Carl Hayden Bee Research Center

Pollen ranges from 2.5% to 61% protein content. Most pollen proteins are likely to be enzymes that function during pollen tube growth and subsequent fertilization, but the vast range of protein quantity may not reflect only pollen–pistil interactions. Because numerous vertebrate and invertebrate floral visitors consume pollen for protein, protein content may influence floral host choice. Additionally, many floral visitors pollinate their host plants. If protein content influences pollinator visitation, then pollinators are hypothesized to select for increased protein content of host plants. We analyzed or gleaned from the literature crude pollen protein concentrations of 377 plant species from 93 plant families. Using this database, we compared pollen protein concentration with (1) pollination mode, (2) pollen collection by bees, and (3) distance from stigma to ovule, after accounting for phylogeny through paired phylogenetic comparisons and a nested ANOVA including taxonomic rank. We found that pollen protein concentrations were highly conserved within plant genera, families, and divisions. We found that bees did not collect pollen that was unusually rich in protein, whether they pollinated or merely robbed their host plant. Plant species with vibratile pollination systems, which require visitation by pollen-collecting bees in order to transfer pollen, tended to have very protein-rich pollen, but it was not clear whether this was due to plant enhancement of pollinator rewards or to the possession of very small pollen grains. We found that zoophilous species were not statistically richer in pollen protein than anemophilous species after accounting for phylogeny, although the three most species-rich anemophilous clades surveyed were generally poor in protein. Plant genera hosting specialist pollen-collecting bees did not have particularly protein-rich pollen. Both mass of protein per pollen grain and pollen grain volume were correlated with stigma–ovule distance. We suggest that the need for growing pollen tubes probably plays a more important role in determining pollen protein content than rewarding pollinators.

As pollinators, bees are cornerstones for terrestrial ecosystem stability and key components in agricultural productivity. All animals, including bees, are associated with a diverse community of microbes, commonly referred to as the microbiome. The bee microbiome is likely to be a crucial factor affecting host health. However, with the exception of a few pathogens, the impacts of most members of the bee microbiome on host health are poorly understood. Further, the evolutionary and ecological forces that shape and change the microbiome are unclear. Here, we discuss recent progress in our understanding of the bee microbiome, and we present challenges associated with its investigation. We conclude that global coordination of research efforts is needed to fully understand the complex and highly dynamic nature of the interplay between the bee microbiome, its host, and the environment. High-throughput sequencing technologies are ideal for exploring complex biological systems, including host-microbe interactions. To maximize their value and to improve assessment of the factors affecting bee health, sequence data should be archived, curated, and analyzed in ways that promote the synthesis of different studies. To this end, the BeeBiome consortium aims to develop an online database which would provide reference sequences, archive metadata, and host analytical resources. The goal would be to support applied and fundamental research on bees and their associated microbes and to provide a collaborative framework for sharing primary data from different research programs, thus furthering our understanding of the bee microbiome and its impact on pollinator health.

Nearly all eukaryotes are host to beneficial or benign bacteria in their gut lumen, either vertically inherited, or acquired from the environment. While bacteria core to the honey bee gut are becoming evident, the influence of the hive and pollination environment on honey bee microbial health is largely unexplored. Here we compare bacteria from floral nectar in the immediate pollination environment, different segments of the honey bee (Apis mellifera) alimentary tract, and food stored in the hive (honey and packed pollen or "beebread"). We used cultivation and sequencing to explore bacterial communities in all sample types, coupled with culture-independent analysis of beebread. We compare our results from the alimentary tract with both culture-dependent and culture-independent analyses from previous studies. Culturing the foregut (crop), midgut and hindgut with standard media produced many identical or highly similar 16S rDNA sequences found with 16S rDNA clone libraries and next generation sequencing of 16S rDNA amplicons. Despite extensive culturing with identical media, our results do not support the core crop bacterial community hypothesized by recent studies. We cultured a wide variety of bacterial strains from 6 of 7 phylogenetic groups considered core to the honey bee hindgut. Our results reveal that many bacteria prevalent in beebread and the crop are also found in floral nectar, suggesting frequent horizontal transmission. From beebread we uncovered a variety of bacterial phylotypes, including many possible pathogens and food spoilage organisms, and potentially beneficial bacteria including Lactobacillus kunkeei, Acetobacteraceae and many different groups of Actinobacteria. Contributions of these bacteria to colony health may include general hygiene, fungal and pathogen inhibition and beebread preservation. Our results are important for understanding the contribution to pollinator health of both environmentally vectored and core microbiota, and the identification of factors that may affect bacterial detection and transmission, colony food storage and disease susceptibility.

