Down-Regulation of Honey Bee IRS Gene Biases Behavior toward Food Rich in Protein

Food choice and eating behavior affect health and longevity. Large-scale research efforts aim to understand the molecular and social/behavioral mechanisms of energy homeostasis, body weight, and food intake. Honey bees (Apis mellifera) could provide a model for these studies since individuals vary in food-related behavior and social factors can be controlled. Here, we examine a potential role of peripheral insulin receptor substrate (IRS) expression in honey bee foraging behavior. IRS is central to cellular nutrient sensing through transduction of insulin/insulin-like signals (IIS). By reducing peripheral IRS gene expression and IRS protein amount with the use of RNA interference (RNAi), we demonstrate that IRS influences foraging choice in two standard strains selected for different food-hoarding behavior. Compared with controls, IRS knockdowns bias their foraging effort toward protein (pollen) rather than toward carbohydrate (nectar) sources. Through control experiments, we establish that IRS does not influence the bees'' sucrose sensory response, a modality that is generally associated with food-related behavior and specifically correlated with the foraging preference of honey bees. These results reveal a new affector pathway of honey bee social foraging, and suggest that IRS expressed in peripheral tissue can modulate an insect''s foraging choice between protein and carbohydrate sources.     

Hormone response to bidirectional selection on social behavior

Behavior is a quantitative trait determined by multiple genes. Some of these genes may have effects from early development and onward by influencing hormonal systems that are active during different life-stages leading to complex associations, or suites, of traits. Honey bees (Apis mellifera) have been used extensively in experiments on the genetic and hormonal control of complex social behavior, but the relationships between their early developmental processes and adult behavioral variation are not well understood. Bidirectional selective breeding on social food-storage behavior produced two honey bee strains, each with several sublines, that differ in an associated suite of anatomical, physiological, and behavioral traits found in unselected wild type bees. Using these genotypes, we document strain-specific changes during larval, pupal, and early adult life-stages for the central insect hormones juvenile hormone (JH) and ecdysteroids. Strain differences correlate with variation in female reproductive anatomy (ovary size), which can be influenced by JH during development, and with secretion rates of ecdysteroid from the ovaries of adults. Ovary size was previously assigned to the suite of traits of honey bee food-storage behavior. Our findings support that bidirectional selection on honey bee social behavior acted on pleiotropic gene networks. These networks may bias a bee's adult phenotype by endocrine effects on early developmental processes that regulate variation in reproductive traits.     

Social context, stress, and plasticity of aging

Positive social contact is an important factor in healthy aging, but our understanding of how social interactions influence senescence is incomplete. As life expectancy continues to increase because of reduced death rates among elderly, the beneficial role of social relationships is emerging as a cross-cutting theme in research on aging and healthspan. There is a need to improve knowledge on how behavior shapes, and is shaped by, the social environment, as well as needs to identify and study biological mechanisms that can translate differences in the social aspects of behavioral efforts, relationships, and stress reactivity (the general physiological and behavioral response-pattern to harmful, dangerous or unpleasant situations) into variation in aging. Honey bees (Apis mellifera) provide a genetic model in sociobiology, behavioral neuroscience, and gerontology that is uniquely sensitive to social exchange. Different behavioral contact between these social insects can shorten or extend lifespan more than 10-fold, and some aspects of their senescence are reversed by social cues that trigger aged individuals to express youthful repertoires of behavior. Here, I summarize how variation in social interactions contributes to this plasticity of aging and explain how beneficial and detrimental roles of social relationships can be traced from environmental and biological effects on honey bee physiology and behavior, to the expression of recovery-related plasticity, stress reactivity, and survival during old age. This system provides intriguing opportunities for research on aging.     

Honey bee PTEN--description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes

Background

Phosphatase and TENsin (PTEN) homolog is a negative regulator that takes part in IIS (insulin/insulin-like signaling) and Egfr (epidermal growth factor receptor) activation in Drosophila melanogaster. IIS and Egfr signaling events are also involved in the developmental process of queen and worker differentiation in honey bees (Apis mellifera). Here, we characterized the bee PTEN gene homologue for the first time and begin to explore its potential function during bee development and adult life.

