is not yet present in the USA. An additional mite species, Kuzinia morsei, was also observed. Molecular screening of common honey bee pathogens in 42 intercepted bees showed a 5% prevalence of trypanosomatid infections and a 2% prevalence of American foulbrood. Additionally, Black queen cell virus (BQCV) and Deformed wing virus B (DWV-B) were detected in 38% and 7% of the samples, respectively. To our knowledge, this is the first detection of T. mercedesae and K. morsei on a vessel bound for the USA, as well as the first interception of a whole swarm of A. dorsata dorsata. Phylogenetic analysis of the CO1 sequence data indicated that the intercepted bee swarm originated from a southern India lineage. Our findings indicate that exotic bee swarms can harbor parasitic mites and multiple bee pathogens, both critical to the apiculture industry. Additionally, our findings reveal that Tropilaelaps mites can survive extended periods associated with adult bees and in the absence of brood, suggesting greater potential for long-distance movement and introduction risk. This study provides new insights into Tropilaelaps dispersal and swarm-mediated pathogen movement, thereby improving our ability to predict establishment and outbreak risk crucial for countering invasive agricultural threats. The findings also emphasize that early detection, through coordinated port and shipboard surveillance, and strong interagency collaboration, is vital for reducing the risk of agricultural pest and pathogen introductions into the USA.

The order Picornavirales is a group of highly diverse RNA viruses that includes many pathogens of significance to human and veterinary health, agriculture, and the wider environment. However, the wide range of viruses assigned to the order, together with their genomic variability, and the recent description of numerous 'picorna-like' viruses derived from metagenomic analyses of environmental samples, challenge the established taxonomic classification of members of the order and the criteria for their classification. Here, we combine the existing gold standard, hallmark RNA-directed RNA-polymerase (RdRP) gene sequence-based analysis with helicase sequence-based phylogeny, RdRP structural prediction through the use of ColabFold and Fold Tree, and analysis of coding-complete genomes using GRAViTy-V2, to genetically classify 525 picornaviral genomes and recently described 'picorna-like' viruses. All analyses were conducted with a bespoke, fully automated pipeline for retrieval of genome sequences, domain prediction and extraction, phylogenetic analysis, and output conditioning, which is available as open-source software. Our results reveal broad support for established families as well as for 6 novel families, and 32 new genera. In instances where inconsistencies were found between classification methods, we demonstrate how examination of the pipeline's output may be used to reconcile differences with respect to the genomic features quantified by the analysis. Automated multimodal taxonomic analysis may save significant resources over manual methods and better define demarcation criteria for families and genera.

In January of 2025, U.S. commercial beekeepers reported unusually high honey bee colony losses as they prepared colonies for almond pollination. Two industry groups launched nationwide surveys to document colony losses between June 2024 and March 2025 across all scales of beekeeping. This study analyzes these survey data to assess colony losses, estimate financial impacts, and identify correlations with beekeeper management practices and geographical locations. Unlike past surveys, commercial beekeepers experienced more severe losses than smaller-scale beekeepers during this period. Respondents, managing over half of U.S. colonies, most frequently cited Varroa mites as the cause for their losses. Varroa mites were followed by pesticides and pathogens in the case of commercial beekeepers and by queen failure and weather in the case of smaller-scale beekeepers. Although Varroa was the most frequently cited cause, losses did not significantly differ between users and non-users of amitraz, suggesting that rising amitraz resistance alone does not explain observed trends. Differences in protein and carbohydrate feeding frequencies also played a role in net losses. While colony loss rates and financial concern varied widely among respondents, commercial beekeepers understandably showed higher sensitivity to financial impacts, with concerns increasing linearly with loss severity. This study highlights the value of beekeeper surveys which, alongside direct analyses of bee samples and longitudinal studies, help identify effective management strategies and environmental risks. Such insights are crucial for addressing the leading causes of colony losses on a national scale, and ultimately aid in safeguarding honey bee health, pollination services, and agricultural production.

