Microbiota-gut-brain axis and behaviour

Joanito Liberti

Assistant Professor

  • T: +41 22 379 67 59
  • office 4049B (Sciences III)

In our laboratory we study the interactions between symbiotic microorganisms and the neurophysiology and behaviour of their animal hosts. We aim to: (1) identify microbes with neuroactive potential, (2) determine the ways in which they influence brain physiology, (3) quantify their effects on animal behaviour, and (4) unravel the evolutionary history of these microbiota – brain interactions. We focus on the gut microbiota of insects. In particular, we study social insects such as bees as their societies express complex (social) behaviours at the individual level and collective behaviours emerge from the interaction between cooperative individuals specializing on distinct tasks. Additionally, we use the fruit fly Drosophila melanogaster to better characterize the molecular mechanisms and neural circuitry affected by gut microbes following cross-species microbiome transplants.

For these studies we use a combination of state-of-the-art technologies including automated behavioural tracking, advanced microscopy techniques, and various -omics approaches such as (meta)genomics, (spatial) transcriptomics, epigenomics and metabolomics. We perform experimental manipulations of the microbiota to produce gnotobiotic animals (i.e., whose gut microbiota is experimentally defined), which allow us to infer causality in microbiota-host interactions and to determine the underlying molecular mechanisms. We rear bees and flies on-site and also collect wild bees in the field.

We are looking for motivated MSc students, PhD students and postdocs interested in applying microbiology, behavioural ecology and -omics approaches to investigate the ultimate and proximate mechanisms of the microbiota-gut-brain axis. We accept applications from molecular biologists, evolutionary biologists, behavioural ecologists, microbiologists, bioinformaticians, engineers and more, interested in working in a dynamic, diverse and cooperative team, and in strengthening their interdisciplinary profile.

A group of bees with a printed patch on their head Description automatically generated

Image credit: Bart Zijlstra

Example of automated behavioural tracking to quantify the social behaviours of bees (adapted from Liberti et al., Nature Ecology & Evolution, 2022, doi: 10.1038/s41559-022-01840-w; Copyright © 2022, The Author(s), under exclusive license to Springer Nature Limited).

  • Gut microbiota influences onset of foraging-related behavior but not physiological hallmarks of division of labor in honeybees.

    mBio 2024 Jul;():e0103424. 10.1128/mbio.01034-24.

    abstract

    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.

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  • Interplay between gut symbionts and behavioral variation in social insects.

    Curr Opin Insect Sci 2024 Jul;65():101233. 10.1016/j.cois.2024.101233. S2214-5745(24)00075-0.

    abstract

    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.

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  • Wound-dependent leg amputations to combat infections in an ant society

    Current biology : CB, S0960-9822(24)00805-4. 25 Jun. 2024, doi:10.1016/j.cub.2024.06.021

    abstract

    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.

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  • Targeted treatment of injured nestmates with antimicrobial compounds in an ant society.

    Nat Commun 2023 Dec;14(1):8446. PMC10756881. 10.1038/s41467-023-43885-w. 10.1038/s41467-023-43885-w.

    abstract

    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.

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  • Social network position is a major predictor of ant behavior, microbiota composition, and brain gene expression.

    PLoS Biol 2023 Jul;21(7):e3002203. PMC10399779. 10.1371/journal.pbio.3002203. PBIOLOGY-D-23-00189.

    abstract

    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.

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  • Deep Divergence and Genomic Diversification of Gut Symbionts of Neotropical Stingless Bees.

    mBio 2023 Apr;14(2):e0353822. PMC10128065. 10.1128/mbio.03538-22.

    abstract

    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.

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  • The gut microbiota affects the social network of honeybees.

    Nat Ecol Evol 2022 Oct;6(10):1471-1479. EMS146375. PMC7613669. 10.1038/s41559-022-01840-w. 10.1038/s41559-022-01840-w.

    abstract

    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.

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  • Mild chronic exposure to pesticides alters physiological markers of honey bee health without perturbing the core gut microbiota.