The honey bee is a key pollinator species in decline worldwide. As part of a commercial operation, bee colonies are exposed to a variety of agricultural ecosystems throughout the year and a multitude of environmental variables that may affect the microbial balance of individuals and the hive. While many recent studies support the idea of a core microbiota in guts of younger in-hive bees, it is unknown whether this core is present in forager bees or the pollen they carry back to the hive. Additionally, several studies hypothesize that the foregut (crop), a key interface between the pollination environment and hive food stores, contains a set of 13 lactic acid bacteria (LAB) that inoculate collected pollen and act in synergy to preserve pollen stores. Here, we used a combination of 454 based 16S rRNA gene sequencing of the microbial communities of forager guts, crops, and corbicular pollen and crop plate counts to show that (1) despite a very different diet, forager guts contain a core microbiota similar to that found in younger bees, (2) corbicular pollen contains a diverse community dominated by hive-specific, environmental or phyllosphere bacteria that are not prevalent in the gut or crop, and (3) the 13 LAB found in culture-based studies are not specific to the crop but are a small subset of midgut or hindgut specific bacteria identified in many recent 454 amplicon-based studies. The crop is dominated by Lactobacillus kunkeei, and Alpha 2.2 (Acetobacteraceae), highly osmotolerant and acid resistant bacteria found in stored pollen and honey. Crop taxa at low abundance include core hindgut bacteria in transit to their primary niche, and potential pathogens or food spoilage organisms seemingly vectored from the pollination environment. We conclude that the crop microbial environment is influenced by worker task, and may function in both decontamination and inoculation.

Abstract A major goal of population biologists involved in restoration work is to restore populations to a level that will allow them to persist over the long term within a dynamic landscape and include the ability to undergo adaptive evolutionary change. We discuss five research areas of particular importance to restoration biology that offer potentially unique opportunities to couple basic research with the practical needs of restorationists. The five research areas are: (1) the influence of numbers of individuals and genetic variation in the initial population on population colonization, establishment, growth, and evolutionary potential; (2) the role of local adaptation and life history traits in the success of restored populations; (3) the influence of the spatial arrangement of landscape elements on metapopulation dynamics and population processes such as migration; (4) the effects of genetic drift, gene flow, and selection on population persistence within an often accelerated, successional time frame; and (5) the influence of interspecific interactions on population dynamics and community development. We also provide a sample of practical problems faced by practitioners, each of which encompasses one or more of the research areas discussed, and that may be solved by addressing fundamental research questions.

A prominent theory states that animal phenotypes arise by evolutionary changes in gene regulation, but the extent to which this theory holds true for behavioral evolution is not known. Because "nature and nurture" are now understood to involve hereditary and environmental influences on gene expression, we studied whether environmental influences on a behavioral phenotype, i.e., aggression, could have evolved into inherited differences via changes in gene expression. Here, with microarray analysis of honey bees, we show that aggression-related genes with inherited patterns of brain expression are also environmentally regulated. There were expression differences in the brain for hundreds of genes between the highly aggressive Africanized honey bee compared with European honey bee (EHB) subspecies. Similar results were obtained for EHB in response to exposure to alarm pheromone (which provokes aggression) and when comparing old and young bees (aggressive tendencies increase with age). There was significant overlap of the gene lists generated from these three microarray experiments. Moreover, there was statistical enrichment of several of the same cis regulatory motifs in promoters of genes on all three gene lists. Aggression shows a remarkably robust brain molecular signature regardless of whether it occurs because of inherited, age-related, or environmental (social) factors. It appears that one element in the evolution of different degrees of aggressive behavior in honey bees involved changes in regulation of genes that mediate the response to alarm pheromone.

The African honey bee subspecies Apis mellifera scutellata has colonized much of the Americas in less than 50 years and has largely replaced European bees throughout its range in the New World. The African bee therefore provides an excellent opportunity to examine the factors that influence invasion success. We provide a synthesis of recent research on the African bee, concentrating on its ability to displace European honey bees. Specifically, we consider (a) the genetic composition of the expanding population and the symmetry of gene flow between African and European bees, (b) the mechanisms that favor the preservation of the African genome, and (c) the possible range and impact of the African bee in the United States.

Microorganisms associated with honey bees, Apis mellifera, and their food include bacteria (Gram-variable pleomorphic bacteria, Bacillus spp., and Enterobacteriaceae), molds (primarily aspergilli and penicillia), and yeasts (mainly Torulopsis spp.). Eggs, prepupae, pupae, and worker bees emerging from cells as adults are usually free of internal microbes. Microorganisms acquired by larvae through ingestion of contaminated food are usually eliminated through the single defecation that occurs at the end of the feeding period prior to pupation. Emerging adult bees acquire intestinal microflora by food exchange with other bees in the colony and through consumption of pollen. Biochemical contributions of microorganisms to honey bees; the role of microorganisms in the conversion, enhancement, and preservation of pollen stored as bee bread in comb cells; and the production of antimycotic substances by molds and Bacillus spp. from honey bee colonies that are resistant to the fungal disease, chalkbrood, are discussed. An association of Bacillus spp. with bees including honey bees, stingless bees, and solitary bees from tropical and temperate zones appears to have evolved in which female bees inoculate food sources with these bacteria whose chemical products contribute to the elaboration and/or protection from spoilage of food that is stored in the nest. This association is ancient based on results from stingless bees preserved in amber for 25–40 million years. It is concluded that bees, their products, and their associated microorganisms are potential sources of bioactive products including antimicrobial compounds.

Honey bee hives are filled with stored pollen, honey, plant resins and wax, all antimicrobial to differing degrees. Stored pollen is the nutritionally rich currency used for colony growth and consists of 40-50% simple sugars. Many studies speculate that prior to consumption by bees, stored pollen undergoes long-term nutrient conversion, becoming more nutritious 'bee bread' as microbes predigest the pollen. We quantified both structural and functional aspects associated with this hypothesis using behavioural assays, bacterial plate counts, microscopy and 454 amplicon sequencing of the 16S rRNA gene from both newly collected and hive-stored pollen. We found that bees preferentially consume fresh pollen stored for <3 days. Newly collected pollen contained few bacteria, values which decreased significantly as pollen were stored >96 h. The estimated microbe to pollen grain surface area ratio was 1:1 000 000 indicating a negligible effect of microbial metabolism on hive-stored pollen. Consistent with these findings, hive-stored pollen grains did not appear compromised according to microscopy. Based on year round 454 amplicon sequencing, bacterial communities of newly collected and hive-stored pollen did not differ, indicating the lack of an emergent microbial community co-evolved to digest stored pollen. In accord with previous culturing and 16S cloning, acid resistant and osmotolerant bacteria like Lactobacillus kunkeei were found in greatest abundance in stored pollen, consistent with the harsh character of this microenvironment. We conclude that stored pollen is not evolved for microbially mediated nutrient conversion, but is a preservative environment due primarily to added honey, nectar, bee secretions and properties of pollen itself.

