The first step in understanding gut microbial ecology is determining the presence and potential niche breadth of associated microbes. While the core gut bacteria of adult honey bees is becoming increasingly apparent, there is very little and inconsistent information concerning symbiotic bacterial communities in honey bee larvae. The larval gut is the target of highly pathogenic bacteria and fungi, highlighting the need to understand interactions between typical larval gut flora, nutrition and disease progression. Here we show that the larval gut is colonized by a handful of bacterial groups previously described from guts of adult honey bees or other pollinators. First and second larval instars contained almost exclusively Alpha 2.2, a core Acetobacteraceae, while later instars were dominated by one of two very different Lactobacillus spp., depending on the sampled site. Royal jelly inhibition assays revealed that of seven bacteria occurring in larvae, only one Neisseriaceae and one Lactobacillus sp. were inhibited. We found both core and environmentally vectored bacteria with putatively beneficial functions. Our results suggest that early inoculation by Acetobacteraceae may be important for microbial succession in larvae. This assay is a starting point for more sophisticated in vitro models of nutrition and disease resistance in honey bee larvae.

Summary Chalkbrood and stonebrood are two fungal diseases associated with honey bee brood. Chalkbrood, caused by Ascosphaera apis, is a common and widespread disease that can result in severe reduction of emerging worker bees and thus overall colony productivity. Stonebrood is caused by Aspergillus spp. that are rarely observed, so the impact on colony health is not very well understood. A major concern with the presence of Aspergillus in honey bees is the production of airborne conidia, which can lead to allergic bronchopulmonary aspergillosis, pulmonary aspergilloma, or even invasive aspergillosis in lung tissues upon inhalation by humans. In the current chapter we describe the honey bee disease symptoms of these fungal pathogens. In addition, we provide research methodologies and protocols for isolating and culturing, in vivo and in vitro assays that are commonly used to study these host pathogen interactions. We give guidelines on the preferred methods used in current research and the application of molecular techniques. We have added photographs, drawings and illustrations to assist bee-extension personnel and bee scientists in the control of these two diseases.

The caste fate of developing female honey bee larvae is strictly socially regulated by adult nurse workers. As a result of this social regulation, nurse‐expressed genes as well as larval‐expressed genes may affect caste expression and evolution. We used a novel transcriptomic approach to identify genes with putative direct and indirect effects on honey bee caste development, and we subsequently studied the relative rates of molecular evolution at these caste‐associated genes. We experimentally induced the production of new queens by removing the current colony queen, and we used RNA sequencing to study the gene expression profiles of both developing larvae and their caregiving nurses before and after queen removal. By comparing the gene expression profiles of queen‐destined versus worker‐destined larvae as well as nurses observed feeding these two types of larvae, we identified larval and nurse genes associated with caste development. Of 950 differentially expressed genes associated with caste, 82% were expressed in larvae with putative direct effects on larval caste, and 18% were expressed in nurses with putative indirect effects on caste. Estimated selection coefficients suggest that both nurse and larval genes putatively associated with caste are rapidly evolving, especially those genes associated with worker development. Altogether, our results suggest that indirect effect genes play important roles in both the expression and evolution of socially influenced traits such as caste.