In temperate climates, honey bees rely on stored carbohydrates to sustain them throughout the winter. In nature, honey serves as the bees' source of carbohydrates, but when managed, beekeepers often harvest honey and replace it with cheaper, artificial feed. The effects of alternative carbohydrate sources on colony survival, strength, and individual bee metabolic health are poorly understood. We assessed the impacts of carbohydrate diets (honey, sucrose syrup, high-fructose corn syrup, and invert syrup) on colony winter survival, population size, and worker bee nutritional state (i.e., fat content and gene expression of overwintered bees and emerging callow bees). We observed a nonsignificant trend for greater survival and larger adult population size among colonies overwintered on honey compared to the artificial feeds, with colonies fed high-fructose corn syrup performing particularly poorly. These trends were mirrored in individual bee physiology, with bees from colonies fed honey having significantly larger fat bodies than those from colonies fed high-fructose corn syrup. For bees fed honey or sucrose, we also observed gene expression profiles consistent with a higher nutritional state, associated with physiologically younger individuals. That is, there was significantly higher expression of vitellogenin and insulin-like peptide 2 and lower expression of insulin-like peptide 1 and juvenile hormone acid methyltransferase in the brains of bees that consumed honey or sucrose syrup relative to those that consumed invert syrup or high-fructose corn syrup. These findings further our understanding of the physiological implications of carbohydrate nutrition in honey bees and have applied implications for colony management.

Honey bees are important pollinators of wild plants and crops. MicroRNAs (miRNAs) are endogenous regulators of gene expression. In this study, we initially determined that the lethal concentration 50 (LC50) of dinotefuran was 0.773 mg/l. Then, the expression profiles and differentially expressed miRNAs (DE miRNAs) in honey bee brains after 1, 5, and 10 d of treatment with the lethal concentration 10 (LC10) of dinotefuran were explored via deep small-RNA sequencing and bioinformatics. In total, 2, 23, and 27 DE miRNAs were identified after persistent exposure to the LC10 of dinotefuran for 1, 5, and 10 d, respectively. Some abundant miRNAs, such as ame-miR-375-3p, ame-miR-281-5p, ame-miR-3786-3p, ame-miR-10-5p, and ame-miR-6037-3p, were extremely significantly differentially expressed. Enrichment analysis suggested that the candidate target genes of the DE miRNAs are involved in the regulation of biological processes, cellular processes, and behaviors. These results expand our understanding of the regulatory roles of miRNAs in honey bee Apis mellifera (Hymenopptera: Apidae) responses to neonicotinoid insecticides and facilitate further studies on the functions of miRNAs in honey bees.

Chlorothalonil is a broad spectrum chloronitrile fungicide that has been identified as one of the most common pesticide contaminants found in managed honey bees (Hymenoptera: Apidae: Apis mellifera L.), their food stores, and the hive environment. While not acutely toxic to honey bees, several studies have identified potential sublethal effects, especially in larvae, but comprehensive information regarding the impact of chlorothalonil on adults is lacking. The goal of this study was to investigate the effects of exposure to a field relevant level of chlorothalonil on honey bee antiviral immunity and biochemical markers of general and social immunity, as well as macronutrient markers of nutrition and morphological markers of growth and development. Chlorothalonil exposure was found to have an effect on 1) honey bee resistance and/or tolerance to viral infection by decreasing the survival of bees following a viral challenge, 2) social immunity, by increasing the level of glucose oxidase activity, 3) nutrition, by decreasing levels of total carbohydrate and protein, and 4) development, by decreasing the total body weight, head width, and wing length of adult nurse and forager bees. Although more research is required to better understand how chlorothalonil interacts with bee physiology to increase mortality associated with viral infections, this study clearly illustrates the sublethal effects of chlorothalonil exposure on bee immunity, nutrition, and development.

