The evolution of caste-biasing symbionts in the social hymenoptera.

The separation of individuals into reproductive and worker castes is the defining feature of insect societies. However, caste determination is itself a complex phenomenon, dependent on interacting genetic and environmental factors. It has been suggested by some authors that widespread maternally transmitted symbionts such as Wolbachia may be selected to interfere with caste determination, whilst others have discounted this possibility on theoretical grounds. We argue that there are in fact three distinct evolutionary scenarios in which maternally transmitted symbionts might be selected to influence the process of caste determination in a social hymenopteran host. Each of these scenarios generate testable predictions which we outline here. Given the increasing recognition of the complexity and multi-faceted nature of caste determination in social insects, we argue that maternally transmitted symbionts should also be considered as possible factors influencing the development of social hymenopterans.

The gut microbiome is associated with behavioural task in honey bees.

The gut microbiome is recognised as playing an integral role in the health and ecology of a wide variety of animal taxa. However, the relationship between social behavioural traits and the microbial community has received little attention. Honey bees are highly social and the workers perform different behavioural tasks in the colony that cause them to be exposed to different local environments. Here we examined whether the gut microbial community composition of worker honey bees is associated with the behavioural tasks they perform, and therefore also the local environment they are exposed to. We set up five observation hives, in which all workers were matched in age and observed the behaviour of marked bees in each colony over 4 days. The gut bacterial communities of bees seen performing predominantly foraging or predominantly in nest tasks were then characterised and compared based on amplicon sequencing of the 16S rRNA gene. Our results show that some core members of the unique honey bee gut bacterial community are represented in different relative abundances in bees performing different behavioural tasks. The differentially represented bacterial taxa include some thought to be important in carbohydrate metabolism and transport, and also linked to bee health. The results suggest an influence of task-related local environment exposure and diet on the honey bee gut microbial community and identify focal core taxa for further functional analyses.

The effects of disturbance threat on leaf-cutting ant colonies: a laboratory study.

The flexibility of organisms to respond plastically to their environment is fundamental to their fitness and evolutionary success. Social insects provide some of the most impressive examples of plasticity, with individuals exhibiting behavioral and sometimes morphological adaptations for their specific roles in the colony, such as large soldiers for nest defense. However, with the exception of the honey bee model organism, there has been little investigation of the nature and effects of environmental stimuli thought to instigate alternative phenotypes in social insects. Here, we investigate the effect of repeated threat disturbance over a prolonged (17 month) period on both behavioral and morphological phenotypes, using phenotypically plastic leaf-cutting ants (Atta colombica) as a model system. We found a rapid impact of threat disturbance on the behavioral phenotype of individuals within threat-disturbed colonies becoming more aggressive, threat responsive, and phototactic within as little as 2 weeks. We found no effect of threat disturbance on morphological phenotypes, potentially, because constraints such as resource limitation outweighed the benefit for colonies of producing larger individuals. The results suggest that plasticity in behavioral phenotypes can enable insect societies to respond to threats even when constraints prevent alteration of morphological phenotypes.

The tooth, the whole tooth and nothing but the tooth: tooth shape and ontogenetic shift dynamics in the white shark Carcharodon carcharias.

Results from this study of the white shark Carcharodon carcharias include measurements obtained using a novel photographic method that reveal significant differences between the sexes in the relationship between tooth cuspidity and shark total length, and a novel ontogenetic change in male tooth shape. Males exhibit broader upper first teeth and increased distal inclination of upper third teeth with increasing length, while females do not present a consistent morphological change. Substantial individual variation, with implications for pace of life syndrome, was present in males and tooth polymorphism was suggested in females. Sexual differences and individual variation may play major roles in ontogenetic changes in tooth morphology in C. carcharias, with potential implications for their foraging biology. Such individual and sexual differences should be included in studies of ontogenetic shift dynamics in other species and systems.

Behavioural development, fat reserves and their association with productivity in Lasius flavus founding queens.

Reproduction-related behaviours are key components determining individual fitness. Many behavioural traits are linked, and such trait associations often affect fitness. Here, we combine behavioural and physiological data during two critical time points of founding queens (early and late nest-founding stage) in the claustral ant Lasius flavus to assess how these factors affect their initial productivity. We show that most behavioural traits, except brood care behaviour, are plastic during queen development and demonstrate that there are alternative behavioural pathways to achieve high productivity under standardised conditions. These results indicate that queens can utilise multiple behavioural trait combinations to maximise reproductive output at the earliest, and arguably most critical, time of colony foundation.

