First large-scale study reveals important losses of managed honey bee and stingless bee colonies in Latin America.

Over the last quarter century, increasing honey bee colony losses motivated standardized large-scale surveys of managed honey bees (Apis mellifera), particularly in Europe and the United States. Here we present the first large-scale standardized survey of colony losses of managed honey bees and stingless bees across Latin America. Overall, 1736 beekeepers and 165 meliponiculturists participated in the 2-year survey (2016-2017 and 2017-2018). On average, 30.4% of honey bee colonies and 39.6% of stingless bee colonies were lost per year across the region. Summer losses were higher than winter losses in stingless bees (30.9% and 22.2%, respectively) but not in honey bees (18.8% and 20.6%, respectively). Colony loss increased with operation size during the summer in both honey bees and stingless bees and decreased with operation size during the winter in stingless bees. Furthermore, losses differed significantly between countries and across years for both beekeepers and meliponiculturists. Overall, winter losses of honey bee colonies in Latin America (20.6%) position this region between Europe (12.5%) and the United States (40.4%). These results highlight the magnitude of bee colony losses occurring in the region and suggest difficulties in maintaining overall colony health and economic survival for beekeepers and meliponiculturists.

The role of temperature on the development of circadian rhythms in honey bee workers.

Circadian rhythms in honey bees are involved in various processes that impact colony survival. For example, young nurses take care of the brood constantly throughout the day and lack circadian rhythms. At the same time, foragers use the circadian clock to remember and predict food availability in subsequent days. Previous studies exploring the ontogeny of circadian rhythms of workers showed that the onset of rhythms is faster in the colony environment (~2 days) than if workers were immediately isolated after eclosion (7-9 days). However, which specific environmental factors influenced the early development of worker circadian rhythms remained unknown. We hypothesized that brood nest temperature plays a key role in the development of circadian rhythmicity in young workers. Our results show that young workers kept at brood nest-like temperatures (33-35 °C) in the laboratory develop circadian rhythms faster and in greater proportion than bees kept at lower temperatures (24-26 °C). In addition, we examined if the effect of colony temperature during the first 48 h after emergence is sufficient to increase the rate and proportion of development of circadian rhythmicity. We observed that twice as many individuals exposed to 35 °C during the first 48 h developed circadian rhythms compared to individuals kept at 25 °C, suggesting a critical developmental period where brood nest temperatures are important for the development of the circadian system. Together, our findings show that temperature, which is socially regulated inside the hive, is a key factor that influences the ontogeny of circadian rhythmicity of workers.

Effects of lithium on locomotor activity and circadian rhythm of honey bees.

Lithium has been considered a potential acaricidal agent against the honey bee (Apis mellifera) parasite Varroa. It is known that lithium suppresses elevated activity and regulates circadian rhythms and light response when administered to humans as a primary therapeutic chemical for bipolar disorder and to other bipolar syndrome model organisms, given the crucial role of timing in the bee's foraging activity and the alternating sunlight vs dark colony environment bees are exposed, we explored the influence of lithium on locomotor activity (LMA) and circadian rhythm of honey bees. We conducted acute and chronic lithium administration experiments, altering light conditions and lithium doses to assess LMA and circadian rhythm changes. We fed bees one time 10 µl sucrose solution with 0, 50, 150, and 450 mM LiCl in the acute application experiment and 0, 1, 5, and 10 mmol/kg LiCl ad libitum in bee candy in the chronic application experiment. Both acute and chronic lithium treatments significantly decreased the induced LMA under constant light. Chronic lithium treatment disrupted circadian rhythmicity in constant darkness. The circadian period was lengthened by lithium treatment under constant light. We discuss the results in the context of Varroa control and lithium's effect on bipolar disorder.

Gut microbiota variation of a tropical oil-collecting bee species far exceeds that of the honeybee.

