Chronic Bee Paralysis Virus and Nosema ceranae Experimental Co-Infection of Winter Honey Bee Workers (Apis mellifera L.)

Chronic bee paralysis virus (CBPV) is an important viral disease of adult bees which induces significant losses in honey bee colonies. Despite comprehensive research, only limited data is available from experimental infection for this virus. In the present study winter worker bees were experimentally infected in three different experiments. Bees were first inoculated per os (p/o) or per cuticle (p/c) with CBPV field strain M92/2010 in order to evaluate the virus replication in individual bees. In addition, potential synergistic effects of co-infection with CBPV and Nosema ceranae (N. ceranae) on bees were investigated. In total 558 individual bees were inoculated in small cages and data were analyzed using quantitative real time RT-PCR (RT-qPCR). Our results revealed successful replication of CBPV after p/o inoculation, while it was less effective when bees were inoculated p/c. Dead bees harbored about 1,000 times higher copy numbers of the virus than live bees. Co-infection of workers with CBPV and N. ceranae using either method of virus inoculation (p/c or p/o) showed increased replication ability for CBPV. In the third experiment the effect of inoculation on bee mortality was evaluated. The highest level of bee mortality was observed in a group of bees inoculated with CBPV p/o, followed by a group of workers simultaneously inoculated with CBPV and N. ceranae p/o, followed by the group inoculated with CBPV p/c and the group with only N. ceranae p/o. The experimental infection with CBPV showed important differences after p/o or p/c inoculation in winter bees, while simultaneous infection with CBPV and N. ceranae suggesting a synergistic effect after inoculation.     

Molecular Identification of Chronic Bee Paralysis Virus Infection in Apis mellifera Colonies in Japan

Chronic bee paralysis virus (CBPV) infection causes chronic paralysis and loss of workers in honey bee colonies around the world. Although CBPV shows a worldwide distribution, it had not been molecularly detected in Japan. Our investigation of Apis mellifera and Apis cerana japonica colonies with RT-PCR has revealed CBPV infection in A. mellifera but not A. c. japonica colonies in Japan. The prevalence of CBPV is low compared with that of other viruses: deformed wing virus (DWV), black queen cell virus (BQCV), Israel acute paralysis virus (IAPV), and sac brood virus (SBV), previously reported in Japan. Because of its low prevalence (5.6%) in A. mellifera colonies, the incidence of colony losses by CBPV infection must be sporadic in Japan. The presence of the (-) strand RNA in dying workers suggests that CBPV infection and replication may contribute to their symptoms. Phylogenetic analysis demonstrates a geographic separation of Japanese isolates from European, Uruguayan, and mainland US isolates. The lack of major exchange of honey bees between Europe/mainland US and Japan for the recent 26 years (1985-2010) may have resulted in the geographic separation of Japanese CBPV isolates.     

Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity

In most animals, longevity is achieved at the expense of fertility, but queen honey bees do not show this tradeoff. Queens are both long-lived and fertile, whereas workers, derived from the same genome, are both relatively short-lived and normally sterile. It has been suggested, on the basis of results from workers, that vitellogenin (Vg), best known as a yolk protein synthesized in the abdominal fat body, acts as an antioxidant to promote longevity in queen bees. We explored this hypothesis, as well as related roles of insulin-IGF-1 signaling and juvenile hormone. Vg was expressed in thorax and head fat body cells in an age-dependent manner, with old queens showing much higher expression than workers. In contrast, Vg expression in worker head was much lower. Queens also were more resistant to oxidative stress than workers. These results support the hypothesis that caste-specific differences in Vg expression are involved in queen longevity. Consistent with predictions from Drosophila, old queens had lower head expression of insulin-like peptide and its putative receptors than did old workers. Juvenile hormone affected the expression of Vg and insulin-IGF-1 signaling genes in opposite directions. These results suggest that conserved and species-specific mechanisms interact to regulate queen bee longevity without sacrificing fecundity.     

Interactions in Colonies of Primitively Social Bees: Artificial Colonies of Lasioglossum zephyrum

Lasioglossum zephyrum usually lives in small colonies but is facultatively solitary. Lone bees and colonies produced from female pupae of the same generation were established in artificial indoor nests. Both the length of the prereproductive period and the number of cells produced per bee per day decreased with increasing colony size. In most colonies, ovarially and behaviorally recognized castes arose, a queen and workers, but with all intergradations. The mean size of queens was larger than that of workers. Nearly all queens mated although few workers did so in rooms with a few males, but mating had no effect on subsequent behavior or ovarian development. In groups of diverse age there was a tendency for the oldest bees to be queens; queens also were larger on the average than workers. In groups of equal age, the largest bee was most often queen. As would be expected for a scarcely social species, mechanisms of social integration (resulting in division of labor and differentiation of castes) mostly appear to involve behavioral features of the solitary ancestors and accidental results of joint occupancy of nests. There is no evidence of direct food or pheromone transfer among adult bees.     

