Evaluating Effects of a Critical Micronutrient (24-Methylenecholesterol) on Honey Bee Physiology.

Although poor nutrition is cited as one of the crucial factors in global pollinator decline, the requirements and role of several important nutrients (especially micronutrients) in honey bees are not well understood. Micronutrients, viz. phytosterols, play a physiologically vital role in insects as precursors of important molting hormones and building blocks of cellular membranes. There is a gap in comprehensive understanding of the impacts of dietary sterols on honey bee physiology. In the present study, we investigated the role of 24-methylenecholesterol-a key phytosterol-in honey bee nutritional physiology. Artificial diets with varying concentrations of 24-methylenecholesterol (0%, 0.1%. 0.25%, 0.5%, 0.75%, and 1% dry diet weight) were formulated and fed to honey bees in a laboratory cage experiment. Survival, diet consumption, head protein content, and abdominal lipid contents were significantly higher in dietary sterol-supplemented bees. Our findings provide additional insights regarding the role of this important sterol in honey bee nutritional physiology. The insights gleaned from this study could also advance the understanding of sterol metabolism and regulation in other bee species that are dependent on pollen for sterols, and assist in formulation of a more complete artificial diet for honey bees (Apis mellifera Linnaeus, 1758) (Hymenoptera: Apidae).

Novel Insights into Dietary Phytosterol Utilization and Its Fate in Honey Bees (Apis mellifera L.).

Poor nutrition is an important factor in global bee population declines. A significant gap in knowledge persists regarding the role of various nutrients (especially micronutrients) in honey bees. Sterols are essential micronutrients in insect diets and play a physiologically vital role as precursors of important molting hormones and building blocks of cellular membranes. Sterol requirements and metabolism in honey bees are poorly understood. Among all pollen sterols, 24-methylenecholesterol is considered the key phytosterol required by honey bees. Nurse bees assimilate this sterol from dietary sources and store it in their tissues as endogenous sterol, to be transferred to the growing larvae through brood food. This study examined the duration of replacement of such endogenous sterols in honey bees. The dietary 13C-labeled isotopomer of 24-methylenecholesterol added to artificial bee diet showed differential, progressive in vivo assimilation across various honey bee tissues. Significantly higher survival, diet consumption, head protein content and abdominal lipid content were observed in the dietary sterol-supplemented group than in the control group. These findings provide novel insights into phytosterol utilization and temporal pattern of endogenous 24-methylenecholesterol replacement in honey bees.

The omics approach to bee nutritional landscape.

BACKGROUND: Significant annual honey bee colony losses have been reported in the USA and across the world over the past years. Malnutrition is one among several causative factors for such declines. Optimal nutrition serves as the first line of defense against multiple stressors such as parasites/pathogens and pesticides. Given the importance of nutrition, it is imperative to understand bee nutrition holistically, identifying dietary sources that may fulfill bee nutritional needs. Pollen is the primary source of protein for bees and is critical for brood rearing and colony growth. Currently, there is significant gap in knowledge regarding the chemical and nutritional composition of pollen. METHODS: Targeted sterol analysis and untargeted metabolomics were conducted on five commercially available crop pollens, three bee-collected crop pollens, three vegetable oils (often added to artificial protein supplements by beekeepers), and one commonly used artificial protein supplement. RESULTS: This study reports key phytosterols and metabolites present across a spectrum of bee diets, including some of the major bee-pollinated crop pollens in the western United States. Significant differences were observed in sterol concentrations among the dietary sources tested. Among all quantified sterols, the highest concentrations were observed for 24-methylenecholesterol and further, pollen samples exhibited the highest 24-methylenecholesterol among all diet sources that were tested. Also, 236 metabolites were identified across all dietary sources examined. CONCLUSION: Information gleaned from this study is crucial in understanding the nutritional landscape available to all bee pollinators and may further assist in future efforts to develop comprehensive database of nutrients and metabolites present in all bee diets.

Impacts of Different Winter Storage Conditions on the Physiology of Diutinus Honey Bees (Hymenoptera: Apidae).

Global decline in insect pollinators, especially bees, have resulted in extensive research into understanding the various causative factors and formulating mitigative strategies. For commercial beekeepers in the United States, overwintering honey bee colony losses are significant, requiring tactics to overwinter bees in conditions designed to minimize such losses. This is especially important as overwintered honey bees are responsible for colony expansion each spring, and overwintered bees must survive in sufficient numbers to nurse the spring brood and forage until the new 'replacement' workers become fully functional. In this study, we examined the physiology of overwintered (diutinus) bees following various overwintering storage conditions. Important physiological markers, i.e., head proteins and abdominal lipid contents were higher in honey bees that overwintered in controlled indoor storage facilities, compared with bees held outdoors through the winter months. Our findings provide new insights into the physiology of honey bees overwintered in indoor and outdoor environments and have implications for improved beekeeping management.

Field rates of Sivanto™ (flupyradifurone) and Transform® (sulfoxaflor) increase oxidative stress and induce apoptosis in honey bees (Apis mellifera L.).