Introduction: Interest for bee microbiota has recently been rising, alleviating the gap in knowledge in regard to drivers of solitary bee gut microbiota. However, no study has addressed the microbial acquisition routes of tropical solitary bees. For both social and solitary bees, the gut microbiota has several essential roles such as food processing and immune responses. While social bees such as honeybees maintain a constant gut microbiota by direct transmission from individuals of the same hive, solitary bees do not have direct contact between generations. They thus acquire their gut microbiota from the environment and/or the provision of their brood cell. To establish the role of life history in structuring the gut microbiota of solitary bees, we characterized the gut microbiota of Centris decolorata from a beach population in Mayagüez, Puerto Rico. Females provide the initial brood cell provision for the larvae, while males patrol the nest without any contact with it. We hypothesized that this behavior influences their gut microbiota, and that the origin of larval microbiota is from brood cell provisions. Methods: We collected samples from adult females and males of C. decolorata (n = 10 each, n = 20), larvae (n = 4), and brood cell provisions (n = 10). For comparison purposes, we also sampled co-occurring female foragers of social Apis mellifera (n = 6). The samples were dissected, their DNA extracted, and gut microbiota sequenced using 16S rRNA genes. Pollen loads of A. mellifera and C. decolorata were analyzed and interactions between bee species and their plant resources were visualized using a pollination network. Results: While we found the gut of A. mellifera contained the same phylotypes previously reported in the literature, we noted that the variability in the gut microbiota of solitary C. decolorata was significantly higher than that of social A. mellifera. Furthermore, the microbiota of adult C. decolorata mostly consisted of acetic acid bacteria whereas that of A. mellifera mostly had lactic acid bacteria. Among C. decolorata, we found significant differences in alpha and beta diversity between adults and their brood cell provisions (Shannon and Chao1 p < 0.05), due to the higher abundance of families such as Rhizobiaceae and Chitinophagaceae in the brood cells, and of Acetobacteraceae in adults. In addition, the pollination network analysis indicated that A. mellifera had a stronger interaction with Byrsonima sp. and a weaker interaction with Combretaceae while interactions between C. decolorata and its plant resources were constant with the null model. Conclusion: Our data are consistent with the hypothesis that behavioral differences in brood provisioning between solitary and social bees is a factor leading to relatively high variation in the microbiota of the solitary bee.

Antibiotics Alter the Expression of Genes Related to Behavioral Development in Honey Bees (Hymenoptera: Apidae).

Honey bees, as many species of social insects, display a division of labor among colony members based on behavioral specializations related to age. Adult worker honey bees perform a series of tasks in the hive when they are young (such as brood care or nursing) and at ca. 2-3 wk of age, shift to foraging for nectar and pollen outside the hive. The transition to foraging involves changes in metabolism and neuroendocrine activities. These changes are associated with a suite of developmental genes. It was recently demonstrated that antibiotics influence behavioral development by accelerating or delaying the onset of foraging depending on timing of antibiotic exposure. To understand the mechanisms of these changes, we conducted a study on the effects of antibiotics on expression of candidate genes known to regulate behavioral development. We demonstrate a delay in the typical changes in gene expression over the lifetime of the individuals that were exposed to antibiotics during immature stage and adulthood. Additionally, we show an acceleration in the typical changes in gene expression on individuals that were expose to antibiotics only during immature stage. These results show that timing of antibiotic exposure alter the typical regulation of behavioral development by metabolic and neuroendocrine processes.

Tissue-specific transcriptional patterns underlie seasonal phenotypes in honey bees (Apis mellifera).

Faced with adverse conditions, such as winter in temperate regions or hot and dry conditions in tropical regions, many insect species enter a state of diapause, a period of dormancy associated with a reduction or arrest of physical activity, development and reproduction. Changes in common physiological pathways underlie diapause phenotypes in different insect species. However, most transcriptomic studies of diapause have not simultaneously evaluated and compared expression patterns in different tissues. Honey bees (Apis mellifera) represent a unique model system to study the mechanisms underpinning diapause-related phenotypes. In winter, honey bees exhibit a classic diapause phenotype, with reduced metabolic activity, increased physiological nutritional resources and altered hormonal profiles. However, winter bees actively heat their colony by vibrating their wing muscles; thus, this tissue is not quiescent. Here, we evaluated the transcriptional profiles of flight muscle tissue and fat body tissue (involved in nutrient storage, metabolism and immune function) of winter bees. We also evaluated two behavioural phenotypes of summer bees: nurses, which exhibit high nutritional stores and low flight activity, and foragers, which exhibit low nutritional stores and high flight activity. We found winter bees and nurses have similar fat body transcriptional profiles, whereas winter bees and foragers have similar flight muscle transcriptional profiles. Additionally, differentially expressed genes were enriched in diapause-related gene ontology terms. Thus, honey bees exhibit tissue-specific transcriptional profiles associated with seasonal phenotypes, laying the groundwork for future studies evaluating the mechanisms, evolution and consequences of this tissue-specific regulation.

The Role of Colony Temperature in the Entrainment of Circadian Rhythms of Honey Bee Foragers.

