The importance of scale in comparative microbiome research: New insights from the gut and glands of captive and wild lemurs.

Research on animal microbiomes is increasingly aimed at determining the evolutionary and ecological factors that govern host-microbiome dynamics, which are invariably intertwined and potentially synergistic. We present three empirical studies related to this topic, each of which relies on the diversity of Malagasy lemurs (representing a total of 19 species) and the comparative approach applied across scales of analysis. In Study 1, we compare gut microbial membership across 14 species in the wild to test the relative importance of host phylogeny and feeding strategy in mediating microbiome structure. Whereas host phylogeny strongly predicted community composition, the same feeding strategies shared by distant relatives did not produce convergent microbial consortia, but rather shaped microbiomes in host lineage-specific ways, particularly in folivores. In Study 2, we compare 14 species of wild and captive folivores, frugivores, and omnivores, to highlight the importance of captive populations for advancing gut microbiome research. We show that the perturbational effect of captivity is mediated by host feeding strategy and can be mitigated, in part, by modified animal management. In Study 3, we examine various scent-gland microbiomes across three species in the wild or captivity and show them to vary by host species, sex, body site, and a proxy of social status. These rare data provide support for the bacterial fermentation hypothesis in olfactory signal production and implicate steroid hormones as mediators of microbial community structure. We conclude by discussing the role of scale in comparative microbial studies, the links between feeding strategy and host-microbiome coadaptation, the underappreciated benefits of captive populations for advancing conservation research, and the need to consider the entirety of an animal's microbiota. Ultimately, these studies will help move the field from exploratory to hypothesis-driven research.

Social odours covary with bacterial community in the anal secretions of wild meerkats.

The fermentation hypothesis for animal signalling posits that bacteria dwelling in an animal's scent glands metabolize the glands' primary products into odorous compounds used by the host to communicate with conspecifics. There is, however, little evidence of the predicted covariation between an animal's olfactory cues and its glandular bacterial communities. Using gas chromatography-mass spectrometry, we first identified the volatile compounds present in 'pure' versus 'mixed' anal-gland secretions ('paste') of adult meerkats (Suricata suricatta) living in the wild. Low-molecular-weight chemicals that likely derive from bacterial metabolism were more prominent in mixed than pure secretions. Focusing thereafter on mixed secretions, we showed that chemical composition varied by sex and was more similar between members of the same group than between members of different groups. Subsequently, using next-generation sequencing, we identified the bacterial assemblages present in meerkat paste and documented relationships between these assemblages and the host's sex, social status and group membership. Lastly, we found significant covariation between the volatile compounds and bacterial assemblages in meerkat paste, particularly in males. Together, these results are consistent with a role for bacteria in the production of sex- and group-specific scents, and with the evolution of mutualism between meerkats and their glandular microbiota.

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird.

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host.

Microbes and animal olfactory communication: Where do we go from here?

We know that microbes contribute to the production of odors that some animals use to communicate, but how common is this phenomenon? Recent studies capitalizing on new molecular technologies are uncovering fascinating associations between microbes and odors of wild animals, but causality is difficult to ascertain. Fundamental questions about the nature of these unique host-microbe interactions also remain unanswered. For instance, do microbes benefit from signaling associations with hosts? How does microbial community structure influence signal production? How do hosts regulate microbes in order to generate appropriate signals? Here, we review the current state of knowledge on microbially produced signals in animals and discuss key research foci that can advance our understanding of microbial-based signaling in the animal world.

Symbiotic bacteria appear to mediate hyena social odors.

All animals harbor beneficial microbes. One way these microbes can benefit their animal hosts is by increasing the diversity and efficacy of communication signals available to the hosts. The fermentation hypothesis for mammalian chemical communication posits that bacteria in the scent glands of mammals generate odorous metabolites used by their hosts for communication and that variation in host chemical signals is a product of underlying variation in the bacterial communities inhabiting the scent glands. An effective test of this hypothesis would require accurate surveys of the bacterial communities in mammals' scent glands and complementary data on the odorant profiles of scent secretions--both of which have been historically lacking. Here we use next-generation sequencing to survey deeply the bacterial communities in the scent glands of wild spotted and striped hyenas. We show that these communities are dominated by fermentative bacteria and that the structures of these communities covary with the volatile fatty acid profiles of scent secretions in both hyena species. The bacterial and volatile fatty acid profiles of secretions differ between spotted and striped hyenas, and both profiles vary with sex and reproductive state among spotted hyenas within a single social group. Our results strongly support the fermentation hypothesis for chemical communication, suggesting that symbiotic bacteria underlie species-specific odors in both spotted and striped hyenas and further underlie sex and reproductive state-specific odors among spotted hyenas. We anticipate that the fermentation hypothesis for chemical communication will prove broadly applicable among scent-marking mammals as others use the technical and analytical approaches used here.