Hygienic food to reduce pathogen risk to bumblebees.

Bumblebees are ecologically and economically important pollinators, and the value of bumblebees for crop pollination has led to the commercial production and exportation/importation of colonies on a global scale. Commercially produced bumblebee colonies can carry with them infectious parasites, which can both reduce the health of the colonies and spillover to wild bees, with potentially serious consequences. The presence of parasites in commercially produced bumblebee colonies is in part because colonies are reared on pollen collected from honey bees, which often contains a diversity of microbial parasites. In response to this threat, part of the industry has started to irradiate pollen used for bumblebee rearing. However, to date there is limited data published on the efficacy of this treatment. Here we examine the effect of gamma irradiation and an experimental ozone treatment on the presence and viability of parasites in honey bee pollen. While untreated pollen contained numerous viable parasites, we find that gamma irradiation reduced the viability of parasites in pollen, but did not eliminate parasites entirely. Ozone treatment appeared to be less effective than gamma irradiation, while an artificial pollen substitute was, as expected, entirely free of parasites. The results suggest that the irradiation of pollen before using it to rear bumblebee colonies is a sensible method which will help reduce the incidence of parasite infections in commercially produced bumblebee colonies, but that further optimisation, or the use of a nutritionally equivalent artificial pollen substitute, may be needed to fully eliminate this route of disease entry into factories.

Genetic diversity, virulence and fitness evolution in an obligate fungal parasite of bees.

Within-host competition is predicted to drive the evolution of virulence in parasites, but the precise outcomes of such interactions are often unpredictable due to many factors including the biology of the host and the parasite, stochastic events and co-evolutionary interactions. Here, we use a serial passage experiment (SPE) with three strains of a heterothallic fungal parasite (Ascosphaera apis) of the Honey bee (Apis mellifera) to assess how evolving under increasing competitive pressure affects parasite virulence and fitness evolution. The results show an increase in virulence after successive generations of selection and consequently faster production of spores. This faster sporulation, however, did not translate into more spores being produced during this longer window of sporulation; rather, it appeared to induce a loss of fitness in terms of total spore production. There was no evidence to suggest that a greater diversity of competing strains was a driver of this increased virulence and subsequent fitness cost, but rather that strain-specific competitive interactions influenced the evolutionary outcomes of mixed infections. It is possible that the parasite may have evolved to avoid competition with multiple strains because of its heterothallic mode of reproduction, which highlights the importance of understanding parasite biology when predicting disease dynamics.

Horizontal transmission of a parasite is influenced by infected host phenotype and density.

Transmission is a key determinant of parasite fitness, and understanding the dynamics of transmission is fundamental to the ecology and evolution of host-parasite interactions. Successful transmission is often reliant on contact between infected individuals and susceptible hosts. The social insects consist of aggregated groups of genetically similar hosts, making them particularly vulnerable to parasite transmission. Here we investigate how the ratio of infected to susceptible individuals impacts parasite transmission, using the honey bee, Apis mellifera and its microsporidian parasite Nosema ceranae. We used 2 types of infected hosts found simultaneously in colonies; sterile female workers and sexual males. We found a higher ratio of infected to susceptible individuals in groups resulted in a greater proportion of susceptibles becoming infected, but this effect was non-linear and interestingly, the ratio also affected the spore production of infected individuals. The transmission level was much greater in an experiment where the infected individuals were drones than in an experiment where they were workers, suggesting drones may act as intracolonial 'superspreaders'. Understanding the subtleties of transmission and how it is influenced by the phenotype of the infected/susceptible individuals is important for understanding pathogen transmission at population level, and for optimum targeting of parasite control strategies.

A carbohydrate-rich diet increases social immunity in ants.

