Bee pollen substitute1/17/2024 This experiment was performed twice because the CV group died in the first round (n = 8 bees from 2 cups per condition, repeated twice). We then dissected abdomens, extracted RNA, and used this to synthesize cDNA and run qPCR to examine developmental gene expression. We placed the bees into cup cages with sterile pollen and sugar syrup for 14 days. These sterile workers were either inoculated with gut homogenate from adult bees (conventionalized = CV), with co-cultured gut bacterial isolates (defined community = DC), or kept uninoculated (microbiota deficient = MD). We pulled late-stage pupae from brood frames and allowed them to emerge as adults in sterile conditions. (C) Effect of microbiota on expression of developmental genes. After 14 days we dissected the bees’ abdomens and performed RNA extraction and developmental gene assays as above (n = 8 bees per condition). We placed the bees into cup cages and fed each cup a different proportion of pollen mixed with an artificial diet (percentage of pollen in cups = 100%, 50%, 25%, or 0%). We allowed bees to emerge on a brood frame overnight and then conventionalized them by feeding them a gut homogenate prepared from adult workers. (B) Effect of proportion pollen in diet on expression of developmental genes. We used RNA to make cDNA and used qPCR to examine transcript abundance of developmental and dietary genes. We used these nucleic acids to perform 16S rRNA gene or taxon-specific gene sequencing, as well as quantitative polymerase chain reaction (qPCR) of total copies of 16S rRNA genes. After 14 days we dissected the bees’ abdomens and extracted DNA and RNA from 7 workers sampled from 4 cups per condition per site. We placed them into cup cages and fed them either pollen (POL), an artificial diet (AD), or an artificial diet that included hemicellulose and pectin additives (ADA). We allowed worker bees to emerge from a frame overnight and inoculated them with conventional honey bee gut microbiota. (A) Effect of diet on the bee gut microbiota and gene expression. The work is made available under the Creative Commons CC0 public domain dedication. Our findings demonstrate that a pollen-free diet significantly impacts the gut microbiota and gene expression of honey bees, indicating the importance of natural pollen as a primary protein source.Ĭopyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Lastly, we demonstrated that for bees inoculated with a defined gut microbiota, those raised on an artificial diet were less able to suppress infection from a bacterial pathogen than those that were fed natural pollen. In subsequent experiments, we showed that these shifts in gene expression may be linked to colonization by the gut microbiome. Furthermore, the pollen-free diet sharply reduced the expression of genes central to honey bee development. Our experiments on the impact of different diets showed that a simplified pollen-free diet formulated to resemble the macronutrient profile of a monofloral pollen source resulted in larger microbial communities with reduced diversity, reduced evenness, and reduced levels of potentially beneficial hive-associated bacteria. The crude proteins in these supplemental feeds are typically byproducts from food manufacturing processes and are rarely derived from pollen. During periods of reduced availability of floral pollen, supplemental protein sources are frequently provided to managed honey bee colonies. It also includes complex polysaccharides in its outer coat, which are largely indigestible by bees but can be metabolized by bacterial species within the gut microbiota. Pollen is the primary source of dietary protein for honey bees.
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