Queen presence mediates the relationship between collective behaviour and disease susceptibility in ant colonies.

The success of social living can be explained, in part, by a group's ability to execute collective behaviours unachievable by solitary individuals. However, groups vary in their ability to execute these complex behaviours, often because they vary in their phenotypic composition. Group membership changes over time due to mortality or emigration, potentially leaving groups vulnerable to ecological challenges in times of flux. In some societies, the loss of important individuals (e.g. leaders, elites and queens) may have an especially detrimental effect on groups' ability to deal with these challenges. Here, we test whether the removal of queens in colonies of the acorn ant Temnothorax curvispinosus alters their ability to execute important collective behaviours and survive outbreaks of a generalist entomopathogen. We employed a split-colony design where one half of a colony was maintained with its queen, while the other half was separated from the queen. We then tested these subcolonies' performance in a series of collective behaviour assays and finally exposed colonies to the entomopathogenic fungus Metarhizium robertsii by exposing two individuals from the colony and then sealing them back into the nest. We found that queenright subcolonies outperformed their queenless counterparts in nearly all collective behaviours. Queenless subcolonies were also more vulnerable to mortality from disease. However, queenless groups that displayed more interactions with brood experienced greater survivorship, a trend not present in queenright subcolonies. Queenless subcolonies that engage in more brood interactions may have had more resources available to cope with two physiological challenges (ovarian development after queen loss and immune activation after pathogen exposure). Our results indicate that queen presence can play an integral role in colony behaviour, survivorship and their relationship. They also suggest that interactions between workers and brood are integral to colonies survival. Overall, a social group's history of social reorganization may have strong consequences on their collective behaviours and their vulnerability to disease outbreaks.

The transcriptomic and evolutionary signature of social interactions regulating honey bee caste development.

The caste fate of developing female honey bee larvae is strictly socially regulated by adult nurse workers. As a result of this social regulation, nurse-expressed genes as well as larval-expressed genes may affect caste expression and evolution. We used a novel transcriptomic approach to identify genes with putative direct and indirect effects on honey bee caste development, and we subsequently studied the relative rates of molecular evolution at these caste-associated genes. We experimentally induced the production of new queens by removing the current colony queen, and we used RNA sequencing to study the gene expression profiles of both developing larvae and their caregiving nurses before and after queen removal. By comparing the gene expression profiles of queen-destined versus worker-destined larvae as well as nurses observed feeding these two types of larvae, we identified larval and nurse genes associated with caste development. Of 950 differentially expressed genes associated with caste, 82% were expressed in larvae with putative direct effects on larval caste, and 18% were expressed in nurses with putative indirect effects on caste. Estimated selection coefficients suggest that both nurse and larval genes putatively associated with caste are rapidly evolving, especially those genes associated with worker development. Altogether, our results suggest that indirect effect genes play important roles in both the expression and evolution of socially influenced traits such as caste.

Mixed infections reveal virulence differences between host-specific bee pathogens.

Dynamics of host-pathogen interactions are complex, often influencing the ecology, evolution and behavior of both the host and pathogen. In the natural world, infections with multiple pathogens are common, yet due to their complexity, interactions can be difficult to predict and study. Mathematical models help facilitate our understanding of these evolutionary processes, but empirical data are needed to test model assumptions and predictions. We used two common theoretical models regarding mixed infections (superinfection and co-infection) to determine which model assumptions best described a group of fungal pathogens closely associated with bees. We tested three fungal species, Ascosphaera apis, Ascosphaera aggregata and Ascosphaera larvis, in two bee hosts (Apis mellifera and Megachile rotundata). Bee survival was not significantly different in mixed infections vs. solo infections with the most virulent pathogen for either host, but fungal growth within the host was significantly altered by mixed infections. In the host A. mellifera, only the most virulent pathogen was present in the host post-infection (indicating superinfective properties). In M. rotundata, the most virulent pathogen co-existed with the lesser-virulent one (indicating co-infective properties). We demonstrated that the competitive outcomes of mixed infections were host-specific, indicating strong host specificity among these fungal bee pathogens.

Ant colonies prefer infected over uninfected nest sites.

During colony relocation, the selection of a new nest involves exploration and assessment of potential sites followed by colony movement on the basis of a collective decision making process. Hygiene and pathogen load of the potential nest sites are factors worker scouts might evaluate, given the high risk of epidemics in group-living animals. Choosing nest sites free of pathogens is hypothesized to be highly efficient in invasive ants as each of their introduced populations is often an open network of nests exchanging individuals (unicolonial) with frequent relocation into new nest sites and low genetic diversity, likely making these species particularly vulnerable to parasites and diseases. We investigated the nest site preference of the invasive pharaoh ant, Monomorium pharaonis, through binary choice tests between three nest types: nests containing dead nestmates overgrown with sporulating mycelium of the entomopathogenic fungus Metarhizium brunneum (infected nests), nests containing nestmates killed by freezing (uninfected nests), and empty nests. In contrast to the expectation pharaoh ant colonies preferentially (84%) moved into the infected nest when presented with the choice of an infected and an uninfected nest. The ants had an intermediate preference for empty nests. Pharaoh ants display an overall preference for infected nests during colony relocation. While we cannot rule out that the ants are actually manipulated by the pathogen, we propose that this preference might be an adaptive strategy by the host to "immunize" the colony against future exposure to the same pathogenic fungus.

Standard methods for fungal brood disease research.

Chalkbrood and stonebrood are two fungal diseases associated with honey bee brood. Chalkbrood, caused by Ascosphaera apis, is a common and widespread disease that can result in severe reduction of emerging worker bees and thus overall colony productivity. Stonebrood is caused by Aspergillus spp. that are rarely observed, so the impact on colony health is not very well understood. A major concern with the presence of Aspergillus in honey bees is the production of airborne conidia, which can lead to allergic bronchopulmonary aspergillosis, pulmonary aspergilloma, or even invasive aspergillosis in lung tissues upon inhalation by humans. In the current chapter we describe the honey bee disease symptoms of these fungal pathogens. In addition, we provide research methodologies and protocols for isolating and culturing, in vivo and in vitro assays that are commonly used to study these host pathogen interactions. We give guidelines on the preferred methods used in current research and the application of molecular techniques. We have added photographs, drawings and illustrations to assist bee-extension personnel and bee scientists in the control of these two diseases.

Microbial gut diversity of Africanized and European honey bee larval instars.

The first step in understanding gut microbial ecology is determining the presence and potential niche breadth of associated microbes. While the core gut bacteria of adult honey bees is becoming increasingly apparent, there is very little and inconsistent information concerning symbiotic bacterial communities in honey bee larvae. The larval gut is the target of highly pathogenic bacteria and fungi, highlighting the need to understand interactions between typical larval gut flora, nutrition and disease progression. Here we show that the larval gut is colonized by a handful of bacterial groups previously described from guts of adult honey bees or other pollinators. First and second larval instars contained almost exclusively Alpha 2.2, a core Acetobacteraceae, while later instars were dominated by one of two very different Lactobacillus spp., depending on the sampled site. Royal jelly inhibition assays revealed that of seven bacteria occurring in larvae, only one Neisseriaceae and one Lactobacillus sp. were inhibited. We found both core and environmentally vectored bacteria with putatively beneficial functions. Our results suggest that early inoculation by Acetobacteraceae may be important for microbial succession in larvae. This assay is a starting point for more sophisticated in vitro models of nutrition and disease resistance in honey bee larvae.

Virulence of mixed fungal infections in honey bee brood.

INTRODUCTION: Honey bees, Apis mellifera, have a diverse community of pathogens. Previous research has mostly focused on bacterial brood diseases of high virulence, but milder diseases caused by fungal pathogens have recently attracted more attention. This interest has been triggered by partial evidence that co-infection with multiple pathogens has the potential to accelerate honey bee mortality. In the present study we tested whether co-infection with closely related fungal brood-pathogen species that are either specialists or non-specialist results in higher host mortality than infections with a single specialist. We used a specially designed laboratory assay to expose honey bee larvae to controlled infections with spores of three Ascosphaera species: A. apis, the specialist pathogen that causes chalkbrood disease in honey bees, A. proliperda, a specialist pathogen that causes chalkbrood disease in solitary bees, and A. atra, a saprophytic fungus growing typically on pollen brood-provision masses of solitary bees. RESULTS: We show for the first time that single infection with a pollen fungus A. atra may induce some mortality and that co-infection with A. atra and A. apis resulted in higher mortality of honey bees compared to single infections with A. apis. However, similar single and mixed infections with A. proliperda did not increase brood mortality. CONCLUSION: Our results show that co-infection with a closely related fungal species can either increase or have no effect on host mortality, depending on the identity of the second species. Together with other studies suggesting that multiple interacting pathogens may be contributing to worldwide honey bee health declines, our results highlight the importance of studying effects of multiple infections, even when all interacting species are not known to be specialist pathogens.