Increased potential for disease transmission among nest-mates means living in groups has inherent costs. This increased potential is predicted to select for disease resistance mechanisms that are enhanced by cooperative exchanges among group members, a phenomenon known as social immunity. One potential mediator of social immunity is diet nutritional balance because traits underlying immunity can require different nutritional mixtures. Here, we show how dietary protein-carbohydrate balance affects social immunity in ants. When challenged with a parasitic fungus Metarhizium anisopliae, workers reared on a high-carbohydrate diet survived approximately 2.8× longer in worker groups than in solitary conditions, whereas workers reared on an isocaloric, high-protein diet survived only approximately 1.3× longer in worker groups versus solitary conditions. Nutrition had little effect on social grooming, a potential mechanism for social immunity. However, experimentally blocking metapleural glands, which secrete antibiotics, completely eliminated effects of social grouping and nutrition on immunity, suggesting a causal role for secretion exchange. A carbohydrate-rich diet also reduced worker mortality rates when whole colonies were challenged with Metarhizium. These results provide a novel mechanism by which carbohydrate exploitation could contribute to the ecological dominance of ants and other social groups.

Size and asymmetry: are there costs to winning the royalty race?

Body size and morphology are key fitness-determining traits that can vary genotypically. They are likely to be important in social insect queens, which mate in swarms and found colonies independently, but genetic influences on queen morphology have been little investigated. Here, we show that the body size and morphology of queens are influenced by their genotype in the leaf-cutting ant Acromyrmex echinatior, a species in which certain lineages (patrilines) bias their development towards reproductive queens rather than sterile workers. We found no relationship between the queen-worker skew of patrilines and the size or morphology of queens, but there was a significant relationship with fluctuating asymmetry, which was greater in more queen-biased patrilines. Our results suggest that queen-biased patrilines do not incur a fitness cost in terms of body size, but may face more subtle costs in developmental stability. Such costs may constrain the evolution of royal cheating in social insects.

Caste-specific expression of genetic variation in the size of antibiotic-producing glands of leaf-cutting ants.

Social insect castes represent some of the most spectacular examples of phenotypic plasticity, with each caste being associated with different environmental conditions during their life. Here we examine the level of genetic variation in different castes of two polyandrous species of Acromyrmex leaf-cutting ant for the antibiotic-producing metapleural gland, which has a major role in defence against parasites. Gland size increases allometrically. The small workers that play the main role in disease defence have relatively large glands compared with larger workers, while the glands of gynes are substantially larger than those of any workers, for their body size. The gland size of large workers varies significantly between patrilines in both Acromyrmex echinatior and Acromyrmex octospinosus. We also examined small workers and gynes in A. echinatior, again finding genetic variation in gland size in these castes. There were significant positive relationships between the gland sizes of patrilines in the different castes, indicating that the genetic mechanism underpinning the patriline variation has remained similar across phenotypes. The level of expressed genetic variation decreased from small workers to large workers to gynes. This is consistent with the hypothesis that there is individual selection on disease defence in founding queens and colony-level selection on disease defence in the worker castes.

Multiple gains and losses of Wolbachia symbionts across a tribe of fungus-growing ants.

Although the intracellular bacterium Wolbachia is ubiquitous in insects, it has a unique relationship with New World ants on which particular bacterial strains have specialized. However, data are from distantly related hosts and detailed phylogenetic information which could reveal transmission dynamics are lacking. Here, we investigate host-Wolbachia relationships in the monophyletic fungus-growing ant tribe Attini, screening 23 species and using multilocus sequence typing to reliably identify Wolbachia strains. This technique reduces the significant problem of recombination seen using traditional single gene techniques. The relationship between Wolbachia and the fungus-growing ants appears complex and dynamic. There is evidence of co-cladogenesis, supporting vertical transmission; however, this is incomplete, demonstrating that horizontal transmission has also occurred. Importantly, the infection prevalence is frequently different between closely related taxa, with the Acromyrmex leaf-cutting ants appearing particularly prone to infection and there being no consistent relationship with any of the major life history transitions. We suggest that infection loss and horizontal transmission have driven epidemics or selective sweeps of Wolbachia, resulting in multiple gains and losses of infection across the fungus-growing ants.

Multi-level selection for hygienic behaviour in honeybees.

Disease is one of the main factors driving both natural and artificial selection. It is a particularly important and increasing threat to the managed honeybee colonies, which are vital in crop pollination. Artificial selection for disease-resistant honeybee genotypes has previously only been carried out at the colony-level, that is, by using queens or males reared from colonies that show resistance. However, honeybee queens mate with many males and so each colony consists of multiple patrilines that will vary in heritable traits, such as disease resistance. Here, we investigate whether response to artificial selection for a key resistance mechanism, hygienic behaviour, can be improved using multi-level selection, that is, by selecting not only among colonies as normal but also among patrilines within colonies. Highly hygienic colonies were identified (between-colony selection), and the specific patrilines within them responsible for most hygienic behaviour were determined using observation hives. Queens reared from these hygienic patrilines (within-colony selection) were identified using DNA microsatellite analysis of a wing-tip tissue sample and then mated to drones from a third highly hygienic colony. The resulting colonies headed by queens from hygienic patrilines showed approximately double the level of hygienic behaviour of colonies headed by sister queens from non-hygienic patrilines. The results show that multi-level selection can significantly improve the success of honeybee breeding programs.

Multiple paternity or multiple queens: two routes to greater intracolonial genetic diversity in the eusocial Hymenoptera.

Understanding the evolution of multiple mating by females (polyandry) is an important question in behavioural ecology. Most leading explanations for polyandry by social insect queens are based upon a postulated fitness benefit from increased intracolonial genetic diversity, which also arises when colonies are headed by multiple queens (polygyny). An indirect test of the genetic diversity hypotheses is therefore provided by the relationship between polyandry and polygyny across species, which should be negative if the genetic diversity hypotheses are correct. Here, we conduct a powerful comparative investigation of the relationship between polyandry and polygyny for 241 species of eusocial Hymenoptera (ants, bees and wasps). We find a clear and significant negative relationship between polyandry and polygyny after controlling for phylogeny. These results strongly suggest that fitness benefits resulting from increased intracolonial genetic diversity have played an important role in the evolution of polyandry, and possibly polygyny, in social insects.

Sex, size and isotopes: cryptic trophic ecology of an apex predator, the white shark Carcharodon carcharias.

Demographic differences in resource use are key components of population and species ecology across the animal kingdom. White sharks (Carcharodon carcharias) are migratory, apex predators, which have undergone significant population declines across their range. Understanding their ecology is key to ensuring that management strategies are effective. Here, we carry out the first stable isotope analyses of free-swimming white sharks in South Africa. Biopsies were collected in Gansbaai (34.5805°S, 19.3518°E) between February and July 2015. We used Stable Isotope Bayesian Ellipsis in R and traditional statistical analyses to quantify and compare isotopic niches of male and female sharks of two size classes, and analyse relationships between isotopic values and shark length. Our results reveal cryptic trophic differences between the sexes and life stages. Males, but not females, were inferred to feed in more offshore or westerly habitats as they grow larger, and only males exhibited evidence of an ontogenetic niche shift. Lack of relationship between δ13C, δ15N and female shark length may be caused by females exhibiting multiple migration and foraging strategies, and a greater propensity to travel further north. Sharks < 3 m had much wider, and more diverse niches than sharks > 3 m, drivers of which may include individual dietary specialisation and temporal factors. The differences in migratory and foraging behaviour between sexes, life stages, and individuals will affect their exposure to anthropogenic threats, and should be considered in management strategies.

Symmetry breaking in mass-recruiting ants: extent of foraging biases depends on resource quality.

ABSTRACT: The communication involved in the foraging behaviour of social insects is integral to their success. Many ant species use trail pheromones to make decisions about where to forage. The strong positive feedback caused by the trail pheromone is thought to create a decision between two or more options. When the two options are of identical quality, this is known as symmetry breaking, and is important because it helps colonies to monopolise food sources in a competitive environment. Symmetry breaking is thought to increase with the quantity of pheromone deposited by ants, but empirical studies exploring the factors affecting symmetry breaking are limited. Here, we tested if (i) greater disparity between two food sources increased the degree to which a higher quality food source is favoured and (ii) if the quality of identical food sources would affect the degree of symmetry breaking that occurs. Using the mass-recruiting Pharaoh ant, Monomorium pharaonis, we carried out binary choice tests to investigate how food quality affects the choice and distribution of colony foraging decisions. We found that colonies could coordinate foraging to exploit food sources of greater quality, and a greater contrast in quality between the food sources created a stronger collective decision. Contrary to prediction, we found that symmetry breaking decreased as the quality of two identical food sources increased. We discuss how stochastic effects might lead to relatively strong differences in the amount of pheromone on alternative routes when food source quality is low. SIGNIFICANCE STATEMENT: Pheromones used by social insects should guide a colony via positive feedback to distribute colony members at resources in the most adaptive way given the current environment. This study shows that when food resources are of equal quality, Pharaoh ant foragers distribute themselves more evenly if the two food sources are both of high quality compared to if both are of low quality. The results highlight the way in which individual ants can modulate their response to pheromone trails which may lead colonies to exploiting resources more evenly when in a resource rich environment.