Honey bees utilize their circadian rhythms to accurately predict the time of day. This ability allows foragers to remember the specific timing of food availability and its location for several days. Previous studies have provided strong evidence toward light/dark cycles being the primary Zeitgeber for honey bees. Work in our laboratory described large individual variation in the endogenous period length of honey bee foragers from the same colony and differences in the endogenous rhythms under different constant temperatures. In this study, we further this work by examining the temperature inside the honey bee colony. By placing temperature and light data loggers at different locations inside the colony we measured temperature at various locations within the colony. We observed significant oscillations of the temperature inside the hive, that show seasonal patterns. We then simulated the observed temperature oscillations in the laboratory and found that using the temperature cycle as a Zeitgeber, foragers present large individual differences in the phase of locomotor rhythms for temperature. Moreover, foragers successfully synchronize their locomotor rhythms to these simulated temperature cycles. Advancing the cycle by six hours, resulting in changes in the phase of activity in some foragers in the assay. The results are shown in this study highlight the importance of temperature as a potential Zeitgeber in the field. Future studies will examine the possible functional and evolutionary role of the observed phase differences of circadian rhythms.

Parallel mechanisms of visual memory formation across distinct regions of the honey bee brain.

Visual learning is vital to the behavioral ecology of the Western honey bee (Apis mellifera). Honey bee workers forage for floral resources, a behavior that requires the learning and long-term memory of visual landmarks, but how these memories are mapped to the brain remains poorly understood. To address this gap in our understanding, we collected bees that successfully learned visual associations in a conditioned aversion paradigm and compared gene expression correlates of memory formation in the mushroom bodies, a higher-order sensory integration center classically thought to contribute to learning, as well as the optic lobes, the primary visual neuropil responsible for sensory transduction of visual information. We quantified expression of CREB and CaMKII, two classical genetic markers of learning, and fen-1, a gene specifically associated with punishment learning in vertebrates. As expected, we found substantial involvement of the mushroom bodies for all three markers but additionally report the involvement of the optic lobes across a similar time course. Our findings imply the molecular involvement of a sensory neuropil during visual associative learning parallel to a higher-order brain region, furthering our understanding of how a tiny brain processes environmental signals.

Antibiotics in hives and their effects on honey bee physiology and behavioral development.

Recurrent honeybee losses make it critical to understand the impact of human interventions, such as antibiotic use in apiculture. Antibiotics are used to prevent or treat bacterial infections in colonies. However, little is known about their effects on honeybee development. We studied the effect of two commercial beekeeping antibiotics on the bee physiology and behavior throughout development. Our results show that antibiotic treatments have an effect on amount of lipids and rate of behavioral development. Lipid amount in treated bees was higher than those not treated. Also, the timing of antibiotic treatment had distinct effects for the age of onset of behaviors, starting with cleaning, then nursing and lastly foraging. Bees treated during larva-pupa stages demonstrated an accelerated behavioral development and loss of lipids, while bees treated from larva to adulthood had a delay in behavioral development and loss of lipids. The effects were shared across the two antibiotics tested, TerramycinR (oxytetracycline) and TylanR (tylosin tartrate). These effects of antibiotic treatments suggest a role of microbiota in the interaction between the fat body and brain that is important for honeybee behavioral development.This paper has an associated First Person interview with the first author of the article.

Genomic regions influencing aggressive behavior in honey bees are defined by colony allele frequencies.

For social animals, the genotypes of group members affect the social environment, and thus individual behavior, often indirectly. We used genome-wide association studies (GWAS) to determine the influence of individual vs. group genotypes on aggression in honey bees. Aggression in honey bees arises from the coordinated actions of colony members, primarily nonreproductive "soldier" bees, and thus, experiences evolutionary selection at the colony level. Here, we show that individual behavior is influenced by colony environment, which in turn, is shaped by allele frequency within colonies. Using a population with a range of aggression, we sequenced individual whole genomes and looked for genotype-behavior associations within colonies in a common environment. There were no significant correlations between individual aggression and specific alleles. By contrast, we found strong correlations between colony aggression and the frequencies of specific alleles within colonies, despite a small number of colonies. Associations at the colony level were highly significant and were very similar among both soldiers and foragers, but they covaried with one another. One strongly significant association peak, containing an ortholog of the Drosophila sensory gene dpr4 on linkage group (chromosome) 7, showed strong signals of both selection and admixture during the evolution of gentleness in a honey bee population. We thus found links between colony genetics and group behavior and also, molecular evidence for group-level selection, acting at the colony level. We conclude that group genetics dominates individual genetics in determining the fatal decision of honey bees to sting.

