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Intragenic DNA methylation, also called gene body methylation, is an evolutionarily-conserved epigenetic mechanism in animals and plants. In social insects, gene body methylation is thought to contribute to behavioral plasticity, for example between foragers and nurse workers, by modulating gene expression. However, recent studies have suggested that the majority of DNA methylation is sequence-specific, and therefore cannot act as a flexible mediator between environmental cues and gene expression. To address this paradox, we examined whole-genome methylation patterns in the brains and ovaries of young honey bee workers that had been subjected to divergent social contexts: the presence or absence of the queen. Although these social contexts are known to bring about extreme changes in behavioral and reproductive traits through differential gene expression, we found no significant differences between the methylomes of workers from queenright and queenless colonies. In contrast, thousands of regions were differentially methylated between colonies, and these differences were not associated with differential gene expression in a subset of genes examined. Methylation patterns were highly similar between brain and ovary tissues and only differed in nine regions. These results strongly indicate that DNA methylation is not a driver of differential gene expression between tissues or behavioral morphs. Finally, despite the lack of difference in methylation patterns, queen presence affected the expression of all four DNA methyltransferase genes, suggesting that these enzymes have roles beyond DNA methylation. Therefore, the functional role of DNA methylation in social insect genomes remains an open question.

Male honey bees trapped at Drone Congregation Areas (DCAs) can be used to infer the number of colonies from which drones were derived, and thereby colony density in the environment. Crucial to the accuracy of this method is precise grouping of males into brother groups based on genetic markers, and a sample size that is sufficient so that all colonies in the area are included in the sample. The optimal sample size is a trade-off between cost and accuracy and cannot be known prior to sampling. Therefore, follow-up surveys may be necessary if the data indicate that the first sample was too small. However, the effect of multiple sampling on allele frequency estimates and the accuracy of the method is poorly understood. Here we trapped drones from two independent DCAs every month over 2 and 2.5 years. We analysed our data using the sibship grouping programme COLONY in three ways: (i) using data from the entire year, and counting the number of colonies identified in each month; (ii) using monthly data with allele frequencies from the entire season; and (iii) using data from each month separately. Although there were significant changes in allele frequencies over the year, these changes had no material effect on classifications of drones into families. Therefore, multiple samples can provide more robust estimates of family groupings due to the larger sample size and can be used with confidence where required.

The evolutionary significance of epigenetic inheritance is controversial. While epigenetic marks such as DNA methylation can affect gene function and change in response to environmental conditions, their role as carriers of heritable information is often considered anecdotal. Indeed, near-complete DNA methylation reprogramming, as occurs during mammalian embryogenesis, is a major hindrance for the transmission of nongenetic information between generations. Yet it remains unclear how general DNA methylation reprogramming is across the tree of life. Here we investigate the existence of epigenetic inheritance in the honey bee. We studied whether fathers can transfer epigenetic information to their daughters through DNA methylation. We performed instrumental inseminations of queens, each with four different males, retaining half of each male’s semen for whole genome bisulfite sequencing. We then compared the methylation profile of each father’s somatic tissue and semen with the methylation profile of his daughters. We found that DNA methylation patterns were highly conserved between tissues and generations. There was a much greater similarity of methylomes within patrilines (i.e., father-daughter subfamilies) than between patrilines in each colony. Indeed, the samples’ methylomes consistently clustered by patriline within colony. Samples from the same patriline had twice as many shared methylated sites and four times fewer differentially methylated regions compared to samples from different patrilines. Our findings indicate that there is no DNA methylation reprogramming in bees and, consequently, that DNA methylation marks are stably transferred between generations. This points to a greater evolutionary potential of the epigenome in invertebrates than there is in mammals.

SUMMARYInsect sociality is a major evolutionary transition based on the suppression of worker reproduction in favor of the reproductive monopoly of the queen. In the honey bee (Apis mellifera) model organism, the development of the two female caste phenotypes, queen and worker, is triggered by differences in their larval diets. However, the mechanistic details underlying their respective developmental trajectories, as well as the maintenance of sterility in the adult workers, are still not fully understood. Here we show that the long non-coding RNA lncov1 interacts with the Tudor staphylococcus nuclease (Tudor-SN) protein to form a regulatory module that promotes apoptosis in the ovaries of worker larvae. In adult workers, the lncov1/Tudor-SN module responds positively to environmental cues that suppress reproductive capacity. As lncov1 is considerably conserved in the Apidae, we propose that, by promoting worker sterility, the lncov1/Tudor-SN module has likely played critical roles in the social evolution of bees.

