Priority effects alter microbiome composition and increase abundance of probiotic taxa in treefrog tadpoles.

Host-associated microbial communities, like other ecological communities, may be impacted by the colonization order of taxa through priority effects. Developing embryos and their associated microbiomes are subject to stochasticity during colonization by bacteria. For amphibian embryos, often developing externally in bacteria-rich environments, this stochasticity may be particularly impactful. For example, the amphibian microbiome can mitigate lethal outcomes from disease for their hosts; however, this may depend on microbiome composition. Here, we examined the assembly of the bacterial community in spring peeper (Pseudacris crucifer) embryos and tadpoles. First, we reared embryos from identified mating pairs in either lab or field environments to examine the relative impact of environment and parentage on embryo and tadpole bacterial communities. Second, we experimentally inoculated embryos to determine if priority effects (i) could be used to increase the relative abundance of Janthinobacterium lividum, an amphibian-associated bacteria capable of preventing fungal infection, and (ii) would lead to observed differences in the relative abundances of two closely related bacteria from the genus Pseudomonas. Using 16S rRNA gene amplicon sequencing, we observed differences in community composition based on rearing location and parentage in embryos and tadpoles. In the inoculation experiment, we found that priority inoculation could increase the relative abundance of J. lividum, but did not find that either Pseudomonas isolate was able to prevent colonization by the other when given priority. These results highlight the importance of environmental source pools and parentage in determining microbiome composition, while also providing novel methods for the administration of a known amphibian probiotic. IMPORTANCE: Harnessing the functions of host-associated bacteria is a promising mechanism for managing disease outcomes across different host species. In the case of amphibians, certain frog-associated bacteria can mitigate lethal outcomes of infection by the fungal pathogen Batrachochytrium dendrobatidis. Successful probiotic applications require knowledge of community assembly and an understanding of the ecological mechanisms that structure these symbiotic bacterial communities. In our study, we show the importance of environment and parentage in determining bacterial community composition and that community composition can be influenced by priority effects. Further, we provide support for the use of bacterial priority effects as a mechanism to increase the relative abundance of target probiotic taxa in a developing host. While our results show that priority effects are not universally effective across all host-associated bacteria, our ability to increase the relative abundance of specific probiotic taxa may enhance conservation strategies that rely on captive rearing of endangered vertebrates.

Nonrandom foraging and resource distributions affect the relationships between host density, contact rates and parasite transmission.

Nonrandom foraging can cause animals to aggregate in resource dense areas, increasing host density, contact rates and pathogen transmission, but when should nonrandom foraging and resource distributions also have density-independent effects? Here, we used a factorial experiment with constant resource and host densities to quantify host contact rates across seven resource distributions. We also used an agent-based model to compare pathogen transmission when host movement was based on random foraging, optimal foraging or something between those states. Nonrandom foraging strongly depressed contact rates and transmission relative to the classic random movement assumptions used in most epidemiological models. Given nonrandom foraging in the agent-based model and experiment, contact rates and transmission increased with resource aggregation and average distance to resource patches due to increased host movement in search of resources. Overall, we describe three density-independent mechanisms by which host behaviour and resource distributions alter contact rate functions and pathogen transmission.

Comparative genomics of bacteria from amphibian skin associated with inhibition of an amphibian fungal pathogen, Batrachochytrium dendrobatidis.

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is a skin disease associated with worldwide amphibian declines. Symbiotic microbes living on amphibian skin interact with Bd and may alter infection outcomes. We completed whole genome sequencing of 40 bacterial isolates cultured from the skin of four amphibian species in the Eastern US. Each isolate was tested in vitro for the ability to inhibit Bd growth. The aim of this study was to identify genomic differences among the isolates and generate hypotheses about the genomic underpinnings of Bd growth inhibition. We identified sixty-five gene families that were present in all 40 isolates. Screening for common biosynthetic gene clusters revealed that this set of isolates contained a wide variety of clusters; the two most abundant clusters with potential antifungal activity were siderophores (N=17 isolates) and Type III polyketide synthases (N=22 isolates). We then examined various subsets of the 22 isolates in the phylum Proteobacteria for genes encoding specific compounds that may inhibit fungal growth, including chitinase and violacein. We identified differences in Agrobacterium and Sphingomonas isolates in the chitinase genes that showed some association with anti-Bd activity, as well as variation in the violacein genes in the Janthinobacterium isolates. Using a comparative genomics approach, we generated several testable hypotheses about differences among bacterial isolates from amphibian skin communities that could contribute to variation in the ability to inhibit Bd growth. Further work is necessary to explore and uncover the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd.

Skin bacterial community differences among three species of co-occurring Ranid frogs.

Skin microbial communities are an essential part of host health and can play a role in mitigating disease. Host and environmental factors can shape and alter these microbial communities and, therefore, we need to understand to what extent these factors influence microbial communities and how this can impact disease dynamics. Microbial communities have been studied in amphibian systems due to skin microbial communities providing some resistance to the amphibian chytrid fungus, Batrachochytrium dendrobatidis. However, we are only starting to understand how host and environmental factors shape these communities for amphibians. In this study, we examined whether amphibian skin bacterial communities differ among host species, host infection status, host developmental stage, and host habitat. We collected skin swabs from tadpoles and adults of three Ranid frog species (Lithobates spp.) at the Mianus River Gorge Preserve in Bedford, New York, USA, and used 16S rRNA gene amplicon sequencing to determine bacterial community composition. Our analysis suggests amphibian skin bacterial communities change across host developmental stages, as has been documented previously. Additionally, we found that skin bacterial communities differed among Ranid species, with skin communities on the host species captured in streams or bogs differing from the communities of the species captured on land. Thus, habitat use of different species may drive differences in host-associated microbial communities for closely-related host species.

Characterization of prophages in bacterial genomes from the honey bee (Apis mellifera) gut microbiome.

The gut of the European honey bee (Apis mellifera) possesses a relatively simple bacterial community, but little is known about its community of prophages (temperate bacteriophages integrated into the bacterial genome). Although prophages may eventually begin replicating and kill their bacterial hosts, they can also sometimes be beneficial for their hosts by conferring protection from other phage infections or encoding genes in metabolic pathways and for toxins. In this study, we explored prophages in 17 species of core bacteria in the honey bee gut and two honey bee pathogens. Out of the 181 genomes examined, 431 putative prophage regions were predicted. Among core gut bacteria, the number of prophages per genome ranged from zero to seven and prophage composition (the compositional percentage of each bacterial genome attributable to prophages) ranged from 0 to 7%. Snodgrassella alvi and Gilliamella apicola had the highest median prophages per genome (3.0 ± 1.46; 3.0 ± 1.59), as well as the highest prophage composition (2.58% ± 1.4; 3.0% ± 1.59). The pathogen Paenibacillus larvae had a higher median number of prophages (