    Sci Rep 2022 Mar;12(1):4281. PMC8917129. 10.1038/s41598-022-08009-2. 10.1038/s41598-022-08009-2.

    abstract

    Recent studies highlighted that exposure to glyphosate can affect specific members of the core gut microbiota of honey bee workers. However, in this study, bees were exposed to relatively high glyphosate concentrations. Here, we chronically exposed newly emerged honey bees to imidacloprid, glyphosate and difenoconazole, individually and in a ternary mixture, at an environmental concentration of 0.1 µg/L. We studied the effects of these exposures on the establishment of the gut microbiota, the physiological status, the longevity, and food consumption of the host. The core bacterial species were not affected by the exposure to the three pesticides. Negative effects were observed but they were restricted to few transient non-core bacterial species. However, in the absence of the core microbiota, the pesticides induced physiological disruption by directly altering the detoxification system, the antioxidant defenses, and the metabolism of the host. Our study indicates that even mild exposure to pesticides can directly alter the physiological homeostasis of newly emerged honey bees and particularly if the individuals exhibit a dysbiosis (i.e. mostly lack the core microbiota). This highlights the importance of an early establishment of a healthy gut bacterial community to strengthen the natural defenses of the honey bee against xenobiotic stressors.

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  • Impact of Chronic Exposure to Sublethal Doses of Glyphosate on Honey Bee Immunity, Gut Microbiota and Infection by Pathogens.

    Microorganisms 2021 Apr;9(4):. PMC8071123. 10.3390/microorganisms9040845. microorganisms9040845.

    abstract

    Glyphosate is the most used pesticide around the world. Although different studies have evidenced its negative effect on honey bees, including detrimental impacts on behavior, cognitive, sensory and developmental abilities, its use continues to grow. Recent studies have shown that it also alters the composition of the honey bee gut microbiota. In this study we explored the impact of chronic exposure to sublethal doses of glyphosate on the honey bee gut microbiota and its effects on the immune response, infection by and Deformed wing virus (DWV) and honey bee survival. Glyphosate combined with infection altered the structure and composition of the honey bee gut microbiota, for example by decreasing the relative abundance of the core members and . Glyphosate increased the expression of some immune genes, possibly representing a physiological response to mitigate its negative effects. However, this response was not sufficient to maintain honey bee health, as glyphosate promoted the replication of DWV and decreased the expression of , which were accompanied by a reduced life span. Infection by also alters honey bee immunity although no synergistic effect with glyphosate was observed. These results corroborate previous findings suggesting deleterious effects of widespread use of glyphosate on honey bee health, and they contribute to elucidate the physiological mechanisms underlying a global decline of pollination services.

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  • The gut microbiota - brain axis of insects.

    Curr Opin Insect Sci 2020 Jun;39():6-13. 10.1016/j.cois.2020.01.004. S2214-5745(20)30018-3.

    abstract

    Research on the connections between gut microbes and the neurophysiology and behavior of their animal hosts has grown exponentially in just a few years. Most studies have focused on mammalian models as their relevance to human health is widely established. However, evidence is accumulating that insect behavior may be governed by molecular mechanisms that are partly homologous to those of mammals, and therefore relevant for the understanding of their behavioral dysfunctions. Social insects in particular may provide experimentally amenable models to disentangle the contributions of individual bacterial symbionts to the gut microbiota - brain axis. In this review, we summarize findings from recent research on the neurological and behavioral effects of the gut microbiota of insects and propose an integrated approach to unravel the extended behavioral phenotypes of gut microbes in the honey bee.

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  • Gut microbiota structure differs between honeybees in winter and summer.

    ISME J 2020 Mar;14(3):801-814. PMC7031341. 10.1038/s41396-019-0568-8. 10.1038/s41396-019-0568-8.

    abstract

    Adult honeybees harbor a specialized gut microbiota of relatively low complexity. While seasonal differences in community composition have been reported, previous studies have focused on compositional changes rather than differences in absolute bacterial loads. Moreover, little is known about the gut microbiota of winter bees, which live much longer than bees during the foraging season, and which are critical for colony survival. We quantified seven core members of the bee gut microbiota in a single colony over 2 years and characterized the community composition in 14 colonies during summer and winter. Our data show that total bacterial loads substantially differ between foragers, nurses, and winter bees. Long-lived winter bees had the highest bacterial loads and the lowest community α-diversity, with a characteristic shift toward high levels of Bartonella and Commensalibacter, and a reduction of opportunistic colonizers. Using gnotobiotic bee experiments, we show that diet is a major contributor to the observed differences in bacterial loads. Overall, our study reveals that the gut microbiota of winter bees is remarkably different from foragers and nurses. Considering the importance of winter bees for colony survival, future work should focus on the role of the gut microbiota in winter bee health and disease.