Viruses and other pathogens can spread rapidly in social insect colonies from close contacts among nestmates, food sharing and periods of confinement. Here we discuss how honey bees decrease the risk of disease outbreaks by a combination of behaviors (social immunity) and individual immune function. There is a relationship between the effectiveness of social and individual immunity and the nutritional state of the colony. Parasitic Varroa mites undermine the relationship because they reduce nutrient levels, suppress individual immune function and transmit viruses. Future research directions to better understand the dynamics of the nutrition-immunity relationship based on levels of stress, time of year and colony demographics are discussed.

Ants dominate many terrestrial ecosystems, yet we know little about their nutritional physiology and ecology. While traditionally viewed as predators and scavengers, recent isotopic studies revealed that many dominant ant species are functional herbivores. As with other insects with nitrogen-poor diets, it is hypothesized that these ants rely on symbiotic bacteria for nutritional supplementation. In this study, we used cloning and 16S sequencing to further characterize the bacterial flora of several herbivorous ants, while also examining the beta diversity of bacterial communities within and between ant species from different trophic levels. Through estimating phylogenetic overlap between these communities, we tested the hypothesis that ecologically or phylogenetically similar groups of ants harbor similar microbial flora. Our findings reveal: (i) clear differences in bacterial communities harbored by predatory and herbivorous ants; (ii) notable similarities among communities from distantly related herbivorous ants and (iii) similar communities shared by different predatory army ant species. Focusing on one herbivorous ant tribe, the Cephalotini, we detected five major bacterial taxa that likely represent the core microbiota. Metabolic functions of bacterial relatives suggest that these microbes may play roles in fixing, recycling, or upgrading nitrogen. Overall, our findings reveal that similar microbial communities are harbored by ants from similar trophic niches and, to a greater extent, by related ants from the same colonies, species, genera, and tribes. These trends hint at coevolved histories between ants and microbes, suggesting new possibilities for roles of bacteria in the evolution of both herbivores and carnivores from the ant family Formicidae.

Israeli acute paralysis virus (IAPV) is a widespread RNA virus of honey bees that has been linked with colony losses. Here we describe the transmission, prevalence, and genetic traits of this virus, along with host transcriptional responses to infections. Further, we present RNAi-based strategies for limiting an important mechanism used by IAPV to subvert host defenses. Our study shows that IAPV is established as a persistent infection in honey bee populations, likely enabled by both horizontal and vertical transmission pathways. The phenotypic differences in pathology among different strains of IAPV found globally may be due to high levels of standing genetic variation. Microarray profiles of host responses to IAPV infection revealed that mitochondrial function is the most significantly affected biological process, suggesting that viral infection causes significant disturbance in energy-related host processes. The expression of genes involved in immune pathways in adult bees indicates that IAPV infection triggers active immune responses. The evidence that silencing an IAPV-encoded putative suppressor of RNAi reduces IAPV replication suggests a functional assignment for a particular genomic region of IAPV and closely related viruses from the Family Dicistroviridae, and indicates a novel therapeutic strategy for limiting multiple honey bee viruses simultaneously and reducing colony losses due to viral diseases. We believe that the knowledge and insights gained from this study will provide a new platform for continuing studies of the IAPV-host interactions and have positive implications for disease management that will lead to mitigation of escalating honey bee colony losses worldwide.

Twenty-five pure pollens plus several blends of pollen were fed as sole protein sources to honey bees, Apis mellifera L., and resultant survival of bees on these diets was measured. Average increase in mean life span of bees on a pollen diet versus sugar water controls was 19.5 d, with a range from 3.9 d less than the controls for Ambrosia to 40.6 d longer, for a five-pollen blend. Actual consumption of test pollen diets also varied dramatically among test pollens, with a mean consumption of 16.5 mg pollen per bee for the first 10 d and a range of 1.9—29.0 mg per bee. Pollens that induced decreased life span included Ambrosia, Uromyces (a rust spore), Typha , and Kallstroemia ; those that induced little increase in life span included Haplopappus, Baccharis , and Taraxacum; and those that induced the greatest increase in life span included Ephedra, Prosopis, Rubus, Populus , and two blends of pollen. Increased life span was not correlated with pollen grain size, grain spininess, or dispersal vector (biotic versus abiotic) and was weakly correlated with season of floral bloom (pollen produced in spring was better than that produced in fall). Pollen from the family Compositae was poorer than average pollen. Major factors affecting life span were amount of pollen consumed, protein concentration in the pollen, and, especially, total amount of pollen protein consumed. By combining information relating to consumption levels and protein intake, reasons why a pollen caused the observed life span could be determined. Reasons included presence or absence of attractants and phagostimulants/deterrents, presence of toxic compounds, and a poor nutrient balance or level.

Dysbiosis, defined as unhealthy shifts in bacterial community composition, can lower the colonization resistance of the gut to intrinsic pathogens. Here, we determined the effect of diet age and type on the health and bacterial community composition of the honeybee (Apis mellifera). We fed newly emerged bees fresh or aged diets, and then recorded host development and bacterial community composition from four distinct regions of the hosts' digestive tract. Feeding fresh pollen or fresh substitute, we found no difference in host mortality, diet consumption, development or microbial community composition. In contrast, bees fed aged diets suffered impaired development, increased mortality and developed a significantly dysbiotic microbiome. The consumption of aged diets resulted in a significant reduction in the core ileum bacterium Snodgrassella alvi and a corresponding increase in intrinsic pathogen Frischella perrara. Moreover, the relative abundance of S. alvi in the ileum was positively correlated with host survival and development. The inverse was true for both F. perrara and Parasacharibacter apium. Collectively, our findings suggest that the early establishment of S. alvi is associated with healthy nurse development and potentially excludes F. perrara and P. apium from the ileum. Although at low abundance, establishment of the common midgut pathogen Nosema spp. was significantly associated with ileum dysbiosis and associated host deficiencies. Moreover, dysbiosis in the ileum was reflected in the rectum, mouthparts and hypopharyngeal glands, suggesting a systemic host effect. Our findings demonstrate that typically occurring alterations in diet quality play a significant role in colony health and the establishment of a dysbiotic gut microbiome.

Spatiotemporal variability in floral resources can have ecological and evolutionary consequences for both plants and the pollinators on which they depend. Seldom, however, can patterns of flower abundance and visitation in the field be linked with the behavioral mechanisms that allow floral visitors to persist when a preferred resource is scarce. To explore these mechanisms better, we examined factors controlling floral preference in the hawkmoth Manduca sexta in the semiarid grassland of Arizona. Here, hawkmoths forage primarily on flowers of the bat-adapted agave, Agave palmeri, but shift to the moth-adapted flowers of their larval host plant, Datura wrightii, when these become abundant. Both plants emit similar concentrations of floral odor, but scent composition, nectar, and flower reflectance are distinct between the two species, and A. palmeri flowers provide six times as much chemical energy as flowers of D. wrightii. Behavioral experiments with both naïve and experienced moths revealed that hawkmoths learn to feed from agave flowers through olfactory conditioning but readily switch to D. wrightii flowers, for which they are the primary pollinator, based on an innate odor preference. Behavioral flexibility and the olfactory contrast between flowers permit the hawkmoths to persist within a dynamic environment, while at the same time to function as the major pollinator of one plant species.

AbstractPart I of this review summarized the initial research on hygienic behaviour of honey bees, Apis mellifera. This early work that concerned hygienic behaviour as a mechanism of resistance to American foulbrood (AFB) has been the foundation for all subsequent research on hygienic behaviour. In Part II, research on hygienic behaviour in relation to other bee diseases and to Varroa jacobsoni and in Apis species and subspecies is reviewed. In addition, techniques to screen bee colonies for the behaviour are detailed, and practical applications of breeding bees for hygienic behaviour are given. A section on neuroethology demonstrates how modern neurobiological techniques are being used to detect the reasons for differences in responses of hygienic and non-hygienic bees to abnormal brood.