Soybean aphid biotype 1 genome: Insights into the invasive biology and adaptive evolution of a major agricultural pest.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae) is a serious pest of the soybean plant, Glycine max, a major world-wide agricultural crop. We assembled a de novo genome sequence of Ap. glycines Biotype 1, from a culture established shortly after this species invaded North America. 20.4% of the Ap. glycines proteome is duplicated. These in-paralogs are enriched with Gene Ontology (GO) categories mostly related to apoptosis, a possible adaptation to plant chemistry and other environmental stressors. Approximately one-third of these genes show parallel duplication in other aphids. But Ap. gossypii, its closest related species, has the lowest number of these duplicated genes. An Illumina GoldenGate assay of 2380 SNPs was used to determine the world-wide population structure of Ap. Glycines. China and South Korean aphids are the closest to those in North America. China is the likely origin of other Asian aphid populations. The most distantly related aphids to those in North America are from Australia. The diversity of Ap. glycines in North America has decreased over time since its arrival. The genetic diversity of Ap. glycines North American population sampled shortly after its first detection in 2001 up to 2012 does not appear to correlate with geography. However, aphids collected on soybean Rag experimental varieties in Minnesota (MN), Iowa (IA), and Wisconsin (WI), closer to high density Rhamnus cathartica stands, appear to have higher capacity to colonize resistant soybean plants than aphids sampled in Ohio (OH), North Dakota (ND), and South Dakota (SD). Samples from the former states have SNP alleles with high FST values and frequencies, that overlap with genes involved in iron metabolism, a crucial metabolic pathway that may be affected by the Rag-associated soybean plant response. The Ap. glycines Biotype 1 genome will provide needed information for future analyses of mechanisms of aphid virulence and pesticide resistance as well as facilitate comparative analyses between aphids with differing natural history and host plant range.

Colonization history and population differentiation of the Honey Bees (Apis mellifera L.) in Puerto Rico.

Honey bees (Apis mellifera L.) are the primary commercial pollinators across the world. The subspecies A. m. scutellata originated in Africa and was introduced to the Americas in 1956. For the last 60 years, it hybridized successfully with European subspecies, previous residents in the area. The result of this hybridization was called Africanized honey bee (AHB). AHB has spread since then, arriving to Puerto Rico (PR) in 1994. The honey bee population on the island acquired a mosaic of features from AHB or the European honey bee (EHB). AHB in Puerto Rico shows a major distinctive characteristic, docile behavior, and is called gentle Africanized honey bees (gAHB). We used 917 SNPs to examine the population structure, genetic differentiation, origin, and history of range expansion and colonization of gAHB in PR. We compared gAHB to populations that span the current distribution of A. mellifera worldwide. The gAHB population is shown to be a single population that differs genetically from the examined populations of AHB. Texas and PR groups are the closest genetically. Our results support the hypothesis that the Texas AHB population is the source of gAHB in Puerto Rico.

Honey bees (Apis mellifera spp.) respond to increased aluminum exposure in their foraging choice, motility, and circadian rhythmicity.

Aluminum is increasingly globally bioavailable with acidification from industrial emissions and poor mining practices. This bioavailability increases uptake by flora, contaminating products such as fruit, pollen, and nectar. Concentrations of aluminum in fruit and pollen have been reported between 0.05 and 670mg/L in North America. This is particularly concerning for pollinators that ingest pollen and nectar. Honey bees represent a globally present species experiencing decline in Europe and North America. Region specific decline may be a result of differential toxicity of exposure between subspecies. We find that European honey bees (Apis mellifera mellifera) may have differential toxicity as compared to two allopatric Mediterranean subspecies (Apis mellifera carnica and Apis mellifera caucasica) which showed no within subspecies exposure differences. European honey bees were then used in a laboratory experiment and exposed to aluminum in their daily water supply to mimic nectar contamination at several concentrations. After approximately 3 weeks of aluminum ingestion these bees showed significantly shorter captive longevity than controls at concentrations as low as 10.4mg/L and showed a possible hormetic response in motility. We also compared European honey bees to Africanized/European hybrid bees (Apis mellifera mellifera/scutellata hybrid) in short-term free-flight experiments. Neither the European honey bee nor the hybrid showed immediate foraging deficits in flight time, color choice, or floral manipulation after aluminum exposure. We conclude that European honey bees are at the greatest risk of aluminum related decline from chronic ingestion as compared to other subspecies and offer new methods for future use in honey bee toxicology.