The gut microbiota-gonadal axis is increasingly recognized, but its reproductive roles remain unclear. Here, we used the Asian honey bee Apis cerana queens as a model to investigate the role of the gut microbiota-gonadal axis on ovary activation. By artificially caging and releasing the mated queens for a short or long period and monitoring the morphological changes of their ovaries, we confirmed that the activation and suppression of the queen ovary could be switched quickly. We found that the ovary weight was positively correlated with the body weight. 16S rRNA sequencing showed ovarian deactivation reduced gut Lactobacillus abundance. Untargeted metabolomics identified purine metabolism as the dominant ovarian pathway, while correlation analyses implicated Lactobacillus in modulating ovarian morphology through purine signaling. This study elucidates microbiota-gonadal crosstalk governing reproduction, providing mechanistic insights with translational potential for reproductive health management.

Social bees, with their specialized gut microbiota and societal transmission between individuals, provide an ideal model for studying host-gut microbiota interactions. While the functional disparities arising from strain-level diversity of gut symbionts and their effects on host health have been studied in Apis mellifera and bumblebees, studies focusing on host-specific investigations of individual strains across different honeybee hosts remain relatively unexplored. In this study, the complete genomic sequences of 17 strains of Gilliamella from A. mellifera, Apis cerana and Bombus terrestris were analyzed. The analysis revealed that the strains of A. mellifera display a more expansive genomic and functional content compared to the strains of A. cerana and B. terrestris. Phylogenetic analysis showed a deep divergence among the Gilliamella strains from different hosts. Additionally, biochemistry tests and antibiotic susceptibility tests revealed that gut strains from A. mellifera exhibited a more extensive pathway for carbohydrate metabolism and a greater resistance to antibiotics than gut strains from A. cerana and B. terrestris. Strains from A. mellifera and A. cerana showed higher colonization efficiency and competitive ability whithin their respective host species, indicating a higher degree of host-specific adaptation of local gut microbiota. In addition, colonization by A. mellifera-derived strain triggers a stronger transcriptional response in the host than A. cerana-derived strain. The variation in the number of differentially expressed genes and the involvement of distinct signaling pathways across these two host species suggest species-specific adaptations to Gilliamella strains. These findings suggest that despite occupying similar niches in the bee gut, strain-level variations can influence microbial functions, and their impact on host physiological functions may vary across different strains.

Microsporidia are disease-causing organisms that can infect invertebrate species. In apiculture, two microsporidians of importance are Vairimorpha (=Nosema) ceranae and Vairimorpha (=Nosema) apis. The taxonomy surrounding the genus assignment of these species has been heavily debated, due to molecular systematic and socio-economic reasons. We provide an update to this debate by developing a 508-gene concatenated protein phylogeny, and a 277-gene concatenated nucleotide phylogeny, to show that these parasites show strong phylogenetic positioning with the Vairimorpha genus and its type species Vairimorpha necatrix. Despite this assignment, we suggest that the terms 'nosema-disease', 'nosemosis' and 'nosematosis' should still be viable for use within apiculture, and be named after the family Nosematidae in which V. ceranae and V. apis sit, instead of the previous genus assignment: Nosema.

Stingless bee-collected pollen, or bee bread (SBB), is a product composed of bee pollen, nectar, and bee salivary enzymes, which undergoes fermentation by microbes in cerumen pots and contains various types of bioactive compounds. This study investigated the carotenoid profiles (using high-performance liquid chromatography, HPLC), and antimicrobial and antioxidant activities of SBB from Tetragonula pagdeni Schwarz. The findings indicated that the predominant carotenoids were beta-cryptoxanthin (2.72 f 0.34-82.94 f 5.62 mu g/g), zeaxanthin (0.40 f 0.14-22.77 f 1.26 mu g/g), and beta-carotene (2.04 f 0.52-15.99 f 0.61 mu g/g). The high total carotenoid and beta-cryptoxanthin contents were highlighted, which suggests SBB as a significant natural carotenoid source. Antimicrobial activities were assessed by determining the minimum bactericidal/fungicidal concentration (MBC/ MFC) and minimum inhibitory concentration (MIC), with average values against the tested pathogens of 15.87 f 2.18 and 7.95 f 1.09 mg/mL (Escherichia coli), 18.86 f 2.82 and 9.45 f 1.41 mg/mL (Methicillin-resistant Staphylococcus aureus; MRSA), and 32.97 f 3.59 and 16.51 f 1.81 mg/mL (Candida albicans), respectively. For the DPPH'assay, the results showed the IC50 values ranged from 6.07 f 0.19 to 21.52 f 0.07 mg/mL, while ABTS'+ IC50 ranged from 1.05 f 0.02 to 3.44 f 0.03 mg/mL. This study represents the first report of carotenoid profiles in SBB samples, supporting its potential as a source of natural carotenoids for functional foods, dietary supplements, and future commercial applications.