International audience; AbstractThe Cape honey bee (Capensis) is unusual in that workers can produce viable female offspring via thelytokous parthenogenesis. In contrast, mated queens never reproduce thelytokously, even though they could benefit from doing so when generating daughter queens. Nonetheless, virgin Capensis queens induced to lay without mating by CO2 narcosis produce a high proportion of thelytokous eggs, and instrumentally inseminated queens produce triploid offspring as the result of the fusion of two egg pronuclei and a sperm nucleus. We show here that thelytoky/triploidy in Capensis queens is not a consequence of CO2 narcosis per se because narcosis of laying queens does not induce thelytokous or triploid progeny. We also show that in artificially inseminated queens, the frequency of thelytoky/triploidy declines with age and is absent 10 months post-insemination. We confirm that the presence of semen in the spermatheca is not the mechanism that prevents thelytoky/triploidy in mated queens.

Tetragonula carbonaria, Tetragonula davenporti, Tetragonula hockingsi and Tetragonula mellipes comprise a species complex of Australian stingless bee species known as the 'Carbonaria' group. The species are difficult to distinguish morphologically and the major species-defining characters relate to comb architecture and nest entrance ornamentation. The taxonomy of the group is further complicated by likely nuclear mitochondrial pseudogenes (numts) and inter-specific hybrids. Here we demonstrate the existence of COI numts and isolate and characterize the 'true' mt-COI gene in T. carbonaria and T. hockingsi. Numts were isolated from enriched-nuclear DNA extraction followed by PCR amplification and Sanger sequencing, and were recognized by the presence of deletions and/or premature stop codons in the translated sequences. The mt-COI sequences were obtained from NGS sequencing using purified mtDNA. In T. carbonaria, two numts (numt1 and numt2) were identified and a third (numt3) was identified in T. hockingsi. Numt2 and numt3 are similar (1.2% sequence divergence), indicating a recent common origin. The genetic distance between the mt-COI of the two Tetragonula species was higher than might be expected for closely related species, 16.5%, corroborating previous studies in which T. carbonaria and T. hockingsi were regarded as separate species. The three numts are more similar to the COI of other stingless bee species, including Australian Austroplebia australis and South American Melipona bicolor (81.7-83.9%) than to the mt-COI of their own species (70-71.4%). This is because the mt-COI of T. carbonaria and T. hockingsi differ greatly from other Meliponinae. Our findings explain some formerly puzzling aspects of Carbonaria biogeography, and misinterpreted amplifications.

International audience; AbstractReliable information on Western honey bee colony density can be important in a variety of contexts including biosecurity responses, determining the sufficiency of pollinators in an agroecosystem and in determining the impacts of feral honey bees on ecosystems. Indirect methods for estimating colony density based on genetic analysis of sampled males are more feasible and cost efficient than direct observation in the field. Microsatellite genotypes of drones caught using Williams drone trap are used to identify the number of colonies (queens) that contributed drones to a mating lek. From the number of colonies, the density of colonies can be estimated based on assumptions about the area from which drones are drawn. This requires reliable estimates of drone flight distance. We estimated average minimum flight distance of drones from feral colonies along two 7-km transects in Southern NSW, Australia. We found that drones from feral colonies flew at least 3.5 km to drone traps. We then determined that the maximum distance that drones flew from a focal colony to a trap was 3.75 km. We conclude that a drone trap samples an area of 44 km2, and that this area should be used to convert estimated colony numbers to colony densities. This area is much greater than has been previously assumed. The densities of honey bee colonies in Grong Grong and Currawarna, NSW, are 1.38–2.73 and 1.31–3.06 colonies/km2 respectively.

DNA methylation is an important epigenetic modification that mediates diverse processes such as cellular differentiation, phenotypic plasticity, and genomic imprinting. Mounting evidence suggests that local DNA sequence variation can be associated with particular DNA methylation states, indicating that the interplay between genetic and epigenetic factors may contribute synergistically to the phenotypic complexity of organisms. Social insects such as ants, bees, and wasps have extensive phenotypic plasticity manifested in their different castes, and this plasticity has been associated with variation in DNA methylation. Yet, the influence of genetic variation on DNA methylation state remains mostly unknown. Here we examine the importance of sequence-specific methylation at the genome-wide level, using whole-genome bisulfite sequencing of the semen of individual honey bee males. We find that individual males harbor unique DNA methylation patterns in their semen, and that genes that are more variable at the epigenetic level are also more likely to be variable at the genetic level. DNA sequence variation can affect DNA methylation by modifying CG sites directly, but can also be associated with local variation in cis that is not CG-site specific. We show that covariation in sequence polymorphism and DNA methylation state contributes to the individual-specificity of epigenetic marks in social insects, which likely promotes their retention across generations, and their capacity to influence evolutionary adaptation.