Individual recognition and learning of queen odors by worker honeybees

A honeybee queen is usually attacked if she is placed among the workers of a colony other than her own. This rejection occurs even if environmental sources of odor, such as food, water, and genetic origin of the workers, are kept constant in laboratory conditions. The genetic similarity of queens determines how similar their recognition characteristics are; inbred sister queens were accepted in 35% of exchanges, outbred sister queens in 12%, and nonsister queens in 0%. Carbon dioxide narcosis results in worker honeybees accepting nonnestmate queens. A learning curve is presented, showing the time after narcosis required by workers to learn to recognize a new queen. In contrast, worker transfers result in only a small percentage of the workers being rejected. The reason for the difference between queens and workers may be because of worker and queen recognition cues having different sources.     

Juvenile hormone, reproduction, and worker behavior in the neotropical social wasp Polistes canadensis

Previous studies of the division of labor in colonies of eusocial Hymenoptera (wasps and bees) have led to two hypotheses regarding the evolution of juvenile hormone (JH) involvement. The novel- or single-function hypothesis proposes that the role of JH has changed from an exclusively reproductive function in primitively eusocial species (those lacking morphologically distinct queen and worker castes), to an exclusively behavioral function in highly eusocial societies (those containing morphologically distinct castes). In contrast, the split-function hypothesis proposes that JH originally functioned in the regulation of both reproduction and behavior in ancestral solitary species. Then, when reproductive and brood-care tasks came to be divided between queens and workers, the effects of JH were divided as well, with JH involved in regulation of reproductive maturation of egg-laying queens, and behavioral maturation, manifested as age-correlated changes in worker tasks, of workers. We report experiments designed to test these hypotheses. After documenting age-correlated changes in worker behavior (age polyethism) in the neotropical primitively eusocial wasp Polistes canadensis, we demonstrate that experimental application of the JH analog methoprene accelerates the onset of guarding behavior, an age-correlated task, and increases the number of foraging females; and we demonstrate that JH titers correlate with both ovarian development of queens and task differentiation in workers, as predicted by the split-function hypothesis. These findings support a view of social insect evolution that sees the contrasting worker and queen phenotypes as derived via decoupling of reproductive and brood-care components of the ancestral solitary reproductive physiology.     

Evolution and mechanisms of long life and high fertility in queen honey bees

Honey bees (Apis mellifera) are eusocial insects that exhibit striking caste-specific differences in longevity. Queen honey bees live on average 1–2 years whereas workers live on average 15–38 days in the summer and 150–200 days in the winter. Previous studies of senescence in the honey bee have focused on establishing the importance of extrinsic mortality factors (predation, weather) and behavior (nursing and foraging) in worker bee longevity. However, few studies have tried to elucidate the mechanisms that allow queen honey bees to achieve their long lifespan without sacrificing fecundity. Here, we review both types of studies and emphasize the importance of understanding both proximate and ultimate causes of the unusual life history of honey bee queens.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Interrogating an insect society

Insect societies such as those of ants, bees, and wasps consist of 1 or a small number of fertile queens and a large number of sterile or nearly sterile workers. While the queens engage in laying eggs, workers perform all other tasks such as nest building, acquisition and processing of food, and brood care. How do such societies function in a coordinated and efficient manner? What are the rules that individuals follow? How are these rules made and enforced? These questions are of obvious interest to us as fellow social animals but how do we interrogate an insect society and seek answers to these questions? In this article I will describe my research that was designed to seek answers from an insect society to a series of questions of obvious interest to us. I have chosen the Indian paper wasp Ropalidia marginata for this purpose, a species that is abundantly distributed in peninsular India and serves as an excellent model system. An important feature of this species is that queens and workers are morphologically identical and physiologically nearly so. How then does an individual become a queen? How does the queen suppress worker reproduction? How does the queen regulate the nonreproductive activities of the workers? What is the function of aggression shown by different individuals? How and when is the queen''s heir decided? I will show how such questions can indeed be investigated and will emphasize the need for a whole range of different techniques of observation and experimentation.     

Queen pheromone modulates brain dopamine function in worker honey bees

Honey bee queens produce a sophisticated array of chemical signals (pheromones) that influence both the behavior and physiology of their nest mates. Most striking are the effects of queen mandibular pheromone (QMP), a chemical blend that induces young workers to feed and groom the queen and primes bees to perform colony-related tasks. But how does this pheromone operate at the cellular level? This study reveals that QMP has profound effects on dopamine pathways in the brain, pathways that play a central role in behavioral regulation and motor control. In young worker bees, dopamine levels, levels of dopamine receptor gene expression, and cellular responses to this amine are all affected by QMP. We identify homovanillyl alcohol as a key contributor to these effects and provide evidence linking QMP-induced changes in the brain to changes at a behavioral level. This study offers exciting insights into the mechanisms through which QMP operates and a deeper understanding of the queen's ability to regulate the behavior of her offspring.     