Results

Honey bee PTEN is alternatively spliced, resulting in three splice variants. Next, we show that the expression of PTEN can be down-regulated by RNA interference (RNAi) in the larval stage, when female caste fate is determined. Relative to controls, we observed that RNAi efficacy is dependent on the amount of PTEN dsRNA that is delivered to larvae. For larvae fed queen or worker diets containing a high amount of PTEN dsRNA, PTEN knockdown was significant at a whole-body level but lethal. A lower dosage did not result in a significant gene down-regulation. Finally, we compared same-aged adult workers with different behavior: nursing vs. foraging. We show that between nurses and foragers, PTEN isoforms were differentially expressed within brain, ovary and fat body tissues. All isoforms were expressed at higher levels in the brain and ovaries of the foragers. In fat body, isoform B was expressed at higher level in the nurse bees.

Conclusion

Our results suggest that PTEN plays a central role during growth and development in queen- and worker-destined honey bees. In adult workers, moreover, tissue-specific patterns of PTEN isoform expression are correlated with differences in complex division of labor between same-aged individuals. Therefore, we propose that knowledge on the roles of IIS and Egfr activity in developmental and behavioral control may increase through studies of how PTEN functions can impact bee social phenotypes.     

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee''s behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.     

Social pleiotropy and the molecular evolution of honey bee vitellogenin

In this issue of Molecular Ecology, Kent et al. (2011) describe the adaptive evolution of honey bee vitellogenin that belongs to a phylogenetically conserved group of egg yolk precursors. This glyco‐lipoprotein leads a double life: it is central to egg production in the reproductive queen caste, and a regulator of social behaviour in the sterile worker caste. Does such social pleiotropy constrain molecular evolution? To the contrary; Kent et al. show that the vitellogenin gene is under strong positive selection in honey bees. Rapid change has taken place in specific protein regions, shedding light on the evolution of novel vitellogenin functions.     

Light exposure leads to reorganization of microglomeruli in the mushroom bodies and influences juvenile hormone levels in the honeybee

Honeybees show a remarkable behavioral plasticity at the transition from nursing inside the hive to foraging for nectar and/or pollen outside. This plasticity is important for age‐related division of labor in honeybee colonies. The behavioral transition is associated with significant volume and synaptic changes in the mushroom bodies (MBs), brain centers for sensory integration, learning, and memory. We tested whether precocious sensory exposure to light leads to changes in the density of synaptic complexes [microglomeruli (MG)] in the MBs. The results show that exposure to light pulses over 3 days induces a significant decrease in the MG density in visual subregions (collar) of the MB. Earlier studies had shown that foragers have increased levels of juvenile hormone (JH) co‐occurring with a decrease of vitellogenin (Vg). Previous work further established that RNAi‐mediated knockdown of vg and ultraspiracle (usp) induced an upregulation of JH levels, which can lead to precocious foraging. By disturbing both Vg and JH pathways using gene knockdown of vg and usp, we tested whether the changes in the hormonal system directly affect MG densities. Our study shows that MG numbers remained unchanged when Vg and JH pathways were perturbed, suggesting no direct hormonal influences on MG densities. However, mass spectrometry detection of JH revealed that precocious light exposure triggered an increase in JH levels in the hemolymph (HL) of young bees. This suggests a dual effect following light exposure via direct effects on MG reorganization in the MB calyx and a possible positive feedback on HL JH levels. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1141–1153, 2014     

Paratransgenesis: an approach to improve colony health and molecular insight in honey bees (Apis mellifera)?

The honey bee (Apis mellifera) is highly valued as a commercial crop pollinator and a model animal in research. Over the past several years, governments, beekeepers, and the general public in the United States and Europe have become concerned by increased losses of honey bee colonies, calling for more research on how to keep colonies healthy while still employing them extensively in agriculture. The honey bee, like virtually all multicellular organisms, has a mutually beneficial relationship with specific microbes. The microbiota of the gut can contribute essential nutrients and vitamins and prevent colonization by non-indigenous and potentially harmful species. The gut microbiota is also of interest as a resource for paratransgenesis; a Trojan horse strategy based on genetically modified symbiotic microbes that express effector molecules antagonizing development or transmission of pathogens. Paratransgenesis was originally engineered to combat human diseases and agricultural pests that are vectored by insects. We suggest an alternative use, as a method to promote health of honey bees and to expand the molecular toolbox for research on this beneficial social insect. The honey bees' gut microbiota contains lactic acid bacteria including the genus Lactobacillus that has paratransgenic potential. We present a strategy for transforming one Lactobacillus species, L. kunkeei, for use as a vector to promote health of honey bees and functional genetic research.