Colony Defense Research Articles

Evolution of obligate asexuality in termites with mixed‐sex societies

AbstractMales are a ubiquitous feature of animals and play crucial roles beyond the contribution of gametes in some species (e.g., paternal care for offspring and nuptial gifts for females). In termites, colonies commonly consist of both male and female reproductives, workers, and soldiers (i.e., mixed‐sex societies), where males and females both play critical roles beyond reproduction (e.g., brood care, foraging, nest construction and maintenance, and colony defense). Male‐specific roles may also exist in termite colonies. While obligate asexuality had not been observed in any termite population until recently, all‐female asexual populations of the termite Glyptotermes nakajimai were reported in 2018. This was the first and only known case of evolutionary transition from mixed‐sex to all‐female asexual societies in animals. Here, asexual reproduction in termites is first reviewed, followed by a discussion of how and why G. nakajimai evolved obligate asexuality. The study of the evolution of obligate asexuality in termites with mixed‐sex societies is important because it may enhance our understanding of the significance of males in animal societies and populations.

Population profile of haemocytes in the subcastes of the leaf‐cutter ant Atta sexdens (Hymenoptera: Formicidae) unveiled by flow cytometry

AbstractThe leaf‐cutter ant Atta sexdens is crucial in ecological balance and economic activities, due to its contribution to the aeration and accumulation of organic matter in the soil, but it may also be an agricultural pest when it defoliates plants for the fungus growth used as a food source. This leaf‐cutter ant exhibits a polymorphic worker caste system with distinct activities in the colony. These functions are performed by workers classified into subcastes, including gardeners, which are responsible for the fungus and brood care; cleaners (waste removers), which remove wastes; foragers, which collect and transport plant parts to the nest; and soldiers, which are responsible for colony defence and the support of foraging activities. In our previous work, we showed that A. sexdens has five types of circulating haemocytes in the haemolymph, which play a vital role in immune defence. Herein, we further investigated the population profile of circulating haemocytes in the haemolymph of different worker subcastes of A. sexdens using flow cytometry. Each subcaste exhibited a single population of haemocytes in the haemolymph. However, a detailed 3D dispersion analysis uncovered five distinct haemocyte subpopulations, two of which presented higher quantities. The soldier displayed haemocytes with high fluorescence intensity, compared with the gardener, waste remover, and forager, along with increased complexity, compared with the gardener subcaste. These results suggest that these cells may be granulocytes and plasmatocytes, which are known for their role in insect immune defence.

Compatibilities of Cyprinus carpio with Varied Colors of Robotic Fish

Visual selection plays a fundamental role in various aspects of animal behavior, such as colony formation, maintenance, defense, and courtship. This study investigated the effect of bionic robot fish color on carp behavior based on physiological characteristics that were observed during behavioral experiments. Through computer image processing and analysis of light attenuation, we observed changes in the number and positioning of carp with bionic robotic fish of different colors (white, red, blue, green, and yellow). The results indicated that (1) the attenuation coefficient of visible light in freshwater was red > yellow > green > blue; (2) the order of the average change in the number of carp responding to different colors of robotic fish was white > red > green > yellow > blue, and carp were more sensitive and responsive to white and red robotic fish; and (3) the order of the distances between different colors of robotic fish and carp was white < yellow < blue < green < red, and white and yellow robotic fish were more attractive to carp. Therefore, the use of white or yellow robotic fish for relevant operations can reduce disturbance to fish schools.

Giant honeybees (Apis dorsata) trade off defensiveness against periodic mass flight activity.

