RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control. Full article

The effects of sublethal pesticide exposure on queen emergence and virus titers were examined. Queen rearing colonies were fed pollen with chlorpyrifos (CPF) alone (pollen-1) and with CPF and the fungicide Pristine^® (pollen-2). Fewer queens emerged when larvae from open foraging (i.e., outside) colonies were reared in colonies fed pollen-1 or 2 compared with when those larvae were reared in outside colonies. Larvae grafted from and reared in colonies fed pollen-2 had lower rates of queen emergence than pollen-1 or outside colonies. Deformed wing virus (DWV) and black queen cell virus were found in nurse bees from colonies fed pollen-1 or 2 and in outside colonies. The viruses also were detected in queen larvae. However, we did not detect virus in emerged queens grafted from and reared in outside colonies. In contrast, DWV was found in all emerged queens grafted from colonies fed pollen-1 or 2 either reared in outside hives or those fed pollen-1 or 2. The results suggest that sublethal exposure of CPF alone but especially when Pristine^® is added reduces queen emergence possibly due to compromised immunity in developing queens. Full article

Honey bees move through a series of in-hive tasks (e.g., “nursing”) to outside tasks (e.g., “foraging”) that are coincident with physiological changes and higher levels of metabolic activity. Social context can cause worker bees to speed up or slow down this process, and foragers may revert back to their earlier in-hive tasks accompanied by reversion to earlier physiological states. To investigate the effects of flight, behavioral state and age on gene expression, we used whole-genome microarrays and real-time PCR. Brain tissue and flight muscle exhibited different patterns of expression during behavioral transitions, with expression patterns in the brain reflecting both age and behavior, and expression patterns in flight muscle being primarily determined by age. Our data suggest that the transition from behaviors requiring little to no flight (nursing) to those requiring prolonged flight bouts (foraging), rather than the amount of previous flight per se, has a major effect on gene expression. Following behavioral reversion there was a partial reversion in gene expression but some aspects of forager expression patterns, such as those for genes involved in immune function, remained. Combined with our real-time PCR data, these data suggest an epigenetic control and energy balance role in honey bee functional senescence. Full article

As key factors in intercellular adhesion processes, cadherins play important roles in a plethora of developmental processes, including gametogenesis. In a previous study on cadherin localization in the gonads of honey bees, performed with heterologous pan-cadherin antibodies, we detected these proteins as (i) associated with cell membranes, (ii) as homogeneously distributed throughout the cytoplasm, and (iii) as nuclear foci in both somatic and germline cells, raising the possibility of alternative functions. To further investigate such unusual intracellular cadherin localization we produced specific antibodies against the N- and C-terminal domains of honey bee N- and E-cadherin. A 160 kDa protein was recognized by the E-cadherin antibodies as well as one of approximately 300 kDa from those raised against N-cadherin. In gonad preparations, both proteins were detected as dispersed throughout the cytoplasm and as nuclear foci in both germline and somatic cells of queen and worker ovarioles, as well as in the testioles of drones. This leads us to infer that cadherins may indeed be involved in certain signaling pathways and/or transcriptional regulation during gametogenesis. In late oogenesis stages, immunolabeling for both proteins was observed at the cell cortex, in conformity with a role in cell adhesion. In testioles, E-cadherin was seen in co-localization with fusomes, indicating a possible role in cyst organization. Taken together, the distribution of N- and E-cadherins in honey bee gonads is suggestive of alternative roles for cadherins in gametogenesis of both sexes. Full article

Since 2006, beekeepers have reported increased losses of Apis mellifera colonies, and one factor that has been potentially implicated in these losses is the microsporidian Nosema ceranae. Since N. ceranae is a fairly recently discovered parasite, there is little knowledge of the variation in infection levels among individual workers within a colony. In this study we examined the levels of infection in individual bees from five colonies over three seasons using both spore counting and quantitative real-time PCR. The results show considerable intra-colony variation in infection intensity among individual workers with a higher percentage of low-level infections detected by PCR than by spore counting. Colonies generally had the highest percentage of infected bees in early summer (June) and the lowest levels in the fall (September). Nosema apis was detected in only 16/705 bees (2.3%) and always as a low-level co-infection with N. ceranae. The results also indicate that intra-colony variation in infection levels could influence the accuracy of Nosema diagnosis. Full article

The insulin signaling pathway has been hypothesized to play a key role in regulation of worker social insect behavior. We tested whether insulin treatment has direct effects on worker honeybee behavior in two contexts, sucrose response thresholds in winter bees and the progression to foraging by summer nurse bees. Treatment of winter worker bees with bovine insulin, used as a proxy for honeybee insulin, increased the bees’ sucrose response threshold. Treatment of summer nurse bees with bovine insulin significantly decreased the age at which foraging was initiated. This work provides further insight into the role of endocrine controls in behavior of in honeybees and insects in general. Full article