Honey bees, as many species of social insects, display a division of labor among colony members based on behavioral specializations related to age. Adult worker honey bees perform a series of tasks in the hive when they are young (such as brood care or nursing) and at ca. 2-3 wk of age, shift to foraging for nectar and pollen outside the hive. The transition to foraging involves changes in metabolism and neuroendocrine activities. These changes are associated with a suite of developmental genes. It was recently demonstrated that antibiotics influence behavioral development by accelerating or delaying the onset of foraging depending on timing of antibiotic exposure. To understand the mechanisms of these changes, we conducted a study on the effects of antibiotics on expression of candidate genes known to regulate behavioral development. We demonstrate a delay in the typical changes in gene expression over the lifetime of the individuals that were exposed to antibiotics during immature stage and adulthood. Additionally, we show an acceleration in the typical changes in gene expression on individuals that were expose to antibiotics only during immature stage. These results show that timing of antibiotic exposure alter the typical regulation of behavioral development by metabolic and neuroendocrine processes.

Plants of Eruca sativa Mill. (Brassicaceae) from desert and Mediterranean populations in Israel differ in flower color and size. In the desert habitat, the population has higher abundance of flowers with cream color and longer petals, whereas in the Mediterranean habitat, the population has higher abundance of flowers with yellow and shorter petals. Choice experiments with honey bee foragers (Apis mellifera Linn., Apidae, Hymenoptera), the main pollinator in the natural habitat in Israel, confirmed that they are more attracted to the yellow flower morph than to the cream one. A proboscis extension response test indicated that honey bees are able to discriminate between flower scents of the desert and Mediterranean populations. Considering the advantage of plants of the yellow morph in attracting pollinators, we further tested in a common garden experiment whether these possess higher fitness than plants of the desert population. Indeed, a significant association was found between flower color and fruit set, and seed mass. In general, our results provide evidence for ecotypic differentiation between populations imposed by pollinators. The advantage of the yellow color morph in attracting pollinators may explain its dominance among plants of the Mediterranean population. We discuss why the cream color morph may be dominant in the desert habitat, considering the possibility of different pollinators, tradeoffs between traits, or pleiotropy.

A major threat to honey bee (Apis mellifera Linnaeus, Hymenoptera: Apidae) health continues to be parasitism by the mite Varroa destructor, which has been linked to high colony losses worldwide. Besides feeding on developing and adult bees, Varroa is also a prolific vector of honey bee-associated viruses. Because they live in unmanaged conditions, wild honey bee colonies are not treated against Varroa, which has enabled the natural selection of more mite-tolerant bees. To date, few studies have explored the prevalence of viruses in unmanaged colonies. The Welder Wildlife Refuge (WWR) in Texas is a unique site to study the viral landscape of unmanaged honey bees in the United States. The goals of this study were to identify and quantify viruses in wild colonies at the WWR, to examine changes in the prevalence of viruses in these colonies over time, and to compare the presence and titers of viruses between wild colonies at the WWR and those from the nearest managed apiary. We collected bees from colonies at the WWR in 2013, 2016, and 2021, and analyzed selected viruses for their presence and titers via quantitative polymerase chain reaction. In 2021, we also sampled bees from the nearest managed apiary for comparison. We found low average virus titers in all wild colonies sampled, and no difference in virus titers between colonies at the WWR and those from the managed apiary. Our study indicates that virus titers in wild colonies at the WWR are similar to those found in nearby colonies, and that these titers fluctuate over time.

In recent decades, some bumble bee species have declined, including in North America. Declines have been reported in species of bumble bees historically present in Ontario, including: yellow bumble bee (Bombus fervidus) (Fabricus, 1798), American bumble bee (Bombus pensylvanicus) (DeGeer, 1773), and yellow-banded bumble bee (Bombus terricola) (Kirby, 1837). Threats contributing to bumble bee population declines include: land-use changes, habitat loss, climate change, pathogen spillover, and pesticide use. A response to the need for action on pollinator preservation in North America has been to encourage 'bee-friendly' plantings. Previous studies show differences in common and at-risk bumble bee foraging; however, similar data are unavailable for Ontario. Our research question is whether there is a difference in co-occurring at-risk and common bumble bee (Bombus spp.) floral use (including nectar and pollen collection) in protected areas in southern Ontario. We hypothesize that common and at-risk species forage differently, predicting that at-risk species forage on a limited selection of host plants. We conducted a field survey of sites in southern Ontario, using observational methods to determine bumble bee foraging by species. The results of a redundancy analysis show a difference in foraging between common and at-risk bumblebee species. At-risk bumble bee species show a preference for foraging on invasive, naturalized Vicia cracca (tufted vetch). This finding raises the question of how to preserve or provide forage for at-risk bumble bees, when they show an association with an invasive species often subject to control in protected areas.

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.

Pesticides have been reported to be one of the major drivers in the global pollinator losses. The large-scale decline in honey bees, an important pollinator group, has resulted in comprehensive studies on honey bee colonies. Lack of information on native wild pollinators has paved the way for this study, which highlights the underlying evolutionary changes occurring in the wild honey bee populations exposed to pesticides along an agricultural intensification landscape. The study reports an increased genetic diversity in native Apis cerana Fabricius (Hymenoptera: Apidae) populations continually exposed to pesticide stress. An increased heterozygosity, evidenced by a higher electrophoretic banding pattern, was observed in the pesticide-exposed populations for two isozymes involved with xenobiotic metabolism-esterase and glucose-6-phosphate dehydrogenase. Differential banding patterns also revealed a higher percentage of polymorphic loci, number of polymorphic bands, Nei's genetic distance, etc. observed in these populations in the Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) experiments using three random decamer primers. Higher heterozygosity, being indicative of a more resistant population, implies population survival within the threshold pesticide stress. This study reports such changes for the first time in native wild Indian honey bee populations exposed to pesticides and has far-reaching implications on the population adaptability under pesticide stress.

The ectoparasitic mite, Varroa destructor, is the most destructive parasite of managed honeybee colonies worldwide. Since V. destructor transfers pathogens to honeybees, it may be adaptive for bees to respond to mite infestation by upregulating their immune responses. Mites, however, may overcome the host's immune responses by suppressing them, which could facilitate the mite's ability to feed on hemolymph. A humoral immune response of bees parasitized by V. destructor may be detected by studying the expression levels of antibacterial peptides, such as abaecin and defensin, known to be immune-responsive. Expression levels for these two antibacterial peptides changed non-linearly with respect to the number of mites parasitizing honeybee pupae. Bees exposed to low or moderate number of mites had fewer immune-related transcripts than pupae that were never parasitized or pupae with high mite loads. Although many of the pupae tested indicated the presence of bacteria, no correlation with mite numbers or immune-response levels existed. All bees tested negative for acute paralysis and Kashmir bee viruses known to be vectored by V. destructor.

Bees of the genus Osmia are being used in crop pollination at an increasing rate. However, a short life expectancy of adult individuals limits the feasibility of their use. Cocoons of the red mason bee, Osmia rufa L. (Hymenoptera: Megachilidae), can be stored at 4° C in a postdiapause state, and adult bees can be used for pollination outside their natural flight period. The period of storage in this form has an unfavorable influence on the survival rate, life expectancy, and fertility of the bee. It was suggested that the negative results are connected with exhaustion of energy reserves. To test this hypothesis, the present study examined the contents of protein, carbohydrates, lipids, and the activities of some enzymes, and their degradation in red mason bees that emerged in spring according to their biological clock and in summer after elongated diapause. It was found that postdiapause artificially elongated by 3 months caused significant decreases in body weight, total sugar, glycogen, lipids, and protein content in O. rufa. Glucose level was highest in bees that emerged in the summer, which was coincident with increased activities of maltase and trehalase. The activities of sucrase and cellobiase were not changed, while amylase activity was considerably decreased. The activities of triacylglycerols lipase and C2, C4, C10 carboxylesterases were highest in bees that emerged in July. Low temperatures restrict O. rufa emergence, and during prolonged postdiapause, metabolic processes lead to significant reductions of structural and energetic compounds.

Immune responsive protein 30 (IRP30) is a Hymenoptera-specific protein first identified from honey bee hemolymph in response to bacterial infection. However, its function remains elusive. Here, we cloned the full-length IRP30 gene and clarified its expression pattern in the bumble bee Bombus lantschouensis (Vogt). The full-length IRP30 gene measures 1443 bp and contains two exons and one intron. The length of the cDNA is 1082 bp, including a 36-bp 5'-UTR and a 218-bp 3'-UTR, and it encodes a putative protein of 275 amino acids. As expected, the sequence of the B. lantschouensis IRP30 protein was clustered with the bumble bee group, which appeared as a single clade next to honey bees. The family shared similar conserved protein domains. Moreover, bumble bee IRP30 belongs to a recently diverged clade that has four leucine-rich repeat (LRR) conserved domains. IRP30 is highly expressed in the worker caste, during pupal developmental stages, and in the head and thorax tissues. Interestingly, its expression increases 20- to 90-fold when female bumble bees (B. lantschouensis) and honey bees (Apis mellifera L.) begin laying eggs. Overall, based on the expression of IRP30 during development and egg laying in female bumble bees, this protein not only responds to immune challenge but also may play an important role in metamorphosis and reproduction.