The giant honeybee Apis dorsata (Fabricius, 1793) is an evolutionarily ancient species that builds its nests in the open. The nest consists of a single honeycomb covered with the bee curtain which are several layers of worker bees that remain almost motionless with their heads up and abdomens down on the nest surface, except for the mouth area, the hub between inner- and outer-nest activities. A colony may change this semi-quiescence several times a day, depending on its reproductive state and ambient temperature, to enter the state of mass flight activity (MFA), in which nest organisation is restructured and defense ability is likely to be suppressed (predicted by the mass-flight-suspend-defensiveness hypothesis). For this study, three episode of MFA (mfa1-3) of a selected experimental nest were analysed in a case study with sequences of >60 000 images at 50 Hz, each comprise a short pre-MFA session, the MFA and the post-MFA phase of further 10 min. To test colony defensiveness under normative conditions, a dummy wasp was cyclically presented with a standardised motion programme (Pd) with intervening sessions without such a presentation (nPd). Motion activity at five selected surveillance zones (sz1-5) on the nest were analysed. In contrast to mfa1,2, in mfa3 the experimental regime started with the cyclic presentation of the dummy wasp only after the MFA had subsided. As a result, the MFA intensity in mfa3 was significantly lower than in mfa1-2, suggesting that a colony is able to perceive external threats during the MFA. Characteristic ripples appear in the motion profiles, which can be interpreted as a start signal for the transition to MFA. Because they are strongest in the mouth zone and shift to higher frequencies on their way to the nest periphery, it can be concluded that MFA starts earlier in the mouth zone than in the peripheral zones, also suggesting that the mouth zone is a control centre for the scheduling of MFA. In Pd phases of pre- and postMFA, the histogram-based motion spectra are biphasic, suggesting two cohorts in the process, one remaining at quiescence and the other involved in shimmering. Under MFA, nPd and Pd spectra were typically Gaussian, suggesting that the nest mates with a uniform workload shifted to higher motion activity. At the end of the MFA, the spectra shift back to the lower motion activities and the Pd spectra form a biphasic again. This happens a few minutes earlier in the peripheral zones than in the mouth zone. Using time profiles of the skewness of the Pd motion spectra, the mass-flight-suspend-defensiveness hypothesis is confirmed, whereby the inhibition of defense ability was found to increase progressively during the MFA. These sawtooth-like time profiles of skewness during MFA show that defense capability is recovered again quite quickly at the end of MFA. Finally, with the help of the Pd motion spectra, clear indications can be obtained that the giant honeybees engage in a decision in the sense of a tradeoff between MFA and collective defensiveness, especially in the regions in the periphery to the mouth zone.

Vairimorpha apis Versus Vairimorpha ceranae, Replacement or Dynamic Prevalence?

Abstract Nosematosis is currently a frequently discussed disease of bees caused by two species of microsporidia: Vairimorpha apis, and Vairimorpha ceranae. Vairimorpha apis represents the original species of Apis mellifera parasite, and Vairimorpha ceranae, is a species introduced from Asia. In the last two decades, epidemiological data on the growth of the prevalence of V. ceranae infection have increased, which has led to its dominant position at the expense of its congener V. apis, practically all over the world. This process is assumed to be the result of asymmetric competition within the host, where V. ceranae, showed a better ability to adapt to higher temperatures, which was first manifested by its spread in warmer climate zones. However, several results of studies from recent years somewhat unexpectedly showed that it is premature to talk about the complete replacement of V. apis by V. ceranae. They pointed to a greater influence of seasonality and average temperatures in winter and summer in a given year on the result of the current prevalence of infection and co-infection of Vairimorpha spp., regardless of the climatic zone in which the colonies were located. Considering the different clinical and subclinical manifestations of infection caused by V. apis, and V. ceranae, and its impact on the defense, survival and productivity of bee colonies, the aim of our work was to analyze the factors affecting the distribution and prevalence of Vairimorpha spp.