Pesticide exposures can have detrimental impacts on bee pollinators, ranging from immediate mortality to sub-lethal impacts. Flupyradifurone is the active ingredient in Sivanto™ and sulfoxaflor is the active ingredient in Transform®. They are both relatively new insecticides developed with an intent to reduce negative effects on bees, when applied to bee-attractive crops. With the growing concern regarding pollinator health and pollinator declines, it is important to have a better understanding of any potential negative impacts, especially sub-lethal, of these pesticides on bees. This study reports novel findings regarding physiological stress experienced by bees exposed to field application rates of these two insecticides via a Potter Tower sprayer. Two contact exposure experiments were conducted-a shorter 6-hour study and a longer 10-day study. Honey bee mortality, sugar syrup and water consumption, and physiological responses (oxidative stress and apoptotic protein assays) were assessed in bees exposed to Sivanto™ and Transform®, and compared to bees in control group. For the longer, 10-day contact exposure experiment, only the Sivanto™ group was compared to the control group, as high mortality recorded in the sulfoxaflor treatment group during the shorter contact exposure experiment, made the latter group unfeasible to test in the longer 10-days experiment. In both the studies, sugar syrup and water consumptions were significantly different between treatment groups and controls. The highest mortality was observed in Transform® exposed bees, followed by the Sivanto™ exposed bees. Estimates of reactive oxygen/nitrogen species indicated significantly elevated oxidative stress in both pesticide treatment groups, when compared to controls. Caspase-3 protein assays, an indicator of onset of apoptosis, was also significantly higher in the pesticide treatment groups. These differences were largely driven by post exposure duration, indicating sub-lethal impacts. Further, our findings also emphasize the need to revisit contact exposure impacts of Sivanto™, given the sub-lethal impacts and mortality observed in our long-term (10-day) contact exposure experiment.

Honey bees consider larval nutritional status rather than genetic relatedness when selecting larvae for emergency queen rearing.

In honey bees and many other social insects, production of queens is a vital task, as colony fitness is dependent on queens. The factors considered by honey bee workers in selecting larvae to rear new queens during emergency queen rearing are poorly understood. Identifying these parameters is critical, both in an evolutionary and apicultural context. As female caste development in honey bees is dependent on larval diet (i.e. nutrition), we hypothesized that larval nutritional state is meticulously assessed and used by workers in selection of larvae for queen rearing. To test this hypothesis, we conducted a series of experiments manipulating the nutritional status of one day old larvae by depriving them of brood food for a four-hour period, and then allowing workers to choose larvae for rearing queens from nutritionally deprived and non-deprived larvae. We simultaneously investigated the role of genetic relatedness in selection of larvae for queen rearing. In all the experiments, significantly greater numbers of non-deprived larvae than deprived larvae were selected for queen rearing irrespective of genetic relatedness. Our results demonstrate that honey bees perceive the nutritional state of larvae and use that information when selecting larvae for rearing queens in the natural emergency queen replacement process.

Effects of pollen dilution on infection of Nosema ceranae in honey bees.

Multiple stressors are currently threatening honey bee health, including pests and pathogens. Among honey bee pathogens, Nosema ceranae is a microsporidian found parasitizing the western honey bee (Apis mellifera) relatively recently. Honey bee colonies are fed pollen or protein substitute during pollen dearth to boost colony growth and immunity against pests and pathogens. Here we hypothesize that N. ceranae intensity and prevalence will be low in bees receiving high pollen diets, and that honey bees on high pollen diets will have higher survival and/or increased longevity. To test this hypothesis we examined the effects of different quantities of pollen on (a) the intensity and prevalence of N. ceranae and (b) longevity and nutritional physiology of bees inoculated with N. ceranae. Significantly higher spore intensities were observed in treatments that received higher pollen quantities (1:0 and 1:1 pollen:cellulose) when compared to treatments that received relatively lower pollen quantities. There were no significant differences in N. ceranae prevalence among different pollen diet treatments. Interestingly, the bees in higher pollen quantity treatments also had significantly higher survival despite higher intensities of N. ceranae. Significantly higher hypopharyngeal gland protein was observed in the control (no Nosema infection, and receiving a diet of 1:0 pollen:cellulose), followed by 1:0 pollen:cellulose treatment that was inoculated with N. ceranae. Here we demonstrate that diet with higher pollen quantity increases N. ceranae intensity, but also enhances the survival or longevity of honey bees. The information from this study could potentially help beekeepers formulate appropriate protein feeding regimens for their colonies to mitigate N. ceranae problems.

Colony Level Prevalence and Intensity of Nosema ceranae in Honey Bees (Apis mellifera L.).

Nosema ceranae is a widely prevalent microsporidian parasite in the western honey bee. There is considerable uncertainty regarding infection dynamics of this important pathogen in honey bee colonies. Understanding the infection dynamics at the colony level may aid in development of a reliable sampling protocol for N. ceranae diagnosis, and provide insights into efficient treatment strategies. The primary objective of this study was to characterize the prevalence (proportion of the sampled bees found infected) and intensity (number of spores per bee) of N. ceranae infection in bees from various age cohorts in a colony. We examined N. ceranae infection in both overwintered colonies that were naturally infected with N. ceranae and in quadruple cohort nucleus colonies that were established and artificially inoculated with N. ceranae. We also examined and quantified effects of N. ceranae infection on hypopharyngeal gland protein content and gut pH. There was no correlation between the prevalence and intensity of N. ceranae infection in composite samples (pooled bee samples used for analysis). Our results indicated that the prevalence and intensity of N. ceranae infection is significantly influenced by honey bee age. The N. ceranae infection prevalence values from composite samples of background bees (unmarked bees collected from four different locations in a colony) were not significantly different from those pertaining to marked-bee age cohorts specific to each sampling date. The foraging-aged bees had a higher prevalence of N. ceranae infection when compared to nurse-aged bees. N. ceranae did not have a significant effect on hypopharyngeal gland protein content. Further, there was no significant difference in mean gut pH of N. ceranae infected bees and non-infected bees. This study provides comprehensive insights into N. ceranae infection dynamics at the colony level, and also demonstrates the effects of N. ceranae infection on hypopharyngeal gland protein content and midgut pH.