United States commercial beekeepers prepare honey bee colonies for almond pollination in California each year in late January to early February. This represents the largest managed pollination event in the world and involves more than half of all U.S. honey bee colonies. In winter 2023, numerous colonies in Florida, which were graded as suitable for almonds (larger than ten frames of bees), dwindled suddenly or altogether died within several weeks, just prior to movement for almonds. The timing of these losses and the resulting morbidity caused severe economic harm to affected operations. This study reports interviews with affected stakeholders, their economic harm, and analyses of pathogens and parasites found in their colonies.

Antibiotics are frequently employed to control bacterial diseases in honeybees, but their broad-spectrum action can disrupt the delicate balance of the gut microbiome, leading to dysbiosis. This imbalance in the gut microbiota of honeybees adversely affects their physiological health and weakens their resistance to pathogens, including viruses that significantly threaten honeybee health. In this study, we investigated whether tetracycline-induced gut microbiome dysbiosis promotes the replication of Israeli acute paralysis virus (IAPV), a key virus associated with colony losses and whether IAPV infection exacerbates gut microbiome dysbiosis. Our results demonstrated that tetracycline-induced gut microbiome dysbiosis increases the susceptibility of honeybees to IAPV infection. The viral titer in worker bees with antibiotic-induced gut microbiome dysbiosis prior to IAPV inoculation was significantly higher than in those merely inoculated with IAPV. Furthermore, we observed a synergistic effect between tetracycline and IAPV on the disruption of the honeybee gut microbiome balance. The progression of IAPV replication could, in turn, exacerbate antibiotic-induced gut microbiome dysbiosis in honeybees. Our research provides novel insights into the role of the gut microbiota in host-virus interactions, emphasizing the complex interplay between antibiotic use, gut microbiome health, and viral susceptibility in honeybees. We highlight the crucial role of a balanced gut microbiota in honey bees for their immune response against pathogens and emphasize the importance of careful, safe antibiotic use in beekeeping to protect these beneficial microbes.

Honey bees and other pollinators face threats from pesticides, imperfect nutrition, and a diverse set of parasites and pathogens. Honey bees are also a research model for development, social behavior, microbiology, and aging. Tackling these questions requires a mix of in-hive and controlled laboratory experiments. We have perfected small-scale, inexpensive, disposable, and rearing arenas for honey bees that have proved useful for hundreds of bioassays with thousands of bees. We describe those arenas here, show their advantages over current hoarding cages, and provide videos demonstrating their many uses.

Honey bees utilize queen mandibular pheromone (QMP) for maintaining social hierarchy and colony development. In controlled cage studies, synthetic QMP is often introduced to mimic natural conditions. However, questions have arisen about the effects of QMP on nosema disease studies. This short report identifies significant early-stage suppression effects of QMP on Nosema (Vairimorpha) ceranae infections. QMP was found to significantly lower infection rates below the reported infectious dose for 50 % infectivity (ID50) and to slow disease development in a dose-independent manner. These effects diminished at doses exceeding ID100. We recommend that studies investigating treatment effects using caged bees avoid QMP to ensure unambiguous results. Additionally, employing multiple infectious doses with shorter incubation times would be useful for evaluating other treatments that may have subtle effects. Furthermore, our findings support previous field studies suggesting that queen replacement reduces nosema disease at levels similar to treatment with fumagillin.

Royal Jelly (RJ) is a natural substance produced by honeybees, serving not only as nutrition for bee brood and queens but also as a functional food due to its health-promoting properties. Despite its well-known broad-spectrum antibacterial activity, the precise molecular mechanism underlying its antibacterial action has remained elusive. In this study, we investigated the impact of RJ on the bacteria model MG1655 at its half-maximal inhibitory concentration, employing LC-MS/MS to analyze proteomic changes. The differentially expressed proteins were found to primarily contribute to the suppression of gene expression processes, specifically transcription and translation, disrupting nutrition and energy metabolism, and inducing oxidative stress. Notably, RJ treatment led to a marked inhibition of superoxide dismutase and catalase activities, resulting in heightened oxidative damage and lipid peroxidation. Furthermore, through a protein-protein interaction network analysis using the STRING database, we identified CRP and IHF as crucial host regulators responsive to RJ. These regulators were found to play a pivotal role in suppressing essential hub genes associated with energy production and antioxidant capabilities. Our findings significantly contribute to the understanding of RJ's antibacterial mechanism, highlighting its potential as a natural alternative to conventional antibiotics. The identification of CRP and IHF as central players highlights the intricate regulatory networks involved in RJ's action, offering new targets for developing innovative antimicrobial strategies.

Cophylogeny has been identified between gut bacteria and their animal host and is highly relevant to host health, but little research has extended to gut bacteriophages. Here we use bee model to investigate host specificity and cophylogeny in the "animal-gut bacteria-phage" tripartite system. Through metagenomic sequencing upon different bee species, the gut phageome revealed a more variable composition than the gut bacteriome. Nevertheless, the bacteriome and the phageome showed a significant association of their dissimilarity matrices, indicating a reciprocal interaction between the two kinds of communities. Most of the gut phages were host generalist at the viral cluster level but host specialist at the viral OTU level. While the dominant gut bacteria Gilliamella and Snodgrassella exhibited matched phylogeny with bee hosts, most of their phages showed a diminished level of cophylogeny. The evolutionary rates of the bee, the gut bacteria and the gut phages showed a remarkably increasing trend, including synonymous and non-synonymous substitution and gene content variation. For all of the three codiversified tripartite members, however, their genes under positive selection and genes involving gain/loss during evolution simultaneously enriched the functions into metabolism of nutrients, therefore highlighting the tripartite coevolution that results in an enhanced ecological fitness for the whole holobiont.

Here, we present a retrospective study on honey bee brood diseases in the USA. The Bee Disease Diagnostic Laboratory (BDDL) in the USDA-ARS Beltsville Bee Research Laboratory received a total of 4790 brood samples between 2015 and 2022 from U.S. State Apiary Inspectors and beekeepers. Samples from 49 states were analyzed by microscopy for the presence and prevalence of two bacteria, Melissococcus plutonius and Paenibacillus larvae, causing European foulbrood (EFB) and American foulbrood (AFB) diseases, respectively. Samples that tested positive for AFB were cultured and subjected to antibiotic susceptibility tests via the agar disc diffusion method to determine their resistance to tetracycline (Terramycin (R)) and tylosin (Tylan (R)) antibiotics. A comprehensive data analysis was conducted at multiple levels, including state, month, year, nationwide, and climate region. Among the 49 states examined, EFB was identified in 44 states, while AFB was found in 31 states. Infection levels vary across states, ranging from 0% to 63.6% for EFB and from 0% to 54.8% for AFB. The national average of EFB (19.2%) was significantly (p < 0.001) higher than AFB (8.6%). Contrary to AFB, significant yearly increases (p < 0.001) were identified for EFB from 2015 to 2022. Furthermore, significant monthly variations were recorded for both brood diseases, with the highest occurrence of EFB observed from April to July. States with the lowest infection rates of both diseases were NV, ND, MS, AK, and AZ. The South U.S. climate region, which comprises six states, had the highest and lowest EFB and ABF infections, respectively. On a nationwide scale, the AFB resistance to tetracycline (38%) was significantly (p < 0.001) higher than the resistance to tylosin (27%).

USDA-ARS Bee Research Laboratory received symptomatic honey bee (Apis mellifera L.) samples across the United States for disease diagnosis. Here, we present a retrospective study and cartography of ectoparasite Varroa destructor and intracellular microsporidia parasite Nosema spp. These two major parasites were identified in the diseased honey bee samples between 2015 and 2022. Varroa infestation level (VIL) was examined by a wash technique (Mites/100 bees) and calculated as a percentage, while Nosema infection was quantified by microscopical spore count (Million Spores/Bee). Data were analyzed by month, year, state, and by nine geographical climate regions described in the U.S. Of adult bee samples (n = 4039) that were analyzed for Varroa mite infestation, the overall VIL in the U.S. ranged between 0.4 and 30.85%, with an overall national VIL and Varroa prevalence of 8.21% and 85.14%, respectively. Overall monthly data showed VIL constantly exceeded the critical level of 4% except from June to September and reached a maximum of 15% in January and December. Nationwide, VIL significantly (p < 0.001) increased from 2015 to 2018 (1.1–4.7%), plateaued from 2018 to 2021 (4.7–4.5%), followed by a significant decrease in 2022 (3.6%). Significant VIL differences (p < 0.001) were recorded among climate regions, with the highest mite infestation levels in the Upper Midwest region (13.9%) and the lowest in the West region (5.1%). Of adult bee samples (n = 2,994) that were analyzed for Nosema infection, Nosema spore count ranged between (1–16.8) million spores per bee among states, with a national average of 6.8 and a prevalence of 99.7%. The lowest and highest Nosema loads were respectively recorded in the South region (3.1) and Upper Midwest (10.5), a significant difference (p < 0.001). No statistical differences were recorded among the six other climate regions. Overall, VIL and Nosema infection correlated significantly (p < 0.001) with a regression coefficient of (R2 = 0.6). Our data, which originated from ailing bee colonies, showed significantly higher rates of maladies compared to data from healthy colonies obtained by the USDA-APHIS National Honey Bee Survey, demonstrating the role of bee diseases caused by Varroa mite and Nosema in honey bee population declines.

Nutritional stress, especially a dearth of pollen, has been linked to honey bee colony losses. Colony-level experiments are critical for understanding the mechanisms by which nutritional stress affects individual honey bee physiology and pushes honey bee colonies to collapse. In this study, we investigated the impact of pollen restriction on key markers of honey bee physiology, main elements of the immune system, and predominant honey bee viruses. To achieve this objective, we uncoupled the effects of behavior, age, and nutritional conditions using a new colony establishment technique designed to control size, demography, and genetic background. Our results showed that the expression of storage proteins, including vitellogenin (vg) and royal jelly major protein 1 (mrjp1), were significantly associated with nursing, pollen ingestion, and older age. On the other hand, genes involved in hormonal regulation including insulin-like peptides (ilp1 and ilp2) and methyl farnesoate epoxidase (mfe), exhibited higher expression levels in young foragers from colonies not experiencing pollen restriction. In contrast, pollen restriction induced higher levels of insulin-like peptides in old nurses. On the other hand, we found a strong effect of behavior on the expression of all immune genes, with higher expression levels in foragers. In contrast, the effects of nutrition and age were significant only the expression of the regulatory gene dorsal. We also found multiple interactions of the experimental variables on viral titers, including higher Deformed wing virus (DWV) titers associated with foraging and age-related decline. In addition, nutrition significantly affected DWV titers in young nurses, with higher titers induced by pollen ingestion. In contrast, higher levels of Black queen cell virus (BQCV) were associated with pollen restriction. Finally, correlation, PCA, and NMDS analyses proved that behavior had had the strongest effect on gene expression and viral titers, followed by age and nutrition. These analyses also support multiple interactions among genes and virus analyzed, including negative correlations between the expression of genes encoding storage proteins associated with pollen ingestion and nursing (vg and mrjp1) with the expression of immune genes and DWV titers. Our results provide new insights into the proximal mechanisms by which nutritional stress is associated with changes in honey bee physiology, immunity, and viral titers.

Deformed wing virus (DWV) is a widespread pathogen of Apis mellifera honey bees, and is considered a major causative factor for the collapse of infected honey bee colonies. DWV can be horizontally transmitted among bees through various oral routes, including via food sharing and by interactions of bees with viral-contaminated solid hive substrates. Cold plasma ionized hydrogen peroxide (iHP) is used extensively by the food production, processing and medical industries to clean surfaces of microbial contaminants. In this study, we investigated the use of iHP to inactivate DWV particles in situ on a solid substrate. iHP-treated DWV sources were ~105-fold less infectious when injected into naïve honey bee pupae compared to DWV receiving no iHP treatment, matching injected controls containing no DWV. iHP treatment also greatly reduced the incidence of overt DWV infections (i.e., pupae having >109 copies of DWV). The level of DWV inactivation achieved with iHP treatment was higher than other means of viral inactivation such as gamma irradiation, and iHP treatment is likely simpler and safer. Treatment of DWV contaminated hive substrates with iHP, even with honey bees present, may be an effective way to decrease the impacts of DWV infection on honey bees.