Cross-kingdom delivery of specific microRNAs to recipient organisms via food ingestion has been reported recently. However, it is unclear if such delivery of microRNAs occurs frequently in animal organisms after typical dietary intake. We found substantial levels of specific microRNAs in diets commonly consumed orally by humans, mice, and honey bees. Yet, after ingestion of fruit replete with plant microRNAs (MIR156a, MIR159a, and MIR169a), a cohort of healthy athletes did not carry detectable plasma levels of those molecules. Similarly, despite consumption of a diet with animal fat replete in endogenous miR-21, negligible expression of miR-21 in plasma or organ tissue was observed in miR-21 −/− recipient mice. Correspondingly, when fed vegetarian diets containing the above plant microRNAs, wild-type recipient mice expressed insignificant levels of these microRNAs. Finally, despite oral uptake of pollen containing these plant microRNAs, negligible delivery of these molecules was observed in recipient honeybees. Therefore, we conclude that horizontal delivery of microRNAs via typical dietary ingestion is neither a robust nor a frequent mechanism to maintain steady-state microRNA levels in a variety of model animal organisms, thus defining the biological limits of these molecules in vivo.

The putative transfer and gene regulatory activities of diet-derived miRNAs in ingesting animals are still debated. Importantly, no study to date has fully examined the role of dietary uptake of miRNA in the honey bee, a critical pollinator in both agricultural and natural ecosystems. After controlled pollen feeding experiments in adult honey bees, we observed that midguts demonstrated robust increases in plant miRNAs after pollen ingestion. However, we found no evidence of biologically relevant delivery of these molecules to proximal or distal tissues of recipient honey bees. Our results, therefore, support the premise that pollen miRNAs ingested as part of a typical diet are not robustly transferred across barrier epithelia of adult honey bees under normal conditions. Key future questions include whether other small RNA species in honey bee diets behave similarly and whether more specialized and specific delivery mechanisms exist for more efficient transport, particularly in the context of stressed barrier epithelia.

The honey bee is of paramount importance to humans in both agricultural and ecological settings. Honey bee colonies have suffered from increased attrition in recent years, stemming from complex interacting stresses. Defining common cellular stress responses elicited by these stressors represents a key step in understanding potential synergies. The proteostasis network is a highly conserved network of cellular stress responses involved in maintaining the homeostasis of protein production and function. Here, we have characterized the Heat Shock Response (HSR), one branch of this network, and found that its core components are conserved. In addition, exposing bees to elevated temperatures normally encountered by honey bees during typical activities results in robust HSR induction with increased expression of specific heat shock proteins that was variable across tissues. Surprisingly, we found that heat shock represses multiple immune genes in the abdomen and additionally showed that wounding the cuticle of the abdomen results in decreased expression of multiple HSR genes in proximal and distal tissues. This mutually antagonistic relationship between the HSR and immune activation is unique among invertebrates studied to date and may promote understanding of potential synergistic effects of disparate stresses in this critical pollinator and social insects more broadly.

The pollination services provided by the honey bee are critical in both natural and agricultural ecosystems. Honey bee colonies in the United States have suffered from an increased rate of die-off in recent years, stemming from a complex set of interacting stresses that remain poorly described. Defining specific common cellular processes and cellular stress responses impacted by multiple stressors represent a key step in understanding these synergies. Proteotoxic stresses negatively impact protein synthesis, folding, and degradation. Diverse proteotoxic stresses induce expression of genes encoding small heat shock proteins (sHSP) of the expanded lethal (2) essential for life (l(2)efl) gene family. In addition to upregulation by the Integrated Stress Response (ISR), the Heat Shock Response (HSR), and the Oxidative Stress Response (OSR), our data provide first evidence that sHSP genes are upregulated by the Unfolded Protein Response (UPR). As these genes appear to be part of a core stress response that could serve as a useful biomarker for cellular stress in honey bees, we designed and tested an RT-LAMP assay to detect increased l(2)efl gene expression in response to heat-stress. While this assay provides a powerful proof of principle, further work will be necessary to link changes in sHSP gene expression to colony-level outcomes, to adapt our preliminary assay into a Point of Care Testing (POCT) assay appropriate for use as a diagnostic tool for use in the field, and to couple assay results to management recommendations.

Foragers facilitate horizontal pathogen transmission in honey bee colonies, yet their systemic immune function wanes during transition to this life stage. In general, the insect immune system can be categorized into mechanisms operating at both the barrier epithelial surfaces and at the systemic level. As proposed by the intergenerational transfer theory of aging, such immunosenescence may result from changes in group resource allocation. Yet, the relative influence of pathogen transmission and resource allocation on immune function in bees from different stages has not been examined in the context of barrier immunity. We find that expression levels of antimicrobial peptides (AMPs) in honey bee barrier epithelia of the digestive tract do not follow a life stage-dependent decrease. In addition, correlation of AMP transcript abundance with microbe levels reveals a number of microbe-associated changes in AMPs levels that are equivalent between nurses and foragers. These results favor a model in which barrier effectors are maintained in foragers as a first line of defense, while systemic immune effectors are dismantled to optimize hive-level resources. These findings have important implications for our understanding of immunosenescence in honey bees and other social insects.

Recent declines in honey bee colonies in the United States have put increased strain on agricultural pollination. Nosema ceranae and Nosema apis, are microsporidian parasites that are highly pathogenic to honey bees and have been implicated as a factor in honey bee losses. While traditional methods for quantifying Nosema infection have high sensitivity and specificity, there is no field-portable device for field measurements by beekeepers. Here we present a field-portable and cost-effective smartphone-based platform for detection and quantification of chitin-positive Nosema spores in honey bees. The handheld platform, weighing only 374 g, consists of a smartphone-based fluorescence microscope, a custom-developed smartphone application, and an easy to perform sample preparation protocol. We tested the performance of the platform using samples at different parasite concentrations and compared the method with manual microscopic counts and qPCR quantification. We demonstrated that this device provides results that are comparable with other methods, having a limit of detection of 0.5 × 10⁶ spores per bee. Thus, the assay can easily identify infected colonies and provide accurate quantification of infection levels requiring treatment of infection, suggesting that this method is potentially adaptable for diagnosis of Nosema infection in the field by beekeepers. Coupled with treatment recommendations, this protocol and smartphone-based optical platform could improve the diagnosis and treatment of nosemosis in bees and provide a powerful proof-of-principle for the use of such mobile diagnostics as useful analytical tools for beekeepers in resource-limited settings.