Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (<em>Apis mellifera</em>) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...

USDA-ARS, Carl Hayden Bee Research Center

Widely considered probiotic organisms, Bifidobacteria are common inhabitants of the alimentary tract of animals including insects. Bifidobacteria identified from the honey bee are found in larval guts and throughout the alimentary tract, but attain their greatest abundance in the adult hind gut. To further understand the role of Bifidobacteria in honey bees, we sequenced two strains of Bifidobacterium cultured from different alimentary tract environments and life stages.

Draft genome sequences of two Bifidobacterium sp. from the honey bee (Apis mellifera).

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. 

Microbial ecology of the hive and pollination landscape: bacterial associates from floral nectar, the alimentary tract and stored food of honey bees (Apis mellifera).

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.

Hive-stored pollen of honey bees: many lines of evidence are consistent with pollen preservation, not nutrient conversion.

Honey bee colonies function as a superorganism, where facultatively sterile female workers perform various tasks that support the hive. Nurse workers undergo numerous anatomical and physiological changes in preparation for brood rearing, including the growth of hypopharyngeal glands (HGs). These glands produce the major protein fraction of a protein- and lipid-rich jelly used to sustain developing larvae. Pollen intake is positively correlated with HG growth, but growth in the first three days is similar regardless of diet, suggesting that initial growth is a pre-determined process while later HG development depends on nutrient availability during a critical window in early adulthood (>3 d). It is unclear whether the resultant size differences in nurse HG are simply due to growth arrest or active degradation of the tissue. To determine what processes cause such differences in HG size, we catalogued the differential expression of both gene transcripts and proteins in the HGs of 8 d old bees that were fed diets containing pollen or no pollen. 3438 genes and 367 proteins were differentially regulated due to nutrition. Of the genes and proteins differentially expressed, undernourished bees exhibited more gene and protein up-regulation compared to well-nourished bees, with the affected processes including salivary gland apoptosis, oogenesis, and hormone signaling. Protein secretion was virtually the only process up-regulated in well-nourished bees. Further assays demonstrated that inhibition of ultraspiracle, one component of the ecdysteroid receptor, in the fat body caused larger HGs. Undernourished bees also had higher acid phosphatase activity, a physiological marker of cell death, compared to well-nourished bees. These results support a connection between poor nutrition, hormonal signaling, and HG degradation.

Transcriptional, translational, and physiological signatures of undernourished honey bees (Apis mellifera) suggest a role for hormonal factors in hypopharyngeal gland degradation.

In many vertebrates, social interactions and nutrition can affect the colonization of gut symbionts across generations. In the highly social honey bee, it is unknown to what extent the hive environment and older worker individuals contribute to the generational transmission of core gut bacteria. We used high-throughput sequencing to investigate the effect of nest materials and social contact on the colonization and succession of core hindgut microbiota in workers. With only brief exposure to hive materials following natural eclosion, gut bacterial communities at 3 and 7 days contained phylotypes typically found in the guts of mature adults regardless of treatment. Continuous exposure to nest materials or direct social interactions with mature adults did not affect the diversity or abundance of gut bacterial communities at the scale examined. Similarly, a common pollen supplement fed by beekeepers during pollen dearth had no effect. A consideration of unique OTUs revealed extensive microbial succession independent of treatment. The dominant Lactobacillus strain at 3 days was largely replaced by a different strain at day 7, revealing the colonization signature of a pioneer species. Similar but less pronounced patterns were evident in less abundant OTU's, many of which may influence community succession via alteration of the gut environment. Our results indicate that the process of bacterial community colonization in the hindgut is resilient to changes in the nutritional, hive, and social environment. Greater taxonomic resolution is needed to accurately resolve questions of ecological succession and typical proportional variation within and between core members of the gut bacterial community. 

Ecological Succession in the Honey Bee Gut: Shift in Lactobacillus Strain Dominance During Early Adult Development.