The Meliponini, also known as stingless bees, are distributed in tropical and subtropical areas of the world and plays an essential role in pollinating many wild plants and crops These bees can build nests in cavities of trees or walls, underground or in associations with ants or termites; interestingly, these nests are sometimes found in aggregations. In order to assess the genetic diversity and structure in aggregates of Nannotrigona testaceicornis (Lepeletier), samples of this species were collected from six aggregations and genetically analyzed for eight specific microsatellite loci. We observed in this analysis that the mean genetic diversity value among aggregations was 0.354, and the mean expected and observed heterozygosity values was 0.414 and 0.283, respectively. The statistically significant Fis value indicated an observed heterozygosity lower than the expected heterozygosity in all loci studied resulting in high homozygosis level in these populations. In addition, the low number of private alleles observed reinforces the absence of structuring that is seen in the aggregates. These results can provide relevant information about genetic diversity in aggregations of N. testaceicornis and contribute to the management and conservation of these bees' species that are critical for the pollination process.

We reported the sequence and characteristics of the complete mitochondrial genome of an ecologically important stingless bee, Lepidotrigona flavibasis (Hymenoptera: Meliponini), that has suffered serious population declines in recent years. A phylogenetic analysis based on complete mitogenomes indicated that L. flavibasis was first clustered with another Lepidotrigona species (L. terminata) and then joined with the other two Melipona (Hymenoptera: Meliponini) stingless bees (M. scutellaris and M. bicolor), forming a single clade of stingless bees. The stingless bee clade has a closer relationship with bumblebees (Bombus) (Hymenoptera: Apidae) than with honeybees (Apis) (Hymenoptera: Apidae). Extremely high gene rearrangements involving tRNAs, rRNAs, D-loop regions, and protein-coding genes were observed in the Lepidotrigona mitogenomes, suggesting an overactive evolutionary status in Lepidotrigona species. These mitogenomic organization variations could provide a good system with which to understand the evolutionary history of Meliponini.

Vitellogenin is a yolk precursor protein in most oviparous females. In the advanced eusocial honeybee, Apis mellifera (Hymenoptera: Apidae), vitellogenin has recently attracted much interest as this protein, in addition to a classical function in oocyte development in the reproductive queen caste, has evolved functions in the facultatively sterile female worker caste not documented in other species. However, research on the spatial dynamics of vitellogenin in various tissues is not easily performed with available tools. Here we present an immunogold staining procedure that visualizes honeybee vitellogenin in resin embedded tissue. To establish the protocol, we used ovaries of worker bees from colonies with and without a queen. Under the first condition, vitellogenin is assumed not to be present in the workers' ovaries. Under the second condition, the ovaries of worker bees become vitellogenic, with abundant opportunities for detection of complex patterns of vitellogenin uptake and storage. By use of this experimental setup, the staining method is shown to be both sensitive and specific. To demonstrate the functional significance of the protocol, it was subsequently used to identify vitellogenin protein in the hypopharyngeal glands (brood food producing head glands) of nursing worker bees and in adjacent head fat body cells for the first time. Localization of vitellogenin in these tissues supports previously hypothesized roles of vitellogenin in social behavior. This protocol thus provides deeper insights into the functions of vitellogenin in the honeybee.

European foulbrood (EFB) is a severe disease of honey bee (Apis mellifera) larvae caused by the bacterium Linnaeus [Hymenoptera: Apidae]) Melissococcus plutonius (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae). Many beekeepers in North America report severe EFB following blueberry pollination, but it is not clear what factors during pollination are related to clinical disease. Additionally, the impact that other factors such as viral load and hygienic behavior have on EFB has not been studied. In Spring of 2020 we enrolled 60 commercial honey bee colonies in a prospective cohort study. Colonies were inspected 3 times over the season with hive metrics and samples taken for viral testing. Each colony was tested for hygienic behavior twice and the score was averaged. Viral loads were determined by qPCR for deformed wing virus (DWV) A and B. We found no statistical difference in the EFB prevalence or severity between the 2 yards at any timepoint; 50% (n = 16) of the colonies in the holding yard and 63% (n = 17) in blueberry developed moderate to severe EFB over the study period. When colonies from both yards were pooled, we found no relationship between viral load or hygienic behavior and development of EFB. These results suggest that other factors may be responsible for driving EFB virulence and hygienic behavior is not likely helpful in managing this disease.