Hey, hold on, we are good guys! Colony defense behavior and notes on nesting of Parachartergus fulgidipennis (de Saussure, 1854) (Vespidae: Polistinae: Epiponini)

Parachartergus wasps are known by having a very aggressive behavior, especially the large black species with white wings tip, which have painful stings. They exhibit a unique and interesting characteristic, the capacity to venom spraying during the colony defense or self-defense. However, for the Amazonian species Parachartergus fulgidipennis (de Saussure, 1854), little is known about its behavior and nesting. In this way, we tracked and described the nesting site, the numbers of combs, and performed an ad libitum method to record behavioral activities. The nest was fixed vertically on a man-made concrete column, 1.2 m above the ground, measuring 25.0 x 6.0 cm and contained four combs with approximately 74 cells, with 31 eggs, seven larvae, 30 pupae, and almost 42 adults. With a flat thin grayish envelope with weak corrugations, the nest is very similar to the color of the substrate which turn in to really cryptic. The colony defense behavior presented by this species is unaggressive and very docile, suggesting that is not similar as observed for other Parachartergus species.

An Updated Review Summarizing the Anticancer Efficacy of Melittin from Bee Venom in Several Models of Human Cancers.

Apitherapy (using bee products) has gained broad recognition in cancer therapeutics globally. Honeybee venom has a broad range of biological potential, and its utilization is rapidly emerging in apitherapy. Bee products have significant potential to strengthen the immune system and improve human health. Thus, this review is targeted toward recapitulating the chemo-preventive potential of melittin (MEL), which constitutes a substantial portion of honeybee venom. Honeybee venom (apitoxin) is produced in the venom gland of the honeybee abdomen, and adult bees utilize it as a primary colony defense mechanism. Apitoxin comprises numerous biologically active compounds, including peptides, enzymes, amines, amino acids, phospholipids, minerals, carbohydrates, and volatile components. We are mainly focused on exploring the potential of melittin (a peptide component) of bee venom that has shown promising potential in the treatment of several human cancers, including breast, stomach, lung, prostate, ovary, kidney, colon, gastric, esophageal, cervical cancers, melanoma, osteosarcoma, and hepatocellular carcinoma. This review has summarized all potential studies related to the anticancerous efficacy of melittin (apitoxin), its formulations, conjugates, and nano-formulations against several human carcinomas, which would further pave the way for future researchers in developing potent drugs for cancer management.

Mating and post-hibernation ovarian development in stylopized and non-stylopized queens of the hornet Vespa analis (Hymenoptera Vespidae)

In social insects, post-hibernation queens of independent-founding species (e.g. bumble bees, some ants and wasps) need to overcome various difficulties for survival and reproduction during the initial period of the colony. Specifically, a single queen must perform all tasks related to colony defense and nursing juveniles alone. In this study, we examined whether the reproductive conditions of post-hibernation solitary queens of the vespine wasp Vespa analis are affected by the following four factors: (1) collection date, (2) mating status, (3) body size and (4) the presence of Xenos parasites. The results showed that ovary development of the queens was associated with collection date and mating status but not with body size or parasitism. The positive effects of the first two factors are reasonable, because they are closely related to life history, but the absence of a link between ovary development of the queens and parasitism was surprising. No association of ovary development of the queens with body size prompts the need for more comparative studies, which might reveal differences in the fecundity-body size relationship among temperate Vespa species.

Olfactory sensitivity differentiates morphologically distinct worker castes in Camponotus floridanus