Hexamerins are storage proteins with primordial functions in insect metamorphosis. They are actively secreted by the larval fat body and stored in the hemolymph. During metamorphosis, they return to the fat body to be processed. For decades, these proteins were thought to exclusively function as an amino acid source for tissue reconstruction during the non-feeding pupal and pharate adult stages and, in some species, for egg production. Recently, new findings have linked the hexamerins to caste polyphenism and gonad development in social insects. To explore the roles of hexamerins during the honey bee metamorphosis, we used specific antibodies in expression analysis by western blot, in situ immunolocalization by confocal laser-scanning microscopy and in vivo injections to lower their endogenous levels. Our expression analysis highlighted the changing expression patterns in the fat body and hemolymph during development, which is consistent with the temporal dynamics of hexamerin secretion, storage and depletion. Confocal microscopy showed hexamerin expression in the cytoplasm of both types of fat body cells, trophocytes and oenocytes. Notably, hexamerin foci were also found in the nuclei of these cells, thus confirming our western blot analysis of fat body nuclear-enriched fractions. We also observed that the decrease in soluble hexamerins in antibody-treated pharate adults led to a precocious adult ecdysis, perhaps in response to the lack (or decrease) in hexamerin-derived amino acids. Taken together, these findings indicate that hexamerins have other functions in addition to their well-established role as amino acid sources for development. Full article

A honey bee queen mates with a number of drones a few days after she emerges as an adult. Spermatozoa of different drones are stored in her spermatheca and used for the rest of the queen’s life to fertilize eggs. Sperm usage is thought to be random, so that the patriline distribution within a honey bee colony would remain constant over time. In this study we assigned the progeny of a naturally mated honey bee queen to patrilines using microsatellite markers at the queen’s age of two, three and four years. No significant changes in patriline distribution occurred within each of two foraging seasons, with samples taken one and five months apart, respectively. Overall and pair-wise comparisons between the three analyzed years reached significant levels. Over the three-year period we found a trend for patrilines to become more equally represented with time. It is important to note that this study was performed with a single queen, and thus individual and population variation in sperm usage patterns must be assessed. We discuss long-term changes in patriline composition due to mixing processes in the queen’s spermatheca, following incomplete mixing of different drones’ sperm after mating. Full article

This work verifies that amitraz and oxalic acid treatment affect honeybee cuticle proteolytic enzymes (CPE). Three bee groups were monitored: oxalic acid treatment, amitraz treatment, control. Electrophoresis of hydrophilic and hydrophobic CPE was performed. Protease and protease inhibitor activities (in vitro) and antifungal/antibacterial efficiencies (in vivo), were analyzed. Amitraz and oxalic acid treatment reduced hydrophobic, but did not affect hydrophilic, protein concentrations and reduced both hydrophilic and hydrophobic body surface asparagine and serine protease activities in relation to most substrates and independently of pH. The activities of natural cuticle inhibitors of acidic, neutral, and alkaline proteases were suppressed as a result of the treatments, corresponding with reduced antifungal and antibacterial activity. Electrophoretic patterns of low-, medium-, and high-molecular-weight proteases and protease inhibitors were also affected by the treatments. Full article

The Small hive beetle, Aethina tumida, is an invasive pest of honey bees. Indigenous to sub-Saharan Africa, it has now become established in North America and Australia. It represents a serious threat to European honey bees. Commercially available entomopathogenic agents were screened for their potential to control beetle larvae. Entomopathogenic fungi investigated had minimal impact. The nematodes Steinernema kraussei and S. carpocapsae provided excellent control with 100% mortality of larvae being obtained. Sequential applications of the nematodes following larvae entering sand to pupate also provided excellent control for up to 3 weeks. The information gained supports the development of contingency plans to deal with A. tumida should it occur in the UK, and is relevant to the management of Small hive beetle where it is already present. Full article

The biological concept of stress originated in mammals, where a “General Adaptation Syndrome” describes a set of common integrated physiological responses to diverse noxious agents. Physiological mechanisms of stress in mammals have been extensively investigated through diverse behavioral and physiological studies. One of the main elements of the stress response pathway is the endocrine hypothalamo-pituitary-adrenal (HPA) axis, which underlies the “fight-or-flight” response via a hormonal cascade of catecholamines and corticoid hormones. Physiological responses to stress have been studied more recently in insects: they involve biogenic amines (octopamine, dopamine), neuropeptides (allatostatin, corazonin) and metabolic hormones (adipokinetic hormone, diuretic hormone). Here, we review elements of the physiological stress response that are or may be specific to honey bees, given the economical and ecological impact of this species. This review proposes a hypothetical integrated honey bee stress pathway somewhat analogous to the mammalian HPA, involving the brain and, particularly, the neurohemal organ corpora cardiaca and peripheral targets, including energy storage organs (fat body and crop). We discuss how this system can organize rapid coordinated changes in metabolic activity and arousal, in response to adverse environmental stimuli. We highlight physiological elements of the general stress responses that are specific to honey bees, and the areas in which we lack information to stimulate more research into how this fascinating and vital insect responds to stress. Full article