The hexagonal structure of the honey bee comb cell has been the source of many studies attempting to understand its structure and function. In the storage area of the comb, only honey is stored and no brood is reared. We predicted that honey bees may construct different hexagonal cells for brood rearing and honey storage. We used quantitative analyses to evaluate the structure and function of the natural comb cell in the Chinese bee, Apis cerana cerana and the Italian bee, A. mellifera ligustica. We made cell molds using a crystal glue solution and measured the structure and inclination of cells. We found that the comb cells of A. c. cerana had both upward-sloping and downward-sloping cells; while the A. m. ligustica cells all tilted upwards. Interestingly, the cells did not conform to the regular hexagonal prism structure and showed irregular diameter sizes. In both species, comb cells also were differentiated into worker, drone and honey cells, differing in their diameter and depth. This study revealed unique differences in the structure and function of comb cells and showed that honey bees design their cells with precise engineering to increase storage capacity, and to create adequate growing room for their brood.

Honey bee larvae are dependent on the social structure of colony for their provisioning and survival. With thousands of larvae being managed collectively by groups of foragers (collecting food resources) and nurse bees (processing food and provisioning larvae), coordination of colony efforts in rearing brood depends on multiple dynamic cues of larval presence and needs. Much of these cues appear to be chemical, with larvae producing multiple pheromones, major being brood ester pheromone (BEP; nonvolatile blend of fatty acid esters) that elicits both short-term releaser effects and long-term primer effects. While BEP can affect colony food collection and processing with the signaling of larval presence, it is unclear if BEP signals individual larval needs. To understand this aspect, in a series of experiments we manipulated larval feeding environment by depriving larvae from adult bee contact for 4-h period and examined (1) nurse bee interactions with contact-deprived and nondeprived larvae and larval extracts; (2) forager bee responses to contact-deprived and nondeprived larval extracts. We also characterized BEP of contact-deprived and nondeprived larvae. We found that nurse honey bees tend to aggregate more over contact-deprived larvae when compared with nondeprived larvae, but that these effects were not found in response to whole hexane extracts. Our analytical results suggest that BEP components changed in both quantity and quality over short period of contact deprivation. These changes affected foraging behavior, but did not appear to directly affect nursing behavior, suggesting that different chemical cues are involved in regulating nursing effort to individual larvae.

Laboratory experiments have advanced our understanding of honey bee (Apis mellifera) responses to environmental factors, but removal from the hive environment may also impact physiology. To examine whether the laboratory environment alters the honey bee gut bacterial community and immune responses, we compared bacterial community structure (based on amplicon sequence variant relative abundance), total bacterial abundance, and immune enzyme (phenoloxidase and glucose oxidase) activity of cohort honey bee workers kept under laboratory and hive conditions. Workers housed in the laboratory showed differences in the relative abundance of their core gut taxa, an increase in total gut bacterial abundance, and reduced phenoloxidase activity, compared to bees housed in hives.

Since the mid-1990s, Bombus occidentalis (Green) has declined from being one of the most common to one of the rarest bumble bee species in the Pacific Northwest of the United States. Although its conservation status is unresolved, a petition to list this species as endangered or threatened was recently submitted to the U.S. Fish and Wildlife Service. To shed light on the conservation situation and inform the U.S. Fish and Wildlife Service decision, we report on the detection and abundance of B. occidentalis following bumble bee collection between 2012 and 2014 across the Pacific Northwest. Collection occurred from the San Juan Islands and Olympic peninsula east to northern Idaho and northeastern Oregon, excluding the arid region in central Washington. B. occidentalis was observed at 23 collection sites out of a total of 234. With the exception of three sites on the Olympic peninsula, all of these were in the southeastern portion of the collection range.