Kin discrimination by worker honey bees in genetically mixed groups

We tested the hypothesis that in a genetically mixed assemblage of worker honey bees, individual workers would behave differently toward unfamiliar sisters than toward unfamiliar nonsisters. Groups of worker honey bees of mixed genetic composition were assembled by collecting pupae from separate colonies and placing the worker bees together on eclosion. A total of 10 workers, 5 from each of two kin groups, were used to form each group. When the workers were 5 days old, a worker of one of the two kin groups was introduced into the mixed group. This worker had previously been held in a group of its sisters, without contact with queen or nonsister bees. The interactions with the introduced bee indicate that in a mixed kin group, individual workers learn the composite identity of the group and do not attack unfamiliar bees differentially on the basis of kinship. However, kinship does influence the total number of interactions in which an introduced bee engages when placed in a genetically mixed group; bees interacted significantly more often with sisters than with nonsisters. There was a trend for bees to be involved in more feeding interactions with sisters. This finding indicates an ability of a bee to learn and use its own cues. In mixed groups, each bee maintains its genotypically correlated identity; the bees' odors do not comingle into a “group” or “gestalt” odor. The significance of these results is discussed in light of the genetic structure of natural colonies of honey bees.     

Metatranscriptome Analysis of Sympatric Bee Species Identifies Bee Virus Variants and a New Virus,Andrena-Associated Bee Virus-1

Bees are important plant pollinators in agricultural and natural ecosystems. High average annual losses of honey bee (Apis mellifera) colonies in some parts of the world, and regional population declines of some mining bee species (Andrena spp.), are attributed to multiple factors including habitat loss, lack of quality forage, insecticide exposure, and pathogens, including viruses. While research has primarily focused on viruses in honey bees, many of these viruses have a broad host range. It is therefore important to apply a community level approach in studying the epidemiology of bee viruses. We utilized high-throughput sequencing to evaluate viral diversity and viral sharing in sympatric, co-foraging bees in the context of habitat type. Variants of four common viruses (i.e., black queen cell virus, deformed wing virus, Lake Sinai virus 2, and Lake Sinai virus NE) were identified in honey bee and mining bee samples, and the high degree of nucleotide identity in the virus consensus sequences obtained from both taxa indicates virus sharing. We discovered a unique bipartite + ssRNA Tombo-like virus, Andrena-associated bee virus-1 (AnBV-1). AnBV-1 infects mining bees, honey bees, and primary honey bee pupal cells maintained in culture. AnBV-1 prevalence and abundance was greater in mining bees than in honey bees. Statistical modeling that examined the roles of ecological factors, including floral diversity and abundance, indicated that AnBV-1 infection prevalence in honey bees was greater in habitats with low floral diversity and abundance, and that interspecific virus transmission is strongly modulated by the floral community in the habitat. These results suggest that land management strategies that aim to enhance floral diversity and abundance may reduce AnBV-1 spread between co-foraging bees.     

Genetic control of social organization in an ant

A central issue in evolutionary biology is the extent to which complex social organization is under genetic control. We have found that a single genomic element marked by the protein-encoding gene Gp-9 is responsible for the existence of two distinct forms of social organization in the fire ant Solenopsis invicta. This genetic factor influences the reproductive phenotypes and behavioral strategies of queens and determines whether workers tolerate a single fertile queen or multiple queens per colony. Furthermore, this factor affects worker tolerance of queens with alternate genotypes, thus explaining the dramatic differences in Gp-9 allele frequencies observed between the two social forms in the wild. These findings reveal how a single genetic factor can have major effects on complex social behavior and influence the nature of social organization.     

Breeding for Virus Resistance and Its Effects on Deformed Wing Virus Infection Patterns in Honey Bee Queens

Viruses, and in particular the deformed wing virus (DWV), are considered as one of the main antagonists of honey bee health. The ‘suppressed in ovo virus infection’ trait (SOV) described for the first time that control of a virus infection can be achieved from genetically inherited traits and that the virus state of the eggs is indicative for this. This research aims to explore the effect of the SOV trait on DWV infections in queens descending from both SOV-positive (QDS+) and SOV-negative (QDS–) queens. Twenty QDS+ and QDS– were reared from each time four queens in the same starter–finisher colony. From each queen the head, thorax, ovaries, spermatheca, guts and eviscerated abdomen were dissected and screened for the presence of the DWV-A and DWV-B genotype using qRT-PCR. Queens descending from SOV-positive queens showed significant lower infection loads for DWV-A and DWV-B as well as a lower number of infected tissues for DWV-A. Surprisingly, differences were less expressed in the reproductive tissues, the ovaries and spermatheca. These results confirm that selection on the SOV trait is associated with increased virus resistance across viral genotypes and that this selection drives DWV towards an increased tissue specificity for the reproductive tissues. Further research is needed to explore the mechanisms underlying the interaction between the antiviral response and DWV.     