Honey bee workers can lay eggs that result in viable males (drones). In queenless colonies, workers activate their ovaries and lay eggs, both in their own colony and in other queenless colonies. While worker reproduction and reproductive parasitism are well demonstrated, the direct contribution of reproductive workers to the gene pool is unclear. Queenless workers mostly lay their eggs in worker-sized brood cells. The resulting drones are smaller than normal drones. We determined two measures of forewing size in drones reared in drone-sized cells (DC) and in worker-sized cells (WC). Forewing length and width were 5.78 ± 0.26 mm (± SD) and 1.27 ± 0.08 mm, respectively, for drones reared in DC, and 5.01 ± 0.21 mm and 1.11 ± 0.05 mm for drones reared in WC. Discriminant function analysis indicated that forewing length alone is sufficient to differentiate DC and WC drones. To determine the contribution of worker-laid drones to the mating population, we sampled males at a natural mating lek using a Williams’ drone trap every month for 12 months. We used the discriminant function to assign drones as being DC or WC based on their forewing length and a strict classification criterion of ≥ 0.99% posterior probability of assignment to one or other group. We estimate that about 0.23% of sampled males were reared in WC and were, therefore, likely to have been laid by workers. Our results suggest that queenless workers and queenless colonies make a small contribution to the male mating population, and that this contribution may be sufficient to provide ongoing selection for worker reproductive parasitism.

In contrast to human societies, where kings and queens can be sources of conflict, we argue that the morphologically distinct queens of insect colonies are central to the minimization of conflict within their societies. Thus, the evolution of irreversible queen and worker castes represents a major transition in social evolution. Queens are selected to become better reproducers, and workers are selected to become better workers. The reproductive success of queens and workers are, therefore, inextricably linked. Workers achieve reproductive success by assisting the queen, whereas the queen needs her workers to provide her with the wherewithal to raise her brood. The tighter the mutual dependence, the lower conflict, and the larger insect societies can become. As the queen becomes a better breeder, workers are selected to become better at raising their siblings. Yet, nothing in nature is ever free of conflict and with the evolution of a true worker caste a new set of conflicts arises. Multiple mating by queens in particular opens the door to a new set of conflicts. Ironically, multiple mating can only evolve once within-colony conflict is reduced by evolving a true worker caste.

The hallmark of eusociality is the division of labour between reproductive (queen) and nonreproductive (worker) females. Yet in many eusocial insects, workers retain the ability to produce haploid male offspring from unfertilized eggs. The reproductive potential of workers has well-documented consequences for the structure and function of insect colonies, but its implications at the population level are less often considered. We show that worker reproduction in honey bees can have an important role in maintaining genetic diversity at the sex locus in invasive populations. The honey bee sex locus is homozygous-lethal, and, all else being equal, a higher allele number in the population lead to higher mean brood survival. In an invasive population of the honey bee Apis cerana in Australia, workers contribute significantly to male production: 38% of male-producing colonies are queenless, and these contribute one-third of all males at mating congregations. Using a model, we show that such male production by queenless workers will increase the number of sex alleles retained in nascent invasive populations following founder events, relative to a scenario in which only queens reproduce. We conclude that by rescuing sex locus diversity that would otherwise be lost, workers' sons help honey bee populations to minimize the negative effects of inbreeding after founder events and so contribute to their success as invaders.

Polyandrous social insects such as the honey bee are prime candidates for parental manipulation of gene expression in offspring. Although there is good evidence for parent-of-origin effects in honey bees the epigenetic mechanisms that underlie these effects remain a mystery. Small RNA molecules such as miRNAs, piRNAs and siRNAs play important roles in transgenerational epigenetic inheritance and in the regulation of gene expression during development. Here we present the first characterisation of small RNAs present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes. We show that semen contains fewer piRNAs relative to eggs and ovaries, and that piRNAs and miRNAs which map antisense to genes involved in DNA regulation and developmental processes are differentially expressed between tissues. tRNA fragments are highly abundant in semen and have a similar profile to those seen in semen in other animals. Intriguingly we find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. We conclude that small RNAs play a fundamental role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of origin-effects on gene expression and reproductive physiology.

In the honey bee (Apis mellifera), queen and worker castes originate from identical genetic templates but develop into different phenotypes. Queens lay up to 2000 eggs daily whereas workers are sterile in the queen's presence. Periodically queens stop laying: during swarming, when resources are scarce in winter, and when they are confined to a cage by beekeepers. We used confocal microscopy and gene expression assays to investigate the control of oogenesis in the ovaries of honey bee queens that were caged inside and outside the colony. We find evidence that queens use a different combination of 'checkpoints' to regulate oogenesis compared to honey bee workers and other insect species. However, both queen and worker castes likely use the same programmed cell death pathways to terminate oocyte development at their caste-specific checkpoints. Our results also suggest that a key factor driving the termination of oogenesis in queens is nutritional stress. Thus, queens may regulate oogenesis via the same regulatory pathways that were utilised by ancestral solitary species but likely have adjusted physiological checkpoints to suit their highly-derived life history.

Soley (1) argues that our study (2) does not prove that “transgenerational [epigenetic] inheritance serv[es] an adaptive role in evolution” in honey bees. This view misrepresents our intent. Our main findings are that in honey bees 1) DNA methylation profiles are much more similar between fathers and daughters than between unrelated males and females of different generations and 2) DNA methylation profiles are conserved across somatic and germline tissues (2). We are agnostic as to whether DNA methylation … [↵][1]1To whom correspondence may be addressed. Email: boris.yagound{at}sydney.edu.au. [1]: #xref-corresp-1-1

Epigenetics is the study of changes in gene activity that can be transmitted through cell divisions but cannot be explained by changes in the DNA sequence. Epigenetic mechanisms are central to gene regulation, phenotypic plasticity, development and the preservation of genome integrity. Epigenetic mechanisms are often held to make a minor contribution to evolutionary change because epigenetic states are typically erased and reset at every generation, and are therefore, not heritable. Nonetheless, there is growing appreciation that epigenetic variation makes direct and indirect contributions to evolutionary processes. First, some epigenetic states are transmitted intergenerationally and affect the phenotype of offspring. Moreover, bona fide heritable ‘epialleles' exist and are quite common in plants. Such epialleles could, therefore, be subject to natural selection in the same way as conventional DNA sequence-based alleles. Second, epigenetic variation enhances phenotypic plasticity and phenotypic variance and thus can modulate the effect of natural selection on sequence-based genetic variation. Third, given that phenotypic plasticity is central to the adaptability of organisms, epigenetic mechanisms that generate plasticity and acclimation are important to consider in evolutionary theory. Fourth, some genes are under selection to be ‘imprinted' identifying the sex of the parent from which they were derived, leading to parent-of-origin-dependent gene expression and effects. These effects can generate hybrid disfunction and contribute to speciation. Finally, epigenetic processes, particularly DNA methylation, contribute directly to DNA sequence evolution, because they act as mutagens on the one hand and modulate genome stability on the other by keeping transposable elements in check. This article is part of the theme issue ‘How does epigenetics influence the course of evolution?'

Haplo-diploidy and the relatedness asymmetries it generates mean that social insects are prime candidates for the evolution of genomic imprinting. In single-mating social insect species, some genes may be selected to evolve genomic mechanisms that enhance reproduction by workers when they are inherited from a female. This situation reverses in multiple mating species, where genes inherited from fathers can be under selection to enhance the reproductive success of daughters. Reciprocal crosses between subspecies of honeybees have shown strong parent-of-origin effects on worker reproductive phenotypes, and this could be evidence of such genomic imprinting affecting genes related to worker reproduction. It is also possible that social insect fathers directly affect gene expression in their daughters, for example, by placing small interfering RNA molecules in semen. Gene expression studies have repeatedly found evidence of parent-specific gene expression in social insects, but it is unclear at this time whether this arises from genomic imprinting, paternal manipulation, an artefact of cyto-nuclear interactions, or all of these. This article is part of the theme issue ‘How does epigenetics influence the course of evolution?’

In the honey bee (Apis mellifera), queen and worker castes originate from identical genetic templates but develop into different phenotypes. Queens lay up to 2,000 eggs daily whereas workers are sterile in the queen’s presence. Periodically queens stop laying; during swarming, when resources are scarce in winter and when they are confined to a cage by beekeepers. We used confocal microscopy and gene expression assays to investigate the control of oogenesis in honey bee queen ovaries. We show that queens use different combination of ‘checkpoints’ to regulate oogenesis compared to honey bee workers and other insect species. However, both queen and worker castes use the same programmed cell death pathways to terminate oocyte development at their caste-specific checkpoints. Our results also suggest that the termination of oogenesis in queens is driven by nutritional stress. Thus, queens may regulate oogenesis via the same regulatory pathways that were utilised by ancestral solitary species but have adjusted physiological checkpoints to suit their highly-derived life history.Summary statementHoney bee queens regulate oogenesis using a different combination of ‘checkpoints’ to workers, but both castes use the same molecular pathways.

When selection favours rare alleles over common ones (balancing selection in the form of negative frequency-dependent selection), a locus may maintain a large number of alleles, each at similar frequency. To better understand how allelic richness is generated and maintained at such loci, we assessed 201 sequences of the complementary sex determiner (csd) of the Asian honeybee (Apis cerana), sampled from across its range. Honeybees are haplodiploid; hemizygotes at csd develop as males and heterozygotes as females, while homozygosity is lethal. Thus, csd is under strong negative frequency-dependent selection because rare alleles are less likely to end up in the lethal homozygous form. We find that in A. cerana, as in other Apis, just a few amino acid differences between csd alleles in the hypervariable region are sufficient to trigger female development. We then show that while allelic lineages are spread across geographical regions, allelic differentiation is high between populations, with most csd alleles (86.3%) detected in only one sample location. Furthermore, nucleotide diversity in the hypervariable region indicates an excess of recently arisen alleles, possibly associated with population expansion across Asia since the last glacial maximum. Only the newly invasive populations of the Austral-Pacific share most of their csd alleles. In all, the geographic patterns of csd diversity in A. cerana indicate that high mutation rates and balancing selection act together to produce high rates of allele genesis and turnover at the honeybee sex locus, which in turn leads to its exceptionally high local and global polymorphism.

In honeybees there are three alleles of cytosolic malate dehydrogenase gene: F, M and S. Allele frequencies are correlated with environmental temperature, suggesting that the alleles have temperature-dependent fitness benefits. We determined the enzyme activity of each allele across a range of temperatures

International audience; AbstractWe examine the origin of honey bee (Apis mellifera) populations in Kangaroo Island (Australia), Norfolk Island (Australia) and the Kingdom of Tonga using a highly polymorphic mitochondrial DNA region and a panel of 37 single nucleotide polymorphisms that assigns ancestry to three evolutionary lineages: Eastern Europe, Western Europe and Africa. We also examine inbreeding coefficients and genetic variation using microsatellites and mitochondrial sequencing. The honey bees of Kangaroo Island have a high proportion of Eastern European ancestry (90.2%), consistent with claims that they are of the subspecies A. m. ligustica. The honey bees of Norfolk Island also had a majority of ancestry from Eastern Europe (73.1%) with some contribution from Western Europe (21.2%). The honey bees of Tonga are mainly of Western European (70.3%) origin with some Eastern European ancestry (27.4%). Despite the suspected severe bottlenecks experienced by these island population, inbreeding coefficients were low.

Pheromones are used by many insects to mediate social interactions. In the highly eusocial honey bee ( Apis mellifera ) queen mandibular pheromone is involved in the regulation of reproduction and the rate of ageing of workers. The molecular mechanisms by which queen mandibular pheromone acts remain largely unknown. Here we investigate how genes responsible for epigenetic modifications to DNA, RNA and histones expressed in the brain of honey bee workers respond to the presence of queen mandibular pheromone. Several of these genes are upregulated in workers exposed to queen mandibular pheromone. This suggests that epigenetic mechanisms are mediated by pheromonal communication between queens and workers, and that the interaction of pheromone signalling and epigenetics may mediate some of the neural plasticity that underlies ageing-associated traits in social insects. We suggest that pheromonal communication systems, such as those used by social insects, evolved to respond to environmental clues by making use of epigenomic machineries.

Inbreeding (the mating between closely related individuals) often has detrimental effects that are associated with loss of heterozygosity at overdominant loci, and the expression of deleterious recessive alleles. However, determining which loci are detrimental when homozygous, and the extent of their phenotypic effects, remains poorly understood. Here, we utilize a unique inbred population of clonal (thelytokous) honey bees, Apis mellifera capensis, to determine which loci reduce individual fitness when homozygous. This asexual population arose from a single worker ancestor approximately 20 years ago and has persisted for at least 100 generations. Thelytokous parthenogenesis results in a 1/3 of loss of heterozygosity with each generation. Yet, this population retains heterozygosity throughout its genome due to selection against homozygotes. Deep sequencing of one bee from each of the three known sub-lineages of the population revealed that 3,766 of 10,884 genes (34%) have retained heterozygosity across all sub-lineages, suggesting that these genes have heterozygote advantage. The maintenance of heterozygosity in the same genes and genomic regions in all three sub-lineages suggests that nearly every chromosome carries genes that show sufficient heterozygote advantage to be selectively detrimental when homozygous.

The haplodiploid system of sex determination of Hymenoptera acts as an exaptation for species to evolve novel forms of asexual reproduction including thelytoky (clonal offspring of the mother). During normal reproduction in Hymenoptera, three of the four products of meiosis that are present in newly-laid eggs are lost as polar bodies, while the remaining pronucleus either develops as a haploid male or fuses with a sperm nucleus to produce a diploid zygote. In contrast, in thelytokous reproduction, which is uncommon but taxonomically widespread, two of the four products of meiosis fuse, as if one acted as a sperm. Queenless workers of Apis mellifera capensis, a subspecies of honey bee from South Africa, routinely reproduce thelytokously. Unmated A. m. capensis queens can also be induced to lay thelytokously by narcosis with carbon dioxide, but mated queens are never thelytokous. We artificially inseminated A. m. capensis queens using CO2 narcosis. Up to 1/3 of offspring workers carried two maternal alleles and an allele of one father whereas no three-allele progeny were seen in control queens of the arrhenotokous (unfertilized eggs result in males) subspecies A. m. scutellata. Flow cytometry of three-allele individuals revealed that they were triploid and arose from the fertilization of a thelytokous fusion nucleus. We then reared six queens from a narcotized A. m. capensis queen and determined the ploidy of the offspring queens based on microsatellites. One of the five daughters was triploid. Following artificial insemination, this queen produced unfertilized thelytokous diploid eggs at high frequency, and unfertilized triploid eggs at much lower frequency. If fertilized, thelytokous diploid eggs were non-viable, even though triploidy in itself does not impede normal development. In contrast, when the rarer triploid eggs were fertilized, a proportion developed into viable tetraploids. Our study highlights the extraordinary developmental flexibility of haplo-diploid systems.

Thelytokous parthenogenesis is the production of females from unfertilized eggs. In this review we categorize the known thelytokous eusocial Hymenopterans (mostly ants) by their modes of worker and queen reproduction. The resultant tabulation reveals that: (1) there are no species in which queens are thelytokous and workers are exclusively arrhenotokous (asexual production of males). (2) When workers are capable of thelytoky, there are no examples of species in which queens are strictly thelytokous. (3) Strict queen thelytoky is only present in species with irreversibly sterile workers. (4) Facultative queen thelytoky and sterile workers can lead to the evolution of androgenesis (males are clonal sons of their fathers). These associations are probably best explained by consideration of differing fitness benefits of thelytoky between workers and queens and suggest that some combinations are unlikely to evolve. We therefore predict that they will hold for all eusocial Hymenoptera. No examples of endobacterium-induced thelytoky are known for the eusocial Hymenoptera, whereas endobacterium-induced thelytoky is widespread in the solitary Hymenoptera. We argue that this is because species in which both queens and workers are thelytokous that are unlikely to persist over evolutionary time. Further, eusocial species have single-locus sex determination, which is not compatible with endobacterium-induced feminization that is typically based on genome duplication. Only two thelytokous eusocial bees are known, and their modes of reproduction are consistent with the associations seen in ants. Thus far, no thelytokous eusocial wasps have been identified.

Across the world, the keeping of stingless bees is increasingly popular, providing commercial pollination, high-value honey and a rewarding pass time. The popularity of stingless beekeeping has resulted in large-scale anthropogenic movements of nests, sometimes from outside their native range. Colony movement has the potential to impact local populations via transfer of parasites and pathogens and gene flow across unnaturally large geographic scales. Tetragonula carbonaria is the most widespread and commonly kept stingless bee species in Australia. Concerns have been raised that large-scale artificial propagation of T. carbonaria colonies by Sydney beekeepers, from a small number of colonies that originated in south-east Queensland, may have two consequences. First, the managed population may be becoming increasingly inbred. Second, the wild population may be experiencing significant introgression of south-east Queensland genotypes, potentially diluting local adaptations to the Sydney environment and resulting in the loss of local alleles. Here we show, based on microsatellite and mitochondrial markers, that both the managed and wild Sydney populations are significantly different from the south-east Queensland population. Nonetheless there is evidence that introgression of south-east Queensland alleles is impacting the genetic structure of both wild and managed Sydney populations. The two Sydney populations are indistinguishable, suggesting two-way gene flow in Sydney consistent with expectations of gene flow via male dispersal. All populations have low inbreeding coefficients, suggesting that they are genetically healthy.