BackgroundCamponotus floridanus ant colonies are comprised of a single reproductive queen and thousands of sterile female offspring that consist of two morphologically distinct castes: smaller minors and larger majors. Minors perform most of the tasks within the colony, including brood care and food collection, whereas majors have fewer clear roles and have been hypothesized to act as a specialized solider caste associated with colony defense. The allocation of workers to these different tasks depends, in part, on the detection and processing of local information including pheromones and other chemical blends such as cuticular hydrocarbons. However, the role peripheral olfactory sensitivity plays in establishing and maintaining morphologically distinct worker castes and their associated behaviors remains largely unexplored.ResultsWe examined the electrophysiological responses to general odorants, cuticular extracts, and a trail pheromone in adult minor and major C. floridanus workers, revealing that the repertoire of social behaviors is positively correlated with olfactory sensitivity. Minors in particular display primarily excitatory responses to olfactory stimuli, whereas major workers primarily manifest suppressed, sub-solvent responses. The notable exception to this paradigm is that both minors and majors display robust, dose-dependent excitatory responses to conspecific, non-nestmate cuticular extracts. Moreover, while both minors and majors actively aggress non-nestmate foes, the larger and physiologically distinct majors display significantly enhanced capabilities to rapidly subdue and kill their adversaries.ConclusionsOur studies reveal the behavioral repertoire of minors and majors aligns with profound shifts in peripheral olfactory sensitivity and odor coding. The data reported here support the hypothesis that minors are multipotential workers with broad excitatory sensitivity, and majors are dedicated soldiers with a highly specialized olfactory system for distinguishing non-nestmate foes. Overall, we conclude that C. floridanus majors do indeed represent a physiologically and behaviorally specialized soldier caste in which caste-specific olfactory sensitivity plays an important role in task allocation and the regulation of social behavior in ant colonies.

Nest camouflage in Metapolybia cingulata and nesting and colony defensive behavior in Metapolybia docilis (Vespidae: Polistinae) in the Brazilian Atlantic Forest

Colony defense by social wasps includes aggressive behavior and camouflage with a high diversity of adaptation of their nests to the environment, including substrate choice. The objective of this study was to present new data on nesting and colony defense by the Neotropical social wasps Metapolybia cingulata and Metapolybia docilis (Hymenoptera: Vespidae). Field observations were made on January 4th and 5th, 2022 at the State Park of Iguaçu, state of Paraná, Brazil, on M. cingulata and M. docilis nests and information on nesting of these wasps obtained in 2014 at the Pandeiros River Wildlife Refuge (REVIS), and in 2012 at the Rio Doce State Park, state of Minas Gerais, Brazil. The types of substrates used by M. cingulata and M. docilis reinforce the importance of nest camouflage as a defense strategy for their colonies. The selection of nesting sites by these species was very variable.

Grenville's Postponement of the Stamp Act Reconsidered*

AbstractIn March 1764 George Grenville announced plans to raise revenues in America for colonial defence with stamp duties. Opinions differ about why Grenville then postponed the Stamp Act until a year hence. Writing in 1950, Edmund S. Morgan found the decision puzzling. Peter D.G. Thomas and John L. Bullion subsequently offered procedural explanations. Grenville envisaged the Stamp Act as a firm precedent for parliament's authority to impose direct taxation (‘inland duties’) in the colonies – a prospect that terrified Americans schooled in the common law, alert to dangerous precedents, and already prone to slippery‐slope reasoning. With the mundane details of the Stamp Act unavailable until after its passage, however, the postponement also raised imperial debate to a level of abstraction from which it never retreated. Grenville's postponement of the Stamp Act foreclosed prospects for compromise and set British imperial policy on a course that ultimately fomented the American Revolution.

Body Size and Symmetry Properties of Termite Soldiers Under Two Intraspecific Competition Scenarios

Single-piece nesting termites live and forage in the same piece of wood throughout their life, which limit their colony size. In certain species, more than one colony thrive in a given piece of wood (multicolonial substrate) and intraspecific competition become important in this limited resource, as has been reported in Zootermopsis nevadensis (Hagen, 1858) and Neotermes chilensis (Blattodea: Kalotermitidae) (Blanchard, 1851). The effects of such competition have been described mainly at population and colony levels rather than at the individual level. In eusocial insects such as termites, intraspecific competition constitutes a stress factor imposed to a colony as a whole and should also cause developmental instability in soldiers produced under such conditions. Investment in the production of soldiers involves a trade-off between colony maintenance costs and defense benefits. Hence, we hypothesize that body size and fluctuating asymmetry, two indicators of developmental instability, will increase when two or more colonies of N. chilensis share a piece of wood (high intraspecific competition scenario). Our results showed that soldiers developing in multicolonial substrates were indeed larger and more asymmetric than soldiers developing in unicolonial substrates. The large body size in a soldier could improve its chance to win a physical contest with a non-nestmate opponent; thus, despite the high cost to produce large soldiers in small colonies, larger soldier production could be an adaptative strategy to avoid being outcompeted. However, the effects of deviations from perfect symmetry on soldier performance are not clear.

