Prof Joanito Liberti

Social insects represent some of the most speciose animal lineages, with biomass surpassing that of other animals by orders of magnitude, and often occupying dominant roles in terrestrial ecosystems. Some species express social organization so complex that they are considered superorganisms. These societies create ecological niches for diverse microorganisms, which associate with their insect hosts across a spectrum of interactions along the parasitism—mutualism continuum. With such complexity, the question arises whether specific aspects of insect social life have driven the evolution of exceptional symbiotic adaptations unseen elsewhere. In this chapter, we review the diversity of microbial symbioses across the social insects, attempting to identify their unique features. We ask whether microbial symbionts have played a role in the evolution of sociality in insects and, in turn, whether sociality exerts specific selective pressures on microbial evolution, transmission, and function. Finally, we review the current knowledge on the relationship between microbial symbionts and insect host sociality, which was recently driven by the development of social insect experimental models allowing for causal inferences in microorganism-host interactions.

The western honeybee, harbours a simple and distinct microbiota that has been linked to various positive outcomes for the host. Among these cited benefits is improved weight gain for bees that have been inoculated with their native microbes. This result has been challenged by recent studies which investigated the impact of the gut microbiota on behavioural maturation and associated physiological changes and revealed no effect of the gut microbiota on weight gain. Therefore, we re-examined the role of the microbiota in weight gain by comparing microbiota-deprived bees with those inoculated with gut homogenate or defined communities composed of isolates representing the major bacterial taxa inhabiting the bee gut. We observed no differences in weight gain of adult bees or of their gut tissues across these groups. Further analysis based on nurse/forager cuticular hydrocarbon status and bacterial composition also revealed no significant changes. These results suggest the need for more nuanced investigations aimed at exploring factors such as the conditions in the hive of origin, including larval nutrition.

The study of the fecal microbiota is crucial for unraveling the pathways through which gut symbionts are acquired and transmitted. While stable gut microbial communities are essential for honey bee health, their modes of acquisition and transmission are yet to be confirmed. The gut of honey bees is colonized by symbiotic bacteria within 5 days after emergence from their wax cells as adults. Few studies have suggested that bees could be colonized in part via contact with fecal matter in the hive. However, the composition of the fecal microbiota is still unknown. It is particularly unclear whether all bacterial species can be found viable in the feces and can therefore be transmitted to newborn nestmates. Using 16S rRNA gene amplicon sequencing, we revealed that the composition of the honey bee fecal microbiota is strikingly similar to the microbiota of entire guts. We found that fecal transplantation resulted in gut microbial communities similar to those obtained from feeding gut homogenates. Our study shows that fecal sampling and transplantation are viable tools for the non-invasive analysis of bacterial community composition and host-microbe interactions. It also implies that contact of young bees with fecal matter in the hive is a plausible route for gut microbiota acquisition.

Gut microbes can impact cognition and behavior, but whether they regulate the division of labor in animal societies is unknown. We addressed this question using honeybees since they exhibit division of labor between nurses and foragers and because their gut microbiota can be manipulated. Using automated behavioral tracking and controlling for co-housing effects, we show that gut microbes influence the age at which bees start expressing foraging-like behaviors in the laboratory but have no effects on the time spent in a foraging arena and number of foraging trips. Moreover, the gut microbiota did not influence hallmarks of behavioral maturation such as body weight, cuticular hydrocarbon profile, hypopharyngeal gland size, gene expression, and the proportion of bees maturing into foragers. Overall, this study shows that the honeybee gut microbiota plays a role in controlling the onset of foraging-related behavior without permanent consequences on colony-level division of labor and several physiological hallmarks of behavioral maturation.

Social insects exhibit a high degree of intraspecific behavioral variation. Moreover, they often harbor specialized microbial communities in their gut. Recent studies suggest that these two characteristics of social insects are interlinked: insect behavioral phenotypes affect their gut microbiota composition, partly through exposure to different environments and diet, and in return, the gut microbiota has been shown to influence insect behavior. Here, we discuss the bidirectional relationship existing between intraspecific variation in gut microbiota composition and behavioral phenotypes in social insects.

Open wounds pose major infection and mortality risks in animals.1,2 To reduce these risks, many animal species apply antimicrobial compounds on their wounds.1,2,3,4 Ant societies use antimicrobial secretions from the metapleural gland to combat pathogens,5,6,7,8,9,10 but this gland has been lost over evolutionary time in several genera, including Camponotus.11 To understand how infected wounds are handled without the use of antimicrobial secretions from the metapleural gland, we conducted behavioral and microbiological experiments in Camponotus floridanus. When we experimentally injured a worker's leg at the femur, nestmates amputated the injured limb by biting the base (trochanter) of the leg until it was severed, thereby significantly increasing survival compared to ants that did not receive amputations. However, when the experimental injury was more distal (at the tibia), nestmates did not amputate the leg and instead directed more wound care to the injury site. Experimental amputations also failed to improve survival in ants with infected tibia injuries unless the leg was amputated immediately after pathogen exposure. Micro-CT scans revealed that the muscles likely responsible for leg hemolymph circulation are predominantly in the femur. Thus, it is likely that femur injuries, by attenuating hemolymph flow, provide sufficient time for workers to perform amputations before pathogen spread. Overall, this study provides the first example of the use of amputations to treat infected individuals in a non-human animal and demonstrates that ants can adapt their type of treatment depending on the location of wounds.

Infected wounds pose a major mortality risk in animals. Injuries are common in the ant Megaponera analis, which raids pugnacious prey. Here we show that M. analis can determine when wounds are infected and treat them accordingly. By applying a variety of antimicrobial compounds and proteins secreted from the metapleural gland to infected wounds, workers reduce the mortality of infected individuals by 90%. Chemical analyses showed that wound infection is associated with specific changes in the cuticular hydrocarbon profile, thereby likely allowing nestmates to diagnose the infection state of injured individuals and apply the appropriate antimicrobial treatment. This study demonstrates that M. analis ant societies use antimicrobial compounds produced in the metapleural glands to treat infected wounds and reduce nestmate mortality.

The physiology and behavior of social organisms correlate with their social environments. However, because social environments are typically confounded by age and physical environments (i.e., spatial location and associated abiotic factors), these correlations are usually difficult to interpret. For example, associations between an individual's social environment and its gene expression patterns may result from both factors being driven by age or behavior. Simultaneous measurement of pertinent variables and quantification of the correlations between these variables can indicate whether relationships are direct (and possibly causal) or indirect. Here, we combine demographic and automated behavioral tracking with a multiomic approach to dissect the correlation structure among the social and physical environment, age, behavior, brain gene expression, and microbiota composition in the carpenter ant Camponotus fellah. Variations in physiology and behavior were most strongly correlated with the social environment. Moreover, seemingly strong correlations between brain gene expression and microbiota composition, physical environment, age, and behavior became weak when controlling for the social environment. Consistent with this, a machine learning analysis revealed that from brain gene expression data, an individual's social environment can be more accurately predicted than any other behavioral metric. These results indicate that social environment is a key regulator of behavior and physiology.

Social bees harbor conserved gut microbiotas that may have been acquired in a common ancestor of social bees and subsequently codiversified with their hosts. However, most of this knowledge is based on studies on the gut microbiotas of honey bees and bumblebees. Much less is known about the gut microbiotas of the third and most diverse group of social bees, the stingless bees. Specifically, the absence of genomic data from their microbiotas presents an important knowledge gap in understanding the evolution and functional diversity of the social bee microbiota. Here, we combined community profiling with culturing and genome sequencing of gut bacteria from six neotropical stingless bee species from Brazil. Phylogenomic analyses show that most stingless bee gut isolates form deep-branching sister clades of core members of the honey bee and bumblebee gut microbiota with conserved functional capabilities, confirming the common ancestry and ecology of their microbiota. However, our bacterial phylogenies were not congruent with those of the host, indicating that the evolution of the social bee gut microbiota was not driven by strict codiversification but included host switches and independent symbiont gain and losses. Finally, as reported for the honey bee and bumblebee microbiotas, we found substantial genomic divergence among strains of stingless bee gut bacteria, suggesting adaptation to different host species and glycan niches. Our study offers first insights into the genomic diversity of the stingless bee microbiota and highlights the need for broader samplings to understand the evolution of the social bee gut microbiota. Stingless bees are the most diverse group of the corbiculate bees and represent important pollinator species throughout the tropics and subtropics. They harbor specialized microbial communities in their gut that are related to those found in honey bees and bumblebees and that are likely important for bee health. Few bacteria have been cultured from the gut of stingless bees, which has prevented characterization of their genomic diversity and functional potential. Here, we established cultures of major members of the gut microbiotas of six stingless bee species and sequenced their genomes. We found that most stingless bee isolates belong to novel bacterial species distantly related to those found in honey bees and bumblebees and encoding similar functional capabilities. Our study offers a new perspective on the evolution of the social bee gut microbiota and presents a basis for characterizing the symbiotic relationships between gut bacteria and stingless bees.

The gut microbiota influences animal neurodevelopment and behaviour but has not previously been documented to affect group-level properties of social organisms. Here, we use honeybees to probe the effect of the gut microbiota on host social behaviour. We found that the microbiota increased the rate and specialization of head-to-head interactions between bees. Microbiota colonization was associated with higher abundances of one-third of the metabolites detected in the brain, including amino acids with roles in synaptic transmission and brain energetic function. Some of these metabolites were significant predictors of the number of social interactions. Microbiota colonization also affected brain transcriptional processes related to amino acid metabolism and epigenetic modifications in a brain region involved in sensory perception. These results demonstrate that the gut microbiota modulates the emergent colony social network of honeybees and suggest changes in chromatin accessibility and amino acid biosynthesis as underlying processes.