The effects of juvenile hormone on Lasius niger reproduction.

Reproduction has been shown to be costly for survival in a wide diversity of taxa. The resulting trade-off, termed the reproduction-survival trade-off, is thought to be one of the most fundamental forces of life-history evolution. In insects the pleiotropic effect of juvenile hormone (JH), antagonistically regulating reproduction and pathogen resistance, is suggested to underlie this phenomenon. In contrast to the majority of insects, reproductive individuals in many eusocial insects defy this trade-off and live both long and prosper. By remodelling the gonadotropic effects of JH in reproductive regulation, the queens of the long-lived black garden ant Lasius niger (living up to 27 years), have circumvented the reproduction-survival trade off enabling them to maximize both reproduction and pathogen resistance simultaneously. In this study we measure fertility, vitellogenin gene expression and protein levels after experimental manipulation of hormone levels. We use these measurements to investigate the mechanistic basis of endocrinological role remodelling in reproduction and determine how JH suppresses reproduction in this species, rather then stimulating it, like in the majority of insects. We find that JH likely inhibits three key aspects of reproduction both during vitellogenesis and oogenesis, including two previously unknown mechanisms. In addition, we document that juvenile hormone, as in the majority of insects, has retained some stimulatory function in regulating vitellogenin expression. We discuss the evolutionary consequences of this complex regulatory architecture of reproduction in L. niger, which might enable the evolution of similar reproductive phenotypes by alternate regulatory pathways, and the surprising flexibility regulatory role of juvenile hormone in this process.

The cost of promiscuity: sexual transmission of Nosema microsporidian parasites in polyandrous honey bees.

Multiple mating (and insemination) by females with different males, polyandry, is widespread across animals, due to material and/or genetic benefits for females. It reaches particularly high levels in some social insects, in which queens can produce significantly fitter colonies by being polyandrous. It is therefore a paradox that two thirds of eusocial hymenopteran insects appear to be exclusively monandrous, in spite of the fitness benefits that polyandry could provide. One possible cost of polyandry could be sexually transmitted parasites, but evidence for these in social insects is extremely limited. Here we show that two different species of Nosema microsporidian parasites can transmit sexually in the honey bee Apis mellifera. Honey bee males that are infected by the parasite have Nosema spores in their semen, and queens artificially inseminated with either Nosema spores or the semen of Nosema-infected males became infected by the parasite. The emergent and more virulent N. ceranae achieved much higher rates of infection following insemination than did N. apis. The results provide the first quantitative evidence of a sexually transmitted disease (STD) in social insects, indicating that STDs may represent a potential cost of polyandry in social insects.

Let your enemy do the work: within-host interactions between two fungal parasites of leaf-cutting ants.

Within-host competition is an important factor in host-parasite relationships, yet most studies consider interactions involving only single parasite species. We investigated the interaction between a virulent obligate entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, and a normally avirulent, opportunistic fungal pathogen, Aspergillus flavus, in their leaf-cutting ant host, Acromyrmex echinatior. Surprisingly, the latter normally out-competed the former in mixed infections and had enhanced fitness relative to when infecting in isolation. The result is most probably due to Metarhizium inhibiting the host's immune defences, which would otherwise normally prevent infections by Aspergillus. With the host defences negated by the virulent parasite, the avirulent parasite was then able to out-compete its competitor. This result is strikingly similar to that seen in immunocompromised vertebrate hosts and indicates that avirulent parasites may play a more important role in host life histories than is generally realized.

Does genetic diversity hinder parasite evolution in social insect colonies?

Polyandry is often difficult to explain because benefits of the behaviour have proved elusive. In social insects, polyandry increases the genetic diversity of workers within a colony and this has been suggested to improve the resistance of the colony to disease. Here we examine the possible impact of host genetic diversity on parasite evolution by carrying out serial passages of a virulent fungal pathogen through leaf-cutting ant workers of known genotypes. Parasite virulence increased over the nine-generation span of the experiment while spore production decreased. The effect of host relatedness upon virulence appeared limited. However, parasites cycled through more genetically diverse hosts were more likely to go extinct during the experiment and parasites cycled through more genetically similar hosts had greater spore production. These results indicate that host genetic diversity may indeed hinder the ability of parasites to adapt while cycling within social insect colonies.