Termite Valkyries: Soldier-Like Alate Termites From the Cretaceous and Task Specialization in the Early Evolution of Isoptera

In several insect eusocial lineages, e.g., some aphids, thrips, ants, some stingless bees, and termites, task specialization is brought to its climax with a sterile soldier caste solely devoted to colony defense. In Isoptera, while the reproductives are defenseless, the soldiers have unique morpho-physiological specializations whose origin and evolution remain unresolved. Here we report on two instances of Cretaceous fossil termite reproductives belonging to different families († Valkyritermes inopinatus gen. et sp. nov. and an unpublished specimen from the Crato Formation), with intriguing phragmotic soldier-like heads and functional wings. These individuals, herein called Valkyries, are the first termite reproductives known with defensive features and suggest that phragmosis arose at least in the Early Cretaceous. Valkyries resemble modern neotenic soldiers except for their complete wings. Their discovery supports the hypothesis that the division between reproductive (indicated by the winged condition of Valkyries) and defensive tasks (indicated by the phragmotic head) has not always been complete in termite history. We explore two alternative scenarios regarding the origin of Valkyries (i.e., relatively recent and convergent origins vs. plesiomorphic condition) and discuss how they might relate to the development of soldiers. We argue that, in both cases, Valkyries likely evolved to face external threats, a selective pressure that could also have favored the origin of soldiers from helpers. Valkyries highlight the developmental flexibility of termites and illustrate the tortuous paths that evolution may follow.

What has been done in the fight against Varroa destructor: from the past to the present

Bees are the major pollinators in natural ecosystems and in the agricultural production of several crops used for human consumption. However, they are exposed to multiple stressors that are causing a serious decline in their population. We highlight a major one among them, the Varroa destructor mite (Varroa) that causes severe impacts on the health of honey bee colonies, transmitting a variety of viruses that can affect the survival ability of individual bees and entire colonies. Diagnosis and mite control methods have been intensively studied in recent decades, with many studies in different areas of knowledge having been conducted. This overview summarizes these studies with a focus on colony defense systems, biological characteristics of the parasite Varroa, diagnostic methods used to establish the infestation level of colonies, and currently used control methods.

Disease defences across levels of biological organization: individual and social immunity in acorn ants

Eusocial insect societies possess complex multilevel disease defences, including individual level protection conferred by physical (e.g. cuticle) and immunological obstacles and colony level protection mediated by collective behaviours (social immunity). It remains unclear whether and how these two levels of disease protection are related to one another in jointly driving colonies’ susceptibility to disease. Here, we examine whether a relationship exists between individual worker survival after exposure to a fungal pathogen (a proxy for immunity) and corpse removal (a colony level social immunity metric) in the acorn ant Temnothorax curvispinosus . Since behavioural avoidance is the first line of defence against infection, we also tested whether individual ants exhibited parasite avoidance behaviour during exploration and whether colonies exhibit avoidance behaviour during foraging. We found that individual level and colony level immunity were negatively correlated: colonies that removed corpses more rapidly contained workers with weaker individual defences. We did not detect parasite avoidance behaviour by individual workers or whole colonies, nor were these two factors related. These data suggest that individual immunity and social immunity may trade off, regulating overall parasite protection. Alternatively, optimized social immunity at the colony level may compensate for disease vulnerability to infection at the individual level, and thus provide a protective benefit in overall colony defence in the absence of pathogen avoidance. • We measured individual and collective disease protection in acorn ants. • Colonies with stronger individual protection had weaker collective protection. • Avoidance was not detected at the individual or collective level. • Multilevel disease protection trade-offs may vary across colonies and environments.

Behavioral flexibility in Wasmannia auropunctata (Hymenoptera: Formicidae).

Worker division of labor is a defining trait in social insects. Many species are characterized by having behavioral flexibility where workers perform non-typical tasks for their age depending on the colony’s needs. Worker division of labor and behavioral flexibility were examined in the little fire ant Wasmannia auropunctata (Roger, 1863), for which age-related division of labor has been found. Young workers perform nursing duties which include tending of brood and queens, and colony defense, while older workers forage. When nurses were experimentally removed from the colony, foragers were observed carrying out nursing and colony defense duties, yet when foragers were removed nurses did not forage precociously. We also administered juvenile hormone analog, methoprene, to workers. When methoprene was applied, foragers increased their nursing and defense activities while nurses became mainly idle. The behavioral flexibility of foragers of the little fire ant may be evidence of an expansion of worker’s repertoires as they age; older workers can perform tasks they have already done in their life while young individuals are not capable of performing tasks ahead of time. This may be an important adaptation associated with the success of this ant as an invasive species.

Queen Pregnancy Increases Group Estradiol Levels in Cooperatively Breeding Naked Mole-Rats.

For cooperative species, there can be great value in the synchronization of physiological states to coordinate group behavioral states. This is evident in naked mole-rats (Heterocephalus glaber), which have the most extreme form of cooperative breeding in mammals. Colonies have a single reproductive female, "the queen," and 1-3 breeding males. These breeders are supported by adult "subordinates," which are all socially suppressed into a pre-pubertal state. Subordinates cooperate in colony maintenance, defense, and alloparental care. Prior work has reported that there may be social sharing of hormones among individuals in the colony because when the queen is pregnant, subordinates of both sexes develop enlarged nipples and female subordinates can develop vaginal perforation. We sought to document the hormonal changes and mechanisms behind these observations. We found that subordinate estradiol levels were elevated during the queen's pregnancy and were correlated with queen levels. To determine if this occurs by direct hormone-sharing, where group members uptake the hormones of conspecifics through excreta or the skin, we then tested whether treating a single subordinate in the colony with estradiol would induce the same effect in other colony members. It did not, which indicates that the influence on group estradiol levels may be specific to cues from the queen. These queen cues may be behavioral in nature, as we found that queens were less aggressive during pregnancy, which prior work has suggested may relax reproductive suppression of subordinates. Yet, levels of queen aggression alone were not associated, or were weakly associated, with their colony's estradiol levels, though our sample size examining this particular relationship was low. This is suggestive that additional queen cues of reproductive status, beyond just aggression, may be relevant in influencing the subordinate hormonal change, or that the relationship between aggression and colony estradiol levels is more subtle and would need to be elucidated with a larger sample size. These results have implications for how cooperative breeders coordinate reproduction and alloparental care, and how social cues can influence individual and group physiology.

Biological and behavioral responses of European honey bee (Apis mellifera) colonies to perfluorooctane sulfonate exposure.

Bees provide pollination services to managed and wild ecosystems but are threatened globally due to multiple stressors, including exposure to contaminants. Perfluorooctane sulfonate (PFOS) is a widely detected and persistent contaminant that accumulates and biomagnifies in food chains. In this exposure effect study, small whole colonies of Apis mellifera (1000 bees) were exposed to PFOS using a purpose-built cage system over a 4-week period. The PFOS exposure concentrations were provided to bees in sugar syrup at concentrations detected in the environment, ranging from 0 to 1.6 mg L-1 . A range of biological and behavioral responses were monitored. Bee tissue, honey, and fecal matter were analyzed using isotope dilution combined with liquid chromatography-tandem mass spectrometry adapted for bee and honey matrix analysis. Bee mortality increased significantly with PFOS exposure at 0.8 mg L-1 or greater, and brood development ceased entirely at 0.02 mg L-1 or greater. Colony activity, temperament, hive maintenance, and defense were adversely affected in all PFOS exposure treatments compared with the control, even at the lowest PFOS exposure of 0.02mg L-1 . Perfluorooctane sulfonate was detected in bee tissue with a mean bioaccumulation factorof 0.3, and it was also identified in honey and in feces collected from the hive cages. These findings provide the first evidence that PFOS exposure adversely affects honey bee colonies and may transfer to honey. With PFOS contaminating thousands of sites worldwide, our study has implications for exposed bee populations under natural conditions, pollination services, the honey industry, and human health. Integr Environ Assess Manag 2021;17:673-683. © 2021 SETAC.

Head and mandible shapes are highly integrated yet represent two distinct modules within and among worker subcastes of the ant genus Pheidole.

Ants use their mandibles for a wide variety of tasks related to substrate manipulation, brood transport, food processing, and colony defense. Due to constraints involved in colony upkeep, ants evolved a remarkable diversity of mandibular forms, often related to specific roles such as specialized hunting and seed milling. Considering these varied functional demands, we focused on understanding how the mandible and head shape vary within and between Pheidole subcastes. Using x‐ray microtomography and 3D geometric morphometrics, we tested whether these structures are integrated and modular, and how ecological predictors influenced these features. Our results showed that mandible and head shape of majors and minor workers tend to vary from robust to slender, with some more complex changes related to the mandibular base. Additionally, we found that head and mandible shapes are characterized by a high degree of integration, but with little correlation with feeding and nesting habits. Our results suggest that a combination of structural (allometric) constraints and the behavioral flexibility conferred by subcaste dimorphism might largely buffer selective pressures that would otherwise lead to a fine‐tuning between ecological conditions and morphological adaptation.

Origin, behaviour, and genetics of reproductive workers in an invasive ant

BackgroundWorker reproduction has an important influence on the social cohesion and efficiency of social insect colonies, but its role in the success of invasive ants has been neglected. We used observations of 233 captive colonies, laboratory experiments, and genetic analyses to investigate the conditions for worker reproduction in the invasive Anoplolepis gracilipes (yellow crazy ant) and its potential cost on interspecific defence. We determined the prevalence of worker production of males and whether it is triggered by queen absence; whether physogastric workers with enlarged abdomens are more likely to be reproductive, how normal workers and physogastric workers compare in their contributions to foraging and defence; and whether worker-produced males and males that could have been queen- or worker-produced differ in their size and heterozygosity.ResultsSixty-six of our 233 captive colonies produced males, and in 25 of these, some males could only have been produced by workers. Colonies with more workers were more likely to produce males, especially for queenless colonies. The average number of days between the first appearance of eggs and adult males in our colonies was 54.1 ± 10.2 (mean ± SD, n = 20). In our laboratory experiment, queen removal triggered an increase in the proportion of physogastric workers. Physogastric workers were more likely to have yolky oocytes (37–54.9%) than normal workers (2–25.6%), which is an indicator of fertile or trophic egg production. Physogastric workers were less aggressive during interspecific aggression tests and foraged less than normal workers. The head width and wing length of worker-produced males were on average 4.0 and 4.3% greater respectively than those of males of undetermined source. Our microsatellite DNA analyses indicate that 5.5% of worker-produced males and 14.3% of males of undetermined source were heterozygous, which suggests the presence of diploid males and/or genetic mosaics in A. gracilipes.ConclusionsOur experimental work provides crucial information on worker reproduction in A. gracilipes and its potential cost to colony defence. The ability of A. gracilipes workers to produce males in the absence of queens may also contribute to its success as an invasive species if intranidal mating can take place between virgin queens and worker-produced males.