Honey bee workers (Apis mellifera) consume a variety of pollens to meet the majority of their requirements for protein and lipids. Recent work indicates that honey bees prefer diets that reflect the proper ratio of nutrients necessary for optimal survival and homeostasis. This idea relies on the precept that honey bees evaluate the nutritional composition of the foods provided to them. While this has been shown in bumble bees, the data for honey bees are mixed. Further, there is controversy as to whether foragers can evaluate the nutritional value of pollens, especially if they do not consume it. Here, we focused on nurse workers, who eat most of the pollen coming into the hive. We tested the hypothesis that nurses prefer diets with higher nutritional value. We first determined the nutritional profile, number of plant taxa (richness), and degree of hypopharyngeal gland (HG) growth conferred by three honey bee collected pollens. We then presented nurses with these same three pollens in paired choice assays and measured consumption. To further test whether nutrition influenced preference, we also presented bees with natural pollens supplemented with protein or lipids and liquid diets with protein and lipid ratios equal to the natural pollens. Different pollens conferred different degrees of HG growth, but despite these differences, nurse bees did not always prefer the most nutritious pollens. Adding protein and/or lipids to less desirable pollens minimally increased pollen attractiveness, and nurses did not exhibit a strong preference for any of the three liquid diets. We conclude that different pollens provide different nutritional benefits, but that nurses either cannot or do not assess pollen nutritional value. This implies that the nurses may not be able to communicate information about pollen quality to the foragers, who regulate the pollens coming into the hive.

Honey bee (Apis mellifera) nurses do not consume pollens based on their nutritional quality.

 displays multiple ecological strategies in its honey bee host. We sequenced the genomes of four strains found in larvae and the adult gut in order to better understand its ecology and relationship to other  The  genome consists of 2,009,892 bp and 1,830 protein-coding genes.

Draft Genome Sequences of Four Parasaccharibacter apium Strains Isolated from Honey Bees.

Free-ranging herbivores have yearly life cycles that generate dynamic resource needs. Honey bee colonies also have a yearly life cycle that might generate nutritional requirements that differ between times of brood rearing and colony expansion in the spring and population contraction and preparation for overwintering in the fall. To test this, we analyzed polyfloral mixes of spring and fall pollens to determine if the nutrient composition differed with season. Next, we fed both types of seasonal pollens to bees reared in spring and fall. We compared the development of brood food glands (i.e., hypopharyngeal glands - HPG), and the expression of genes in the fat body between bees fed pollen from the same (in-season) or different season (out-of-season) when they were reared. Because pathogen challenges often heighten the effects of nutritional stress, we infected a subset of bees with Nosema to determine if bees responded differently to the infection depending on the seasonal pollen they consumed. We found that spring and fall pollens were similar in total protein and lipid concentrations, but spring pollens had higher concentrations of amino and fatty acids that support HPG growth and brood production. Bees responded differently when fed in vs. out of season pollen. The HPG of both uninfected and Nosema-infected spring bees were larger when they were fed spring (in-season) compared to fall pollen. Spring bees differentially regulated more than 200 genes when fed in- vs. out-of-season pollen. When infected with Nosema, approximately 400 genes showed different infection-induced expression patterns in spring bees depending on pollen type. In contrast, HPG size in fall bees was not affected by pollen type, though HPG were smaller in those infected with Nosema. Very few genes were differentially expressed with pollen type in uninfected (4 genes) and infected fall bees (5 genes). Pollen type did not affect patterns of infection-induced expression in fall bees. Our data suggest that physiological responses to seasonal pollens differ between bees reared in the spring and fall with spring bees being significantly more sensitive to pollen type especially when infected with Nosema. This study provides evidence that seasonal pollens may provide levels of nutrients that align with the activities of honey bees during their yearly colony cycle. The findings are important for the planning and establishment of forage plantings to sustain honey bees, and in the development of seasonal nutritional supplements fed to colonies when pollen is unavailable.

Vanessa Corby-Harris

USDA-ARS, Carl Hayden Bee Research Center

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers