Investigating trade-offs between ovary activation and immune protein expression in bumble bee (Bombus impatiens) workers and queens.

Evidence for a trade-off between reproduction and immunity has manifested in many animal species, including social insects. However, investigations in social insect queens present a conundrum: new gynes of many social hymenopterans, such as bumble bees and ants, must first mate, then transition from being solitary to social as they establish their nests, thus experiencing confounding shifts in environmental conditions. Worker bumble bees offer an opportunity to investigate patterns of immune protein expression associated with ovary activation while minimizing extraneous environmental factors and genetic differences. Here, we use proteomics to interrogate the patterns of immune protein expression of female bumble bees (Bombus impatiens) by (i) sampling queens at different stages of their life cycle, then (ii) by sampling workers with different degrees of ovary activation. Patterns of immune protein expression in the haemolymph of queens are consistent with a reproduction-immunity trade-off, but equivalent samples from workers are not. This brings into question whether queen bumble bees really experience a reproduction-immunity trade-off, or if patterns of immune protein expression may actually be due to the selective pressure of the different environmental conditions they are exposed to during their life cycle.

Effects of study design parameters on estimates of bee abundance and richness in agroecosystems: a meta-analysis.

Pollinators are critical for agricultural production and food security, leading to many ongoing surveys of pollinators (especially bees) in crop and adjacent landscapes. These surveys have become increasingly important to better understand the community of potential pollinators, quantify relative insect abundance, and secure crop ecosystem services. However, as some bee populations are declining, there is a need to align and improve bee survey efforts, so that they can best meet research and conservation goals, particularly in light of the logistical and financial constraints of conducting such studies. Here, we mined the existing literature on bee surveys in or around agricultural lands to better understand how sampling methods can be optimized to maximize estimates of 2 key measures of bee communities (abundance and richness). After reviewing 72 papers spanning 20 yr of publication, we found that study duration, number of sites, sampling time, and sampling method most significantly influenced abundance, while the number of trips per year and collection method significantly influenced richness. Our analysis helps to derive thresholds, priorities, and recommendations that can be applied to future studies describing bee communities in agroecosystems.

Feminizer and doublesex knock-outs cause honey bees to switch sexes.

Honey bees are experts at refuting societal norms. Their matriarchal hives are headed by queens, backed by an all-female workforce, and males die soon after copulation. But the biochemical basis of how these distinct castes and sexes (queens, workers, and drones) arise is poorly understood, partly due to a lack of efficient tools for genetic manipulation. Now, Roth and colleagues have used clustered regularly interspaced short palindromic repeats (CRISPR) to knock out two key genes (feminizer and doublesex) that guide sexual development. Their technique yielded remarkably low rates of genetic mosaicism and offers a promising tool for engineering and phenotyping bees for diverse applications.

Impact of Honey Bee Migratory Management on Pathogen Loads and Immune Gene Expression is Affected by Complex Interactions With Environment, Worker Life History, and Season.

The effects of honey bee management, such as intensive migratory beekeeping, are part of the ongoing debate concerning causes of colony health problems. Even though comparisons of disease and pathogen loads among differently managed colonies indicate some effects, the direct impact of migratory practices on honey bee pathogens is poorly understood. To test long- and short-term impacts of managed migration on pathogen loads and immunity, experimental honey bee colonies were maintained with or without migratory movement. Individuals that experienced migration as juveniles (e.g., larval and pupal development), as adults, or both were compared to control colonies that remained stationary and therefore did not experience migratory relocation. Samples at different ages and life-history stages (hive bees or foragers), taken at the beginning and end of the active season, were analyzed for pathogen loads and physiological markers of health. Bees exposed to migratory management during adulthood had increased levels of the AKI virus complex (Acute bee paralysis, Kashmir bee, and Israeli acute bee paralysis viruses) and decreased levels of antiviral gene expression (dicer-like). However, those in stationary management as adults had elevated gut parasites (i.e. trypanosomes). Effects of environment during juvenile development were more complex and interacted with life-history stage and season. Age at collection, life-history stage, and season all influenced numerous factors from viral load to immune gene expression. Although the factors that we examined are not independent, the results illuminate potential factors in both migratory and nonmigratory beekeeping that are likely to contribute to colony stress, and also indicate potential mitigation measures.

Reproductive and Morphological Quality of Commercial Honey Bee (Hymenoptera: Apidae) Drones in the United States.

Exploration into reproductive quality in honey bees (Apis mellifera Linneaus (Hymenoptera: Apidae) largely focuses on factors that affect queens, with drones primarily being considered insofar as they pass on effects of environmental stressors to the queen and subsequent offspring. In those studies that consider drone quality explicitly, a primary focus has been on the dimorphic nature of drones laid in worker cells (either through rare queen error or worker reproduction) as compared to drones laid by the queen in the slightly larger drone cells. The implication from these studies is that that there exists a bimodality of drone morphological quality that is related to reproductive quality and competitive ability during mating. Our study quantifies the presence of such small drones in commercial populations, finding that rates of 'low-quality' drones are far higher than theoretically predicted under optimum conditions. Observations from commercial colonies also show significant inter-colony variation among the size and fecundity of drones produced, prompting speculation as to the mechanisms inducing such variation and the potential use of drone-quality variation for the colony- or apiary-level exposure to nutrition, agrichemical, or parasitic stressors.

Candidate stress biomarkers for queen failure diagnostics.

BACKGROUND: Queen failure is a persistent problem in beekeeping operations, but in the absence of overt symptoms it is often difficult, if not impossible, to ascertain the root cause. Stressors like heat-shock, cold-shock, and sublethal pesticide exposure can reduce stored sperm viability and lead to cryptic queen failure. Previously, we suggested candidate protein markers indicating heat-shock in queens. Here, we further investigate these heat-shock markers and test new stressors to identify additional candidate protein markers. RESULTS: We found that heat-shocking queens for upwards of 1 h at 40 °C was necessary to induce significant changes in the two strongest candidate heat-shock markers, and that relative humidity significantly influenced the degree of activation. In blind heat-shock experiments, we tested the efficiency of these markers at assigning queens to their respective treatment groups and found that one marker was sufficient to correctly assign queens 75% of the time. Finally, we compared cold-shocked queens at 4 °C and pesticide-exposed queens to controls to identify candidate markers for these additional stressors, and compared relative abundances of all markers to queens designated as 'healthy' and 'failing' by beekeepers. Queens that failed in the field had higher expression of both heat-shock and pesticide protein markers, but not cold-shock markers. CONCLUSIONS: This work offers some of the first steps towards developing molecular diagnostic tools to aid in determining cryptic causes of queen failure. Further work will be necessary to determine how long after the stress event a marker's expression remains elevated, and how accurate these markers will be for field diagnoses.

Egg-size plasticity in Apis mellifera: Honey bee queens alter egg size in response to both genetic and environmental factors.

Social evolution has led to distinct life-history patterns in social insects, but many colony-level and individual traits, such as egg size, are not sufficiently understood. Thus, a series of experiments was performed to study the effects of genotypes, colony size and colony nutrition on variation in egg size produced by honey bee (Apis mellifera) queens. Queens from different genetic stocks produced significantly different egg sizes under similar environmental conditions, indicating standing genetic variation for egg size that allows for adaptive evolutionary change. Further investigations revealed that eggs produced by queens in large colonies were consistently smaller than eggs produced in small colonies, and queens dynamically adjusted egg size in relation to colony size. Similarly, queens increased egg size in response to food deprivation. These results could not be solely explained by different numbers of eggs produced in the different circumstances but instead seem to reflect an active adjustment of resource allocation by the queen in response to colony conditions. As a result, larger eggs experienced higher subsequent survival than smaller eggs, suggesting that honey bee queens might increase egg size under unfavourable conditions to enhance brood survival and to minimize costly brood care of eggs that fail to successfully develop, and thus conserve energy at the colony level. The extensive plasticity and genetic variation of egg size in honey bees has important implications for understanding life-history evolution in a social context and implies this neglected life-history stage in honey bees may have trans-generational effects.

Differences in larval pesticide tolerance and esterase activity across honey bee (Apis mellifera) stocks.

Honey bee populations in North America are an amalgamation of diverse progenitor ecotypes experiencing varying levels of artificial selection. Genetic differences between populations can result in variable susceptibility towards environmental stressors, and here we compared pesticide tolerances across breeding stocks using a mixture of seven pesticides frequently found in colonies providing pollination services. We administered the pesticide mixture chronically to in vitro reared larvae at four concentrations of increasing Hazard Quotient (HQ, or cumulative toxicity) and measured mortality during larval development. We found that different stocks had significantly different tolerances to our pesticide mixture as indicated by their median lethal toxicity (HQ50). The intensively selected Pol-Line stock exhibited the greatest pesticide sensitivity while Old World (progenitor) and putatively feral stocks were the most pesticide-tolerant. Furthermore, we found that activity of the detoxification enzyme esterase was positively correlated with pesticide tolerance when measured using two different substrate standards, and confirmed that larvae from the Pol-Line stock had generally lower esterase activity. Consistent with an increased pesticide tolerance, the Old World and putatively feral stocks had higher esterase activities. However, esterases and other detoxification enzymes (CYP450s and GSTs) were found in similar abundances across stocks, suggesting that the differences in enzyme activity we observed might arise from stock-specific single nucleotide polymorphisms or post-translational modifications causing qualitative variation in enzyme activity. These results suggest that selective breeding may inadvertently increase honey bees' sensitivity to pesticides, whereas unselected, putatively feral and Old World stocks have larvae that are more tolerant.

Mitigating effects of pollen during paraquat exposure on gene expression and pathogen prevalence in Apis mellifera L.

Honey bee (Apis mellifera L.) populations have been experiencing notable mortality in Europe and North America. No single cause has been identified for these dramatic losses, but rather multiple interacting factors are likely responsible (such as pesticides, malnutrition, habitat loss, and pathogens). Paraquat is one of the most widely used non-selective herbicides, especially in developing countries. This herbicide is considered slightly toxic to honey bees, despite being reported as a highly effective inducer of oxidative stress in a wide range of living systems. Here, we test the effects of paraquat on the expression of detoxification and antioxidant-related genes, as well as on the dynamics of pathogen titers. Moreover, we tested the effects of pollen as mitigating factor to paraquat exposure. Our results show significant changes in the expression of several antioxidant-related and detoxification-related genes in the presence of paraquat, as well as an increase of pathogens titers. Finally, we demonstrate a mitigating effect of pollen through the up-regulation of specific genes and improvement of survival of bees exposed to paraquat. The presence of pollen in the diet was also correlated with a reduced prevalence of Nosema and viral pathogens. We discuss the importance of honey bees' nutrition, especially the availability of pollen, on colony losses chronically reported in the USA and Europe.

Genetic diversity confers colony-level benefits due to individual immunity.

Several costs and benefits arise as a consequence of eusociality and group-living. With increasing group size, spread of disease among nest-mates poses selective pressure on both individual immunity and group-level mechanisms of disease resistance (social immunity). Another factor known to influence colony-level expression of disease is intracolony genetic diversity, which in honeybees (Apis mellifera) is a direct function of the number of mates of the queen. Colonies headed by queens with higher mating numbers have less variable infections of decreased intensity, though the underlying mechanisms remain unclear. By pathogen-challenging larvae in vitro, we decoupled larval immune response from mechanisms of social immunity. Our results show that baseline immunity and degree of immune response do not vary with genetic diversity. However, intracolony variance in antimicrobial peptide production after pathogen challenge decreases with increasing genetic diversity. This reduction in variability of the larval immune response could drive the mitigation of disease observed in genetically diverse colonies.

Reduced cellular immune response in social insect lineages.

Social living poses challenges for individual fitness because of the increased risk of disease transmission among conspecifics. Despite this challenge, sociality is an evolutionarily successful lifestyle, occurring in the most abundant and diverse group of organisms on earth--the social insects. Two contrasting hypotheses predict the evolutionary consequences of sociality on immune systems. The social group hypothesis posits that sociality leads to stronger individual immune systems because of the higher risk of disease transmission in social species. By contrast, the relaxed selection hypothesis proposes that social species have evolved behavioural immune defences that lower disease risk within the group, resulting in lower immunity at the individual level. We tested these hypotheses by measuring the encapsulation response in 11 eusocial and non-eusocial insect lineages. We built phylogenetic mixed linear models to investigate the effect of behaviour, colony size and body size on cellular immune response. We found a significantly negative effect of colony size on encapsulation response (Markov chain Monte Carlo generalized linear mixed model (mcmcGLMM) p < 0.05; phylogenetic generalized least squares (PGLS) p < 0.05). Our findings suggest that insects living in large societies may rely more on behavioural mechanisms, such as hygienic behaviours, than on immune function to reduce the risk of disease transmission among nest-mates.

Development of the honey bee gut microbiome throughout the queen-rearing process.

The European honey bee (Apis mellifera) is used extensively to produce hive products and for crop pollination, but pervasive concerns about colony health and population decline have sparked an interest in the microbial communities that are associated with these important insects. Currently, only the microbiome of workers has been characterized, while little to nothing is known about the bacterial communities that are associated with queens, even though their health and proper function are central to colony productivity. Here, we provide a large-scale analysis of the gut microbiome of honey bee queens during their developmental trajectory and through the multiple colonies that host them as part of modern queen-rearing practices. We found that queen microbiomes underwent a dramatic shift in size and composition as they aged and encountered different worker populations and colony environments. Queen microbiomes were dominated by enteric bacteria in early life but were comprised primarily of alphaproteobacteria at maturity. Furthermore, queen gut microbiomes did not reflect those of the workers who tended them and, indeed, they lacked many of the bacteria that are considered vital to workers. While worker gut microbiotas were consistent across the unrelated colony populations sampled, the microbiotas of the related queens were highly variable. Bacterial communities in mature queen guts were similar in size to those of mature workers and were characterized by dominant and specific alphaproteobacterial strains known to be associated with worker hypopharyngeal glands. Our results suggest a model in which queen guts are colonized by bacteria from workers' glands, in contrast to routes of maternal inoculation for other animal microbiomes.

Comparative virulence and competition between Nosema apis and Nosema ceranae in honey bees (Apis mellifera).

Honey bees (Apis mellifera) are infected by two species of microsporidia: Nosema apis and Nosemaceranae. Epidemiological evidence indicates that N. ceranae may be replacing N. apis globally in A. mellifera populations, suggesting a potential competitive advantage of N. ceranae. Mixed infections of the two species occur, and little is known about the interactions among the host and the two pathogens that have allowed N. ceranae to become dominant in most geographical areas. We demonstrated that mixed Nosema species infections negatively affected honey bee survival (median survival=15-17days) more than single species infections (median survival=21days and 20days for N. apis and N. ceranae, respectively), with median survival of control bees of 27days. We found similar rates of infection (percentage of bees with active infections after inoculation) for both species in mixed infections, with N. apis having a slightly higher rate (91% compared to 86% for N. ceranae). We observed slightly higher spore counts in bees infected with N. ceranae than in bees infected with N. apis in single microsporidia infections, especially at the midpoint of infection (day 10). Bees with mixed infections of both species had higher spore counts than bees with single infections, but spore counts in mixed infections were highly variable. We did not see a competitive advantage for N. ceranae in mixed infections; N. apis spore counts were either higher or counts were similar for both species and more N. apis spores were produced in 62% of bees inoculated with equal dosages of the two microsporidian species. N. ceranae does not, therefore, appear to have a strong within-host advantage for either infectivity or spore growth, suggesting that direct competition in these worker bee mid-guts is not responsible for its apparent replacement of N. apis.

Metagenomic analysis of the honey bee queen microbiome reveals low bacterial diversity and Caudoviricetes phages.

In eusocial insects, the health of the queens-the colony founders and sole reproductive females-is a primary determinant for colony success. Queen failure in the honey bee Apis mellifera, for example, is a major concern of beekeepers who annually suffer colony losses, necessitating a greater knowledge of queen health. Several studies on the microbiome of honey bees have characterized its diversity and shown its importance for the health of worker bees, the female non-reproductive caste. However, the microbiome of workers differs from that of queens, which, in comparison, is still poorly studied. Thus, direct investigations of the queen microbiome are required to understand colony-level microbiome assembly, functional roles, and evolution. Here, we used metagenomics to comprehensively characterize the honey bee queen microbiome. Comparing samples from different geographic locations and breeder sources, we show that the microbiome of queens is mostly shaped by the environment experienced since early life and is predicted to play roles in the breakdown of the diet and protection from pathogens and xenobiotics. We also reveal that the microbiome of queens comprises only four candidate core bacterial species, Apilactobacillus kunkeei, Lactobacillus apis, Bombella apis, and Commensalibacter sp. Interestingly, in addition to bacteria, we show that bacteriophages infect the queen microbiome, for which Lactobacillaceae are predicted to be the main reservoirs. Together, our results provide the basis to understand the honey bee colony microbiome assemblage, can guide improvements in queen-rearing processes, and highlight the importance of considering bacteriophages for queen microbiome health and microbiome homeostasis in eusocial insects.IMPORTANCEThe queen caste plays a central role in colony success in eusocial insects, as queens lay eggs and regulate colony behavior and development. Queen failure can cause colonies to collapse, which is one of the major concerns of beekeepers. Thus, understanding the biology behind the queen's health is a pressing issue. Previous studies have shown that the bee microbiome plays an important role in worker bee health, but little is known about the queen microbiome and its function in vivo. Here, we characterized the queen microbiome, identifying for the first time the present species and their putative functions. We show that the queen microbiome has predicted nutritional and protective roles in queen association and comprises only four consistently present bacterial species. Additionally, we bring to attention the spread of phages in the queen microbiome, which increased in abundance in failing queens and may impact the fate of the colony.

Genetic diversity affects colony survivorship in commercial honey bee colonies.

Honey bee (Apis mellifera) queens mate with unusually high numbers of males (average of approximately 12 drones), although there is much variation among queens. One main consequence of such extreme polyandry is an increased diversity of worker genotypes within a colony, which has been shown empirically to confer significant adaptive advantages that result in higher colony productivity and survival. Moreover, honey bees are the primary insect pollinators used in modern commercial production agriculture, and their populations have been in decline worldwide. Here, we compare the mating frequencies of queens, and therefore, intracolony genetic diversity, in three commercial beekeeping operations to determine how they correlate with various measures of colony health and productivity, particularly the likelihood of queen supersedure and colony survival in functional, intensively managed beehives. We found the average effective paternity frequency (m e ) of this population of honey bee queens to be 13.6 ± 6.76, which was not significantly different between colonies that superseded their queen and those that did not. However, colonies that were less genetically diverse (headed by queens with m e ≤ 7.0) were 2.86 times more likely to die by the end of the study when compared to colonies that were more genetically diverse (headed by queens with m e > 7.0). The stark contrast in colony survival based on increased genetic diversity suggests that there are important tangible benefits of increased queen mating number in managed honey bees, although the exact mechanism(s) that govern these benefits have not been fully elucidated.

Meta-analysis of genetic diversity and intercolony relatedness among reproductives in commercial honey bee populations.

Honey bee colonies are large kin groups, each with a single mother queen and thousands of female workers. Queen bees are highly polyandrous, each mating with an average of approximately 12 drones from other colonies. We used a meta-analysis approach to compare the pedigree relationships of honey bee reproductives (queens and their mates) across five different studies and to quantify the overall genetic diversity of breeding populations. We compared the inferred genotypes of queens and their mates from microsatellite analyses of worker offspring from a feral Africanized honey bee population (which served as a negative control for inbreeding), an experimentally derived population of sister queens (which served as a positive control for inbreeding), and three separate commercially managed populations. We then compared the relatedness of all drones mated to each queen (mate-mate), all queens within each population (queen-queen), each queen with each of her mates (queen-mate), and all drones within each population (drone-drone). We found, as expected, the lowest levels of genetic similarity in the outcrossed population and highest levels of genetic similarity in the inbred population. Levels of genetic similarity among the managed honey bee populations were intermediate but closer to that of the inbred population. Genetic structuring of the entire breeding population resulted in two major subpopulations, likely deriving from breeders on the east and west coast. The effects that these findings have on the overall population genetic diversity of managed honey bees is discussed.

Group size influences maternal provisioning and compensatory larval growth in honeybees.

Environmental variation selects for the adaptive plasticity of maternal provisioning. Even though developing honeybees find themselves in a protected colony environment, their reproductively specialized queens actively adjust their maternal investment, even among worker-destined eggs. However, the potentially adaptive consequences of this flexible provisioning strategy and their mechanistic basis are unknown. Under natural conditions, we find that the body size of larvae hatching from small eggs in large colonies converges with that of initially larger larvae hatching from large eggs typically produced in small colonies. However, large eggs confer a persistent body size advantage when small and large eggs are cross-fostered in small and large colonies, respectively. We substantiate the increased maternal investment by identifying growth-promoting metabolomes and proteomes in large eggs compared to small eggs, which are primarily enriched in amino acid metabolism and cell maturation. Thus, our study provides a comprehensive adaptive explanation for the worker egg size plasticity of honeybees.

Assessing the mating 'health' of commercial honey bee queens.

Honey bee queens mate with multiple males, which increases the total genetic diversity within colonies and has been shown to confer numerous benefits for colony health and productivity. Recent surveys of beekeepers have suggested that 'poor queens' are a top management concern, thus investigating the reproductive quality and mating success of commercially produced honey bee queens is warranted. We purchased 80 commercially produced queens from large queen breeders in California and measured them for their physical size (fresh weigh and thorax width), insemination success (stored sperm counts and sperm viability), and mating number (determined by patriline genotyping of worker offspring). We found that queens had an average of 4.37 +/- 1.446 million stored sperm in their spermathecae with an average viability of 83.7 +/- 13.33%. We also found that the tested queens had mated with a high number of drones (average effective paternity frequency: 17.0 +/- 8.98). Queen "quality" significantly varied among commercial sources for physical characters but not for mating characters. These findings suggest that it may be more effective to improve overall queen reproductive potential by culling lower-quality queens rather than systematically altering current queen production practices.

Effects of planted pollinator habitat on pathogen prevalence and interspecific detection between bee species.

Shared resources can instigate pathogen spread due to large congregations of individuals in both natural and human modified resources. Of current concern is the addition of pollinator habitat in conservation efforts as it attracts bees of various species, potentially instigating interspecific sharing of pathogens. Common pathogens have been documented across a wide variety of pollinators with shared floral resources instigating their spread in some, but not all, cases. To evaluate the impact of augmented pollinator habitat on pathogen prevalence, we extracted RNA from samples of eight bee species across three families and screened these samples for nine pathogens using RT-qPCR. We found that some habitat characteristics influenced pathogen detection; however, we found no evidence that pathogen detection in one bee species was correlated with pathogen detection in another. In fact, pathogen detection was rare in wild bees. While gut parasites were detected in 6 out of the 8 species included in this study, viruses were only detected in honey bees. Further, virus detection in honey bees was low with a maximum 21% of samples testing positive for BQCV, for example. These findings suggest factors other than the habitat itself may be more critical in the dissemination of pathogens among bee species. However, we found high relative prevalence and copy number of gut parasites in some bee species which may be of concern, such as Bombus pensylvanicus. Long-term monitoring of pathogens in different bee species at augmented pollinator habitat is needed to evaluate if these patterns will change over time.

Variation in the reproductive quality of honey bee males affects their age of flight attempt.

Background: Honey bee males (drones) exhibit life histories that enable a high potential for pre- or post-copulatory sperm competition. With a numerical sex ratio of ∼11,000 drones for every queen, they patrol flyways and congregate aerially to mate on the wing. However, colonies and in fact drones themselves may benefit from a relative lack of competition, as queens are highly polyandrous, and colonies have an adaptive advantage when headed by queens that are multiply mated. Previous research has shown that larger drones are more likely to be found at drone congregation areas, more likely to mate successfully, and obtain a higher paternity share. However, the reproductive quality and size of drones varies widely within and among colonies, suggesting adaptive maintenance of drone quality variation at different levels of selection. Methods: We collected drones from six colony sources over the course of five days. We paint marked and individually tagged drones after taking body measurements at emergence and then placed the drones in one of two foster colonies. Using an entrance cage, we collected drones daily as they attempted flight. We collected 2,420 drones live or dead, analyzed 1,891 for attempted flight, collected emergence data on 207 drones, and dissected 565 upon capture to assess reproductive maturity. We measured drone body mass, head width, and thorax width at emergence, and upon dissection we further measured thorax mass, seminal vesicle length, mucus gland length, sperm count, and sperm viability from the seminal vesicles. Results: We found that drones that were more massive at emergence were larger and more fecund upon capture, suggesting that they are of higher reproductive quality and therefore do not exhibit a trade-off between size and fecundity. However, smaller drones tended to attempt initial flight at a younger age, which suggests a size trade-off not with fecundity but rather developmental maturation. We conclude that smaller drones may take more mating flights, each individually with a lower chance of success but thereby increasing their overall fitness. In doing so, the temporal spread of mating attempts of a single generation of drones within a given colony increases colony-level chances of mating with nearby queens, suggesting an adaptive rationale for high variation among drone reproductive quality within colonies.