SummaryPollination by honey bees plays a key role in the functioning of ecosystems and optimisation of agricultural yields. Severe honey bee colony losses worldwide have raised concerns about the sustainability of these pollination services. In many cases, bee mortality appears to be the product of many interacting factors, but there is a growing consensus that the ectoparasitic mite Varroa destructor plays the role of the major predisposing liability. We argue that the fight against this mite should be a priority for future honey bee health research. We highlight the lack of efficient control methods currently available against the parasite and discuss the need for new approaches. Gaps in our knowledge of the biology and epidemiology of the mite are identified and a research road map towards sustainable control is drawn. Innovative and challenging approaches are suggested in order to stimulate research efforts and ensure that honey bees will be able to sustainably fulfil their role in the ecosystem.

ABSTRACT Several biophysical properties of members of Aleyrodidae and Aphididae were examined in order to explore how homopterous insects fly. Five species of aphids were found to weigh significantly more than five whitefly species (range 1 · 14-7·02×10–4g for aphids vs 3·3-8·0x 10–5g for whiteflies) and to have significantly larger wing surface areas (range 0·0103–0·1106 cm2vs 0·0096-0·0264cm2). As a consequence whiteflies and aphids can be partitioned into two groups with respect to wing loading (range 0·00633-0·01412 g cm–2 for aphids, 1·74-5·23X 10–3gcm–2 for whiteflies). Members of the two families are also separated in terms of wingbeat frequency (range 81·1-123·4Hz for aphids, 165·6–224·2 Hz for whiteflies). Since our animals were much smaller than any insects examined previously for these parameters, values were compared with the same parameters for 149 insect species recorded in the literature. Using these data, we found wingbeat frequency to be significantly correlated with wing loading only in insects weighing more than 0·03 g. Larger insects seem to employ a strategy similar to other flying animals, by compensating for high wing loading with higher wingbeat frequencies. The lack of correlation for these two parameters in insects weighing less than ·03 g probably results from the use of different flying strategies. These include employment of a clap and fling mechanism and the possession by some of exceedingly low wing loading. Also, small insects may have reduced settling velocities because they possess high drag coefficients. Previous studies which failed to establish a relationship between wing loading and wingbeat frequency in larger insects may have considered too few subjects or too great a range of body masses. The mass range is important because smaller insects which employ increased wingbeat frequency must use rates exponentially higher than those of larger insects utilizing the same strategy.

AbstractThere have been very few studies on hygienic behaviour as a mechanism of resistance to American foulbrood since Park, Woodrow, Rothenbuhler, and Rothenbuhler's students published their seminal work. The studies outlined in this part of the review form the core of information from which all later studies on hygienic behaviour have been based.

There is increasing worldwide concern about the impacts of pesticide residues on honey bees and bee colony survival, but how sublethal effects of pesticides on bees might cause colony failure remains highly controversial, with field data giving very mixed results. To explore how trace levels of the neonicotinoid pesticide imidacloprid impacted colony foraging performance, we equipped bees with RFID tags that allowed us to track their lifetime flight behavior. One group of bees was exposed to a trace concentration (5 μg/kg, ppb) of imidacloprid in sugar syrup while in the larval stage. The imidacloprid residues caused bees to start foraging when younger as adults and perform fewer orientation flights, and reduced their lifetime foraging flights by 28%. The magnitude of the effects of a trace imidacloprid concentration delivered only during larval stage highlights the severity of pesticide residues for bee foraging performance. Our data suggest that neonicotinoids could impact colony function by imbalancing the normal age based division of labor in a colony and reducing foraging efficiency. Understanding this mechanism will help the development of interventions to safeguard bee colony health.