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  • Seminal fluid compromises visual perception in honeybee queens reducing their survival during additional mating flights.

    Elife 2019 Sep;8():. PMC6739865. 10.7554/eLife.45009. 45009.

    abstract

    Queens of social insects make all mate-choice decisions on a single day, except in honeybees whose queens can conduct mating flights for several days even when already inseminated by a number of drones. Honeybees therefore appear to have a unique, evolutionarily derived form of sexual conflict: a queen's decision to pursue risky additional mating flights is driven by later-life fitness gains from genetically more diverse worker-offspring but reduces paternity shares of the drones she already mated with. We used artificial insemination, RNA-sequencing and electroretinography to show that seminal fluid induces a decline in queen vision by perturbing the phototransduction pathway within 24-48 hr. Follow up field trials revealed that queens receiving seminal fluid flew two days earlier than sister queens inseminated with saline, and failed more often to return. These findings are consistent with seminal fluid components manipulating queen eyesight to reduce queen promiscuity across mating flights.

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  • Rival seminal fluid induces enhanced sperm motility in a polyandrous ant.

    BMC Evol Biol 2018 Mar;18(1):28. PMC5865361. 10.1186/s12862-018-1144-y. 10.1186/s12862-018-1144-y.

    abstract

    Promiscuous mating and sperm competition often induce arms races between the sexes with detrimental outcomes for females. However, ants with multiply-inseminated queens have only a single time-window for sperm competition and queens are predicted to gain control over the outcome of sperm storage quickly. The seminal fluid of Acromyrmex leaf-cutting ants reduces the viability of rival sperm, but how confrontations between unrelated ejaculates affect sperm storage remains unknown.

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  • Queen reproductive tract secretions enhance sperm motility in ants.

    Biol Lett 2016 Nov;12(11):. PMC5134051. 10.1098/rsbl.2016.0722. rsbl.2016.0722.

    abstract

    Queens of Acromyrmex leaf-cutting ants store sperm of multiple males after a single mating flight, and never remate even though they may live for decades and lay tens of thousands of eggs. Sperm of different males are initially transferred to the bursa copulatrix and compete for access to the long-term storage organ of queens, but the factors determining storage success or failure have never been studied. We used in vitro experiments to show that reproductive tract secretions of Acromyrmex echinatior queens increase sperm swimming performance by at least 50% without discriminating between sperm of brothers and unrelated males. Indiscriminate female-induced sperm chemokinesis makes the likelihood of storage directly dependent on initial sperm viability and thus provides a simple mechanism to secure maximal possible reproductive success of queens, provided that initial sperm motility is an accurate predictor of viability during later egg fertilization.

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  • Bacterial symbiont sharing in Megalomyrmex social parasites and their fungus-growing ant hosts.

    Mol Ecol 2015 Jun;24(12):3151-69. PMC5008137. 10.1111/mec.13216.

    abstract

    Bacterial symbionts are important fitness determinants of insects. Some hosts have independently acquired taxonomically related microbes to meet similar challenges, but whether distantly related hosts that live in tight symbiosis can maintain similar microbial communities has not been investigated. Varying degrees of nest sharing between Megalomyrmex social parasites (Solenopsidini) and their fungus-growing ant hosts (Attini) from the genera Cyphomyrmex, Trachymyrmex and Sericomyrmex allowed us to address this question, as both ant lineages rely on the same fungal diet, interact in varying intensities and are distantly related. We used tag-encoded FLX 454 pyrosequencing and diagnostic PCR to map bacterial symbiont diversity across the Megalomyrmex phylogenetic tree, which also contains free-living generalist predators. We show that social parasites and hosts share a subset of bacterial symbionts, primarily consisting of Entomoplasmatales, Bartonellaceae, Acinetobacter, Wolbachia and Pseudonocardia and that Entomoplasmatales and Bartonellaceae can co-infect specifically associated combinations of hosts and social parasites with identical 16S rRNA genotypes. We reconstructed in more detail the population-level infection dynamics for Entomoplasmatales and Bartonellaceae in Megalomyrmex symmetochus guest ants and their Sericomyrmex amabilis hosts. We further assessed the stability of the bacterial communities through a diet manipulation experiment and evaluated possible transmission modes in shared nests such as consumption of the same fungus garden food, eating of host brood by social parasites, trophallaxis and grooming interactions between the ants, or parallel acquisition from the same nest environment. Our results imply that cohabiting ant social parasites and hosts may obtain functional benefits from bacterial symbiont transfer even when they are not closely related.

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  • Differences in forage-acquisition and fungal enzyme activity contribute to niche segregation in Panamanian leaf-cutting ants.

    PLoS One 2014 ;9(4):e94284. PMC3981799. 10.1371/journal.pone.0094284. PONE-D-13-52597.

    abstract

    The genera Atta and Acromyrmex are often grouped as leaf-cutting ants for pest management assessments and ecological surveys, although their mature colony sizes and foraging niches may differ substantially. Few studies have addressed such interspecific differences at the same site, which prompted us to conduct a comparative study across six sympatric leaf-cutting ant species in Central Panama. We show that foraging rates during the transition between dry and wet season differ about 60 fold between genera, but are relatively constant across species within genera. These differences appear to match overall differences in colony size, especially when Atta workers that return to their nests without leaves are assumed to carry liquid food. We confirm that Panamanian Atta specialize primarily on tree-leaves whereas Acromyrmex focus on collecting flowers and herbal leaves and that species within genera are similar in these overall foraging strategies. Species within genera tended to be spaced out over the three habitat categories that we distinguished (forest, forest edge, open grassland), but each of these habitats normally had only a single predominant Atta and Acromyrmex species. We measured activities of twelve fungus garden decomposition enzymes, belonging to the amylases, cellulases, hemicellulases, pectinases and proteinases, and show that average enzyme activity per unit of fungal mass in Atta gardens is lower than in Acromyrmex gardens. Expression profiles of fungal enzymes in Atta also appeared to be more specialized than in Acromyrmex, possibly reflecting variation in forage material. Our results suggest that species- and genus-level identities of leaf-cutting ants and habitat-specific foraging profiles may give predictable differences in the expression of fungal genes coding for decomposition enzymes.

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  • Chemically armed mercenary ants protect fungus-farming societies.

    Proc Natl Acad Sci U S A 2013 Sep;110(39):15752-7. PMC3785773. 10.1073/pnas.1311654110. 1311654110.

    abstract

    The ants are extraordinary in having evolved many lineages that exploit closely related ant societies as social parasites, but social parasitism by distantly related ants is rare. Here we document the interaction dynamics among a Sericomyrmex fungus-growing ant host, a permanently associated parasitic guest ant of the genus Megalomyrmex, and a raiding agro-predator of the genus Gnamptogenys. We show experimentally that the guest ants protect their host colonies against agro-predator raids using alkaloid venom that is much more potent than the biting defenses of the host ants. Relatively few guest ants are sufficient to kill raiders that invariably exterminate host nests without a cohabiting guest ant colony. We also show that the odor of guest ants discourages raider scouts from recruiting nestmates to host colonies. Our results imply that Sericomyrmex fungus-growers obtain a net benefit from their costly guest ants behaving as a functional soldier caste to meet lethal threats from agro-predator raiders. The fundamentally different life histories of the agro-predators and guest ants appear to facilitate their coexistence in a negative frequency-dependent manner. Because a guest ant colony is committed for life to a single host colony, the guests would harm their own interests by not defending the host that they continue to exploit. This conditional mutualism is analogous to chronic sickle cell anemia enhancing the resistance to malaria and to episodes in human history when mercenary city defenders offered either net benefits or imposed net costs, depending on the level of threat from invading armies.

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