Cellular degradation activity is maintained during aging in long-living queen bees

Queen honeybees (Apis mellifera) have a much longer lifespan than worker bees. Whether cellular degradation activity is involved in the longevity of queen bees is unknown. In the present study, cellular degradation activity was evaluated in the trophocytes and oenocytes of young and old queen bees. The results indicated that (i) 20S proteasome activity and the size of autophagic vacuoles decreased with aging, and (ii) there were no significant differences between young and old queen bees with regard to 20S proteasome expression or efficiency, polyubiquitin aggregate expression, microtubule-associated protein 1 light chain 3-II (LC3-II) expression, 70 kDa heat shock cognate protein (Hsc70) expression, the density of autophagic vacuoles, p62/SQSTM1 expression, the activity or density of lysosomes, or molecular target of rapamycin expression. These results indicate that cellular degradation activity maintains a youthful status in the trophocytes and oenocytes of queen bees during aging and that cellular degradation activity is involved in maintaining the longevity of queen bees.     

Energy-regulated molecules maintain young status in the trophocytes and fat cells of old queen honeybees

Queen honeybees (Apis mellifera) have much longer lifespans than worker bees. Energy-regulated molecules in the trophocytes and fat cells of workers during aging have been determined, but are unknown in queen bees. In the present study, energy-regulated molecules were evaluated in the trophocytes and fat cells of young and old queen bees. Adenosine monophosphate-activated protein kinase α2 (AMPK-α2), phosphorylated AMPK-α2 (pAMPK-α2), and cAMP-specific phosphodiesterases activity increased with aging. The pAMPK-α2/AMPK-α2 ratio and AMPK activity; adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) concentrations; the ADP/ATP ratio and the AMP/ATP ratio; the cyclic adenosine monophosphate concentration; forkhead box protein O expression; Silent information regulator T1 (SirT1) expression and activity; and peroxisome proliferator-activated receptor-α (PPAR-α) expression were not significantly different between young and old queen bees. These results show that energy-regulated molecules maintain a youthful status in the trophocytes and fat cells of queen bees during aging. These cells seem to have longevity-promoting mechanisms and may clarify the secret of longevity in queen bees.     

Extended longevity of queen honey bees compared to workers is associated with peroxidation-resistant membranes

In the honey bee (Apis mellifera), depending on what they are fed, female eggs become either workers or queens. Although queens and workers share a common genome, the maximum lifespan of queens is an order-of-magnitude longer than workers. The mechanistic basis of this longevity difference is unknown. In order to test if differences in membrane composition could be involved we have compared the fatty acid composition of phospholipids of queen and worker honey bees. The cell membranes of both young and old honey bee queens are highly monounsaturated with very low content of polyunsaturates. Newly emerged workers have a similar membrane fatty acid composition to queens but within the first week of hive life, they increase the polyunsaturate content and decrease the monounsaturate content of their membranes, probably as a result of pollen consumption. This means their membranes likely become more susceptible to lipid peroxidation in this first week of hive life. The results support the suggestion that membrane composition might be an important factor in the determination of maximum lifespan. Assuming the same slope of the relationship between membrane peroxidation index and maximum lifespan as previously observed for mammal and bird species, we propose that the 3-fold difference in peroxidation index of phospholipids of queens and workers is large enough to account for the order-of-magnitude difference in their longevity.     

Were Workers of Eusocial Hymenoptera Initially Altruistic or Oppressed?

Studies of a primitively eusocial halictid bee, Lasioglossum zephyrum, strongly suggest that a major factor in originating a worker caste is selection at the individual level for queens that control associated adult females. Even in this scarcely social form, the queen inhibits other adult females from becoming queens, perhaps by her high level of activity and frequent nudging in the nest. Queens are behaviorally less varied than workers and show specialization, particularly in frequency of nudging (which is concentrated on the worker with largest ovaries) and of backing. Backing draws workers, especially those with slender ovaries, down to lower parts of the burrows where the stimuli for cell construction and provisioning probably operate. Eating of worker-laid eggs by queens was also noted. In spite of the suggestion that queens have evolved to control their workers rather than that workers have evolved to help their queens, both may well have occurred, for these processes are not mutually exclusive; moreover, social attributes mutually beneficial to both castes no doubt have arisen.