Although monandry is believed to have facilitated the evolution of eusociality, many highly eusocial insects have since evolved extreme polyandry. The transition to extreme polyandry was likely driven by the benefits of within-colony genetic variance to task specialization and/or disease resistance, but the extent to which it confers secondary benefits, once evolved, is unclear. Here we investigate the consequences of extreme polyandry on the invasive potential of the Asian honey bee, Apis cerana. In honey bees and other Hymenoptera, small newly founded invasive populations must overcome the genetic constraint of their sex determination system that requires heterozygosity at a sex-determining locus to produce viable females. We find A. cerana queens in an invasive population mate with an average of 27 males (range 16–42) that would result in the founding queen/s carrying 75% of their source population’s sex alleles in stored sperm. This mating frequency is similar to native-range Chinese A. cerana (mean 29 males, range 19–46). Simulations reveal that extreme polyandry reduces the risk, relative to monandry or moderate polyandry, that colonies produce a high incidence of inviable brood in populations that have experienced a founder event, that is, when sex allele diversity is low and/or allele frequencies are unequal. Thus, extreme polyandry aids the invasiveness of A. cerana in two ways: (1) by increasing the sex locus allelic richness carried to new populations with each founder, thereby increasing sex locus heterozygosity; and (2) by reducing the population variance in colony fitness following a founder event.

Most studies on collective decision making in honeybees have been performed on the cavity-nesting Western honeybee, Apis mellifera. In more recent years, the open-nesting red dwarf honeybee Apis florea has been developed as a model organism of collective decision making in the context of nest-site selection. These studies have shown that the specifics of the species’ nest-site requirements affect collective decision making. In particular, when potential nesting sites are abundant, as is the case in A. florea, the process of collective decision making can be simplified. Here, we ask if A. florea simply follows the availability of floral resources in their environment when deciding on an area to move into. We determined the locations danced for by three colonies the day before, of and after reproductive swarming. Our results suggest that colonies of A. florea indeed track the availability of forage in their environment and that swarms move in the general direction of forage rather than towards a specific nest site.

International audience; AbstractThe kinship theory of genomic imprinting argues that conflicting reproductive interests between males and females can lead to epigenetic modifications to the genome, altering gene expression in offspring in a parent-of-origin specific manner. The phenomenon is well documented in mammals and angiosperms, while the evidence for imprinting in social insects is steadily increasing. Workers of the South African honey bee, Apis mellifera capensis (Capensis) produce fatherless female offspring via thelytokous parthenogenesis, whereas queens produce female eggs sexually. We examined differences in reproductive phenotype between thelytokously and sexually derived Capensis workers. Workers with a father had significantly more ovarioles than fatherless workers, suggesting that males may imprint genes to enhance the reproductive success of their worker offspring.

International audience; AbstractHaplodiploid insects reproduce both sexually and asexually; haploid males arise from unfertilized eggs, while diploid females arise from fertilized eggs. Some species can also produce female offspring by thelytokous parthenogenesis. For example, queenless workers of the Cape honey bee, Apis mellifera capensis, of South Africa can produce diploid female offspring from unfertilized eggs. Genetic evidence suggests that in A. m. capensis, diploidy is restored in zygotes by the fusion of two maternal pronuclei, the haploid descendants of the two alternate products of meiosis I. Here, we confirm this genetic evidence by direct cytological observation of pronucleus fusion. We also provide a description of how the fusion occurs at 4.5–5 h post oviposition and describe the meiotic events that lead up to and follow the fusion. Finally, we document numerous departures from the typical meiotic patterns, which likely explain some of the anomalous A. m. capensis individuals that have been previously identified genetically.

Summary In honeybees, the ability of workers to produce daughters asexually, i.e., thelytokous parthenogenesis, is restricted to a single subspecies inhabiting the Cape region of South Africa, Apis mellifera capensis. Thelytoky has unleashed new selective pressures and the evolution of traits such as social parasitism, invasiveness, and social cancer. Thelytoky arises from an abnormal meiosis that results in the fusion of two maternal pronuclei, restoring diploidy in newly laid eggs. The genetic basis underlying thelytoky is disputed. To resolve this controversy, we generated a backcross between thelytokous A. m. capensis and non-thelytokous A. m. scutellata from the neighboring population and looked for evidence of genetic markers that co-segregated with thelytokous reproduction in 49 backcross females. We found that markers associated with the gene GB45239 on chromosome 11, including non-synonymous variants, showed consistent co-segregation with thelytoky, whereas no other region did so. Alleles associated with thelytoky were present in all A. m. capensis genomes examined but were absent from all other honeybees worldwide including A. m. scutellata. GB45239 is derived in A. m. capensis and has a putative role in chromosome segregation. It is expressed in ovaries and is downregulated in thelytokous bees, likely because of polymorphisms in the promoter region. Our study reveals how mutations affecting the sequence and/or expression of a single gene can change the reproductive mode of a population.