Genetic variation in virulence among chalkbrood strains infecting honeybees.

Ascosphaera apis causes chalkbrood in honeybees, a chronic disease that reduces the number of viable offspring in the nest. Although lethal for larvae, the disease normally has relatively low virulence at the colony level. A recent study showed that there is genetic variation for host susceptibility, but whether Ascosphaera apis strains differ in virulence is unknown. We exploited a recently modified in vitro rearing technique to infect honeybee larvae from three colonies with naturally mated queens under strictly controlled laboratory conditions, using four strains from two distinct A. apis clades. We found that both strain and colony of larval origin affected mortality rates. The strains from one clade caused 12-14% mortality while those from the other clade induced 71-92% mortality. Larvae from one colony showed significantly higher susceptibility to chalkbrood infection than larvae from the other two colonies, confirming the existence of genetic variation in susceptibility across colonies. Our results are consistent with antagonistic coevolution between a specialized fungal pathogen and its host, and suggest that beekeeping industries would benefit from more systematic monitoring of this chronic stress factor of their colonies.

Morphological differences of symbiotic fungi Smittium culisetae (Harpellales: Legeriomycetaceae) in different Dipteran hosts.

Harpellales (Legeriomycetaceae, Zygomycota) or 'trichomycetes' are fungi that inhabit the digestive tracts of arthropods such as insects, millipedes, and crustaceans. In the current study we examined changes in 5 morphological characters of Smittium culisetae (Harpellales: Legeriomycetaceae) between the two dipteran (mosquito, black fly) hosts reared under 3 different temperatures (17, 22, 30 degrees C). Both host and temperature had a pervasive effect on the linear dimension of trichospores, their generative cells and hyphae width. At 30 degrees C the mean size of all 5 morphological characters were consistently larger in fungus taken from the mosquito host than from the black fly host. At 17 degrees C and 22 degrees C, however, there were no consistent patterns. The effect of host was so pronounced that it could be accurately determined which host S. culisetae colonised based on differences in linear morphology. Such changes in fungal morphology between hosts have important ramifications for the morphologically based taxonomy of this group.

Do different species of Smittium (Harpellales, Legeriomycetaceae) influence each other in the host gut?

Smittium (Harpellales, Legeriomycetaceae) belongs to a cosmopolitan group of filamentous fungi (Trichomycetes, Zygomycota) that live as obligate commensals in the digestive tract of various marine, freshwater, and terrestrial arthropods. The outcome of the paired introductions of three species of Smittium was investigated within the individual hosts of the mosquito Aedes aegypti (Culicidae: Diptera). In the first set of experiments, the host was inoculated with a single species of Smittium to determine whether hyphae location within the host was species specific. In the second experiment the host was exposed to two species of Smittium to determine whether hyphae showed positional displacement when two species of fungi co-inhabited the same host gut. Single species introductions of Smittium resulted in 80-85% of hosts with hyphae present only in the rectum. In all three paired combinations of Smittium species examined, only 40-65% of host larvae had hyphae restricted to the rectum. This is first study to experimentally demonstrate that the microdistribution of Harpellaceae hyphae can be influenced by the presence of a second species of Harpellaceae, suggesting a competition of the symbionts within the host.

The effect of temperature and host species on the development of the trichomycete Smittium culisetae (Zygomycota).

We examined the growth and development of the trichomycete Smittium culisetae (Harpellales: Legeriomycetaceae) in the larval hosts Simulium vittatum Zetterstedt (Diptera: Simuliidae) and Aedes aegypti (L.) (Diptera: Culicidae) at three temperatures, 17, 22 and 30 C. Trichospore maturation of Sm. culicetae external to the host as well as the ability of these trichospores to colonize new hosts also was investigated. Although the development of Sm. culisetae varied with both temperature and host there was a pattern of maximum trichospore production at 48-72 h postinoculation. In addition thalli under laboratory conditions are capable of spore production after extraction from a host and these trichospores can colonize new hosts. Furthermore this was noted to occur in both host species. These results suggest that synchrony between host and symbiont development is not as tightly coupled as previously assumed.