Evidence for a bacterial mechanism for group-specific social odors among hyenas.

Symbiotic microbes can benefit their animal hosts by enhancing the diversity of communication signals available to them. The fermentation hypothesis for chemical recognition posits that 1) fermentative bacteria in specialized mammalian scent glands generate odorants that mammals co-opt to communicate with one another, and 2) that variation in scent gland odors is due to underlying variation in the structure of bacterial communities within scent glands. For example, group-specific social odors are suggested to be due to members of the same social group harboring more similar bacterial communities in their scent glands than do members of different social groups. We used 16S rRNA gene surveys to show that 1) the scent secretions of spotted hyenas are densely populated by fermentative bacteria whose closest relatives are well-documented odor producers, and that 2) these bacterial communities are more similar among hyenas from the same social group than among those from different groups.

Molecular characterization of the microbial communities in the subcaudal gland secretion of the European badger (Meles meles).

Many mammals possess specialized scent glands, which convey information about the marking individual. As the chemical profile of scent marks is likely to be affected by bacteria metabolizing the primary gland products, the variation in bacterial communities between different individuals has been proposed to underpin olfactory communication. However, few studies have investigated the dependency of microbiota residing in the scent organs on the host's individual-specific parameters. Here, we used terminal restriction fragment length polymorphism analysis of the PCR-amplified 16S rRNA gene and clone library construction to investigate the microbial communities in the subcaudal gland secretion of the European badger (Meles meles). As the secretion has been shown to encode individual-specific information, we investigated the correlation of the microbiota with different individual-specific parameters (age, sex, body condition, reproductive status, and season). We discovered a high number of bacterial species (56 operational taxonomic units from four phyla: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes), dominated by Actinobacteria (76.0%). The bacterial communities of cubs and adults differed significantly. Cubs possessed considerably more diverse communities dominated by Firmicutes, while in adults the communities were less diverse and dominated by Actinobacteria, suggesting that the acquisition of a 'mature bacterial community' is an ontogenetic process related to physiological changes during maturation.

Ethyl-branched aldehydes, ketones, and diketones from caimans (Caiman and Paleosuchus; Crocodylia, Reptilia).

Secretions from the paracloacal glands of alligators (Alligator spp.) and caimans (Caiman spp., Melanosuchus niger, and Paleosuchus spp.) were examined by GC-MS. The secretions of the common caiman (C. crocodilus), the broad-snouted caiman (C. latirostris), the yacare caiman (C. yacare), the dwarf caiman (P. palpebrosus), and the smooth-fronted caiman (P. trigonatus) yielded a new family of 43 aliphatic carbonyl compounds that includes aldehydes, ketones, and beta-diketones with an ethyl branch adjacent to the carbonyl group. The identification of these glandular components and the syntheses and stereochemical investigations of selected compounds are described.

Perineal scent gland of wild and domestic cavies: bacterial activity and urine as sources of biologically significant odors.

Several studies were conducted to evaluate the source of biologically relevant odorous substances in male wild cavy (Cavia aperea ) perineal gland secretions. In the first study, using a habituation procedure, male wild cavies distinguished between the urine of two individuals after exposure to the perineal secretions of one of the individuals. However, these animals did not distinguish between the perineal secretions of two individuals after exposure to the urine of one of the individuals. These results suggest that urine is a component of cavy perineal gland secretions as normally found in the perineal sac. Other studies were designed to evaluate the possible role of bacteria in producing biologically relevant odors from cavy perineal gland secretions. Microbiological analyses of secretion that had accumulated in the perineal sacs ("dirty") or been squeezed directly from the glands ("clean") of wild and domestic (C. porcellus) cavies indicated large numbers of bacteria inhabited these secretions. In behavioral studies using two-choice preference tests, male wild cavies spent more time investigating conspecific dirty perineal gland secretions than clean secretions. Clean secretions, however, which had been incubated for 48 hr at 37 degrees C were preferred by these cavies over clean secretions which had been frozen at -60 degrees C during the same time period. In a final experiment it was found that wild cavies preferred conspecific clean secretion that had been sterilized and incubated with bacteria over uninoculated sterilized secretion. Together, these results indicate that urine and bacteria are responsible for components of biologically significant odors of cavy perineal scent marks.