The development of artificial insemination (AI) for use in honey bees in the early half of the 20th century was a turning point for bee breeding programs. AI has since become integral to both breeding and research programs globally. The last overarching review of AI in honey bees was published in 1987. Since then, research has focused on semen storage and handling. Further, advancements in molecular biology have facilitated greater understanding of queen reproductive biology. Here we review the development of the techniques and equipment used in AI of honey bees, as well as the more recent advances. Areas requiring further research are identified, including the optimal duration and timing of narcosis, and the best age for queens at the time of insemination. Finally, having identified some inconsistencies in the terminology and evaluation methods used to describe AI protocols, we suggest guidelines for future papers utilising AI as a research tool.

The Kinship Theory of Genomic Imprinting (KTGI) posits that, in species where females mate with multiple males, there is selection for a male to enhance the reproductive success of his offspring at the expense of other males and his mating partner. Reciprocal crosses between honey bee subspecies show parent-of-origin effects for reproductive traits, suggesting that males modify the expression of genes related to female function in their female offspring. This effect is likely to be greater in the Cape honey bee (Apis mellifera capensis), because a male's daughters have the unique ability to produce female offspring that can develop into reproductive workers or the next queen without mating. We generated reciprocal crosses between Capensis and another subspecies and used RNA-seq to identify transcripts that are over- or underexpressed in the embryos, depending on the parental origin of the gene. As predicted, 21 genes showed expression bias towards the Capensis father's allele in colonies with a Capensis father, with no such bias in the reciprocal cross. A further six genes showed a consistent bias towards expression of the father's allele across all eight colonies examined, regardless of the direction of the cross. Consistent with predictions of the KTGI, six of the 21 genes are associated with female reproduction. No gene consistently showed overexpression of the maternal allele.

The Cape bee (Apis mellifera capensis) is a subspecies of the honeybee, in which workers commonly lay diploid unfertilized eggs via a process known as thelytoky. A recent study aimed to map the genetic basis of this trait in the progeny of a single capensis queen where workers laid either diploid (thelytokous) or haploid (arrhenotokous) eggs. A nonsynonymous single nucleotide polymorphism (SNP) in a gene of unknown function was reported to be strongly associated with thelytoky in this colony. Here, we analyze genome sequences from a global sample of A. mellifera and identify populations where the proposed thelytoky allele at this SNP is common but thelytoky is absent. We also analyze genome sequences of three capensis queens produced by thelytoky and find that, contrary to predictions, they do not carry the proposed thelytoky allele. The proposed SNP is therefore neither sufficient nor required to produce thelytoky in A. mellifera.

Domestication of animal species is often associated with a reduction in genetic diversity. The honey bee, Apis mellifera Linnaeus, 1758, has been managed by beekeepers for millennia for both honey and wax production and for crop pollination. Here we use both microsatellite markers and sequence data from the mitochondrial COI gene to evaluate genetic variation of managed A. mellifera in Thailand, where the species is introduced. Microsatellite analysis revealed high average genetic diversity with expected heterozygosities ranging from 0.620 ± 0.184 to 0.734 ± 0.071 per locus per province. Observed heterozygosities were generally lower than those expected under Hardy–Weinberg equilibrium, both locally and across the population as a whole. Mitochondrial sequencing revealed that the frequency of two evolutionary linages (C—Eastern European and O—Middle Eastern) are similar to those observed in a previous survey 10 yr ago. Our results suggest that Thai beekeepers are managing their A. mellifera in ways that retain overall genetic diversity, but reduce genetic diversity between apiaries.

The ability to clone oneself has clear benefits—no need for mate hunting or dilution of one's genome in offspring. It is therefore unsurprising that some populations of haplo-diploid social insects have evolved thelytokous parthenogenesis—the virgin birth of a female. But thelytokous parthenogenesis has a downside: the loss of heterozygosity (LoH) as a consequence of genetic recombination. LoH in haplo-diploid insects can be highly deleterious because female sex determination often relies on heterozygosity at sex-determining loci. The two female castes of the Cape honeybee,Apis mellifera capensis, differ in their mode of reproduction. While workers always reproduce thelytokously, queens always mate and reproduce sexually. For workers, it is important to reduce the frequency of recombination so as to not produce offspring that are homozygous. Here, we ask whether recombination rates differ between Cape workers and Cape queens that we experimentally manipulated to reproduce thelytokously. We tested our hypothesis that Cape workers have evolved mechanisms that restrain genetic recombination, whereas queens have no need for such mechanisms because they reproduce sexually. Using a combination of microsatellite genotyping and whole-genome sequencing we find that a reduction in recombination is confined to workers only.