Appetitive reversal learning differences of two honey bee subspecies with different foraging behaviors.

We aimed to examine mechanistically the observed foraging differences across two honey bee, Apis mellifera, subspecies using the proboscis extension response assay. Specifically, we compared differences in appetitive reversal learning ability between honey bee subspecies: Apis mellifera caucasica (Pollman), and Apis mellifera syriaca (Skorikov) in a "common garden" apiary. It was hypothesized that specific learning differences could explain previously observed foraging behavior differences of these subspecies: A.m. caucasica switches between different flower color morphs in response to reward variability, and A.m. syriaca does not switch. We suggest that flower constancy allows reduced exposure by minimizing search and handling time, whereas plasticity is important when maximizing harvest in preparation for long winter is at a premium. In the initial or Acquisition phase of the test we examined specifically discrimination learning, where bees were trained to respond to a paired conditioned stimulus with an unconditioned stimulus and not to respond to a second conditioned stimulus that is not followed by an unconditioned stimulus. We found no significant differences among the subspecies in the Acquisition phase in appetitive learning. During the second, Reversal phase of the experiment, where flexibility in association was tested, the paired and unpaired conditioned stimuli were reversed. During the Reversal phase A.m. syriaca showed a reduced ability to learn the reverse association in the appetitive learning task. This observation is consistent with the hypothesis that A.m. syriaca foragers cannot change the foraging choice because of lack of flexibility in appetitive associations under changing contingencies. Interestingly, both subspecies continued responding to the previously rewarded conditioned stimulus in the reversal phase. We discuss potential ecological correlates and molecular underpinnings of these differences in learning across the two subspecies. In addition, in a supplemental experiment we demonstrated that these differences in appetitive reversal learning do not occur in other learning contexts.

A soft selective sweep during rapid evolution of gentle behaviour in an Africanized honeybee.

Highly aggressive Africanized honeybees (AHB) invaded Puerto Rico (PR) in 1994, displacing gentle European honeybees (EHB) in many locations. Gentle AHB (gAHB), unknown anywhere else in the world, subsequently evolved on the island within a few generations. Here we sequence whole genomes from gAHB and EHB populations, as well as a North American AHB population, a likely source of the founder AHB on PR. We show that gAHB retains high levels of genetic diversity after evolution of gentle behaviour, despite selection on standing variation. We observe multiple genomic loci with significant signatures of selection. Rapid evolution during colonization of novel habitats can generate major changes to characteristics such as morphological or colouration traits, usually controlled by one or more major genetic loci. Here we describe a soft selective sweep, acting at multiple loci across the genome, that occurred during, and may have mediated, the rapid evolution of a behavioural trait.

Social signals and aversive learning in honey bee drones and workers.

The dissemination of information is a basic element of group cohesion. In honey bees (Apis mellifera Linnaeus 1758), like in other social insects, the principal method for colony-wide information exchange is communication via pheromones. This medium of communication allows multiple individuals to conduct tasks critical to colony survival. Social signaling also establishes conflict at the level of the individual who must trade-off between attending to the immediate environment or the social demand. In this study we examined this conflict by challenging highly social worker honey bees, and less social male drone honey bees undergoing aversive training by presenting them with a social stress signal (isopentyl acetate, IPA). We utilized IPA exposure methods that caused lower learning performance in appetitive learning in workers. Exposure to isopentyl acetate (IPA) did not affect performance of drones and had a dose-specific effect on worker response, with positive effects diminishing at higher IPA doses. The IPA effects are specific because non-social cues, such as the odor cineole, improve learning performance in drones, and social homing signals (geraniol) did not have a discernible effect on drone or worker performance. We conclude that social signals do generate conflict and that response to them is dependent on signal relevance to the individual as well as the context. We discuss the effect of social signal on learning both related to its social role and potential evolutionary history.

Ethanol-induced effects on sting extension response and punishment learning in the western honey bee (Apis mellifera).

Acute ethanol administration is associated with sedation and analgesia as well as behavioral disinhibition and memory loss but the mechanisms underlying these effects remain to be elucidated. During the past decade, insects have emerged as important model systems to understand the neural and genetic bases of alcohol effects. However, novel assays to assess ethanol's effects on complex behaviors in social or isolated contexts are necessary. Here we used the honey bee as an especially relevant model system since bees are typically exposed to ethanol in nature when collecting standing nectar crop of flowers, and there is recent evidence for independent biological significance of this exposure for social behavior. Bee's inhibitory control of the sting extension response (SER) and a conditioned-place aversion assay were used to study ethanol effects on analgesia, behavioral disinhibition, and associative learning. Our findings indicate that although ethanol, in a dose-dependent manner, increases SER thresholds (analgesic effects), it disrupts the ability of honey bees to inhibit SER and to associate aversive stimuli with their environment. These results suggest that ethanol's effects on analgesia, behavioral disinhibition and associative learning are common across vertebrates and invertebrates. These results add to the use of honey bees as an ethanol model to understand ethanol's effects on complex, socially relevant behaviors.

Measuring individual locomotor rhythms in honey bees, paper wasps and other similar-sized insects.

Circadian rhythms in social insects are highly plastic and are modulated by multiple factors. In addition, complex behaviors such as sun-compass orientation and time learning are clearly regulated by the circadian system in these organisms. Despite these unique features of social insect clocks, the mechanisms as well as the functional and evolutionary relevance of these traits remain largely unknown. Here we show a modification of the Drosophila activity monitoring (DAM) system that allowed us to measure locomotor rhythms of the honey bee, Apis mellifera (three variants; gAHB, carnica and caucasica), and two paper wasps (Polistes crinitus and Mischocyttarus phthisicus). A side-by-side comparison of the endogenous period under constant darkness (free-running period) led us to the realization that these social insects exhibit significant deviations from the Earth's 24 h rotational period as well as a large degree of inter-individual variation compared with Drosophila. Experiments at different temperatures, using honey bees as a model, revealed that testing the endogenous rhythm at 35°C, which is the hive's core temperature, results in average periods closer to 24 h compared with 25°C (23.8 h at 35°C versus 22.7 h at 25°C). This finding suggests that the degree of tuning of circadian temperature compensation varies among different organisms. We expect that the commercial availability, cost-effectiveness and integrated nature of this monitoring system will facilitate the growth of the circadian field in these social insects and catalyze our understanding of the mechanisms as well as the functional and evolutionary relevance of circadian rhythms.

Individual responsiveness to shock and colony-level aggression in honey bees: evidence for a genetic component.

The phenotype of the social group is related to phenotypes of individuals that form that society. We examined how honey bee colony aggressiveness relates to individual response of male drones and foraging workers. Although the natural focus in colony aggression has been on the worker caste, the sterile females engaged in colony maintenance and defense, males carry the same genes. We measured aggressiveness scores of colonies and examined components of individual aggressive behavior in workers and haploid sons of workers from the same colony. We describe for the first time, that males, although they have no stinger, do bend their abdomen (abdominal flexion) in a posture similar to stinging behavior of workers in response to electric shock. Individual worker sting response and movement rates in response to shock were significantly correlated with colony scores. In the case of drones, sons of workers from the same colonies, abdominal flexion significantly correlated but their movement rates did not correlate with colony aggressiveness. Furthermore, the number of workers responding at increasing levels of voltage exhibits a threshold-like response, whereas the drones respond in increasing proportion to shock. We conclude that there are common and caste-specific components to aggressive behavior in honey bees. We discuss implications of these results on social and behavioral regulation and genetics of aggressive response.

Aversive conditioning in honey bees (Apis mellifera anatolica): a comparison of drones and workers.

Honey bees provide a model system to elucidate the relationship between sociality and complex behaviors within the same species, as females (workers) are highly social and males (drones) are more solitary. We report on aversive learning studies in drone and worker honey bees (Apis mellifera anatolica) in escape, punishment and discriminative punishment situations. In all three experiments, a newly developed electric shock avoidance assay was used. The comparisons of expected and observed responses were performed with conventional statistical methods and a systematic randomization modeling approach called object oriented modeling. The escape experiment consisted of two measurements recorded in a master-yoked paradigm: frequency of response and latency to respond following administration of shock. Master individuals could terminate an unavoidable shock triggered by a decrementing 30 s timer by crossing the shuttlebox centerline following shock activation. Across all groups, there was large individual response variation. When assessing group response frequency and latency, master subjects performed better than yoked subjects for both workers and drones. In the punishment experiment, individuals were shocked upon entering the shock portion of a bilaterally wired shuttlebox. The shock portion was spatially static and unsignalled. Only workers effectively avoided the shock. The discriminative punishment experiment repeated the punishment experiment but included a counterbalanced blue and yellow background signal and the side of shock was manipulated. Drones correctly responded less than workers when shock was paired with blue. However, when shock was paired with yellow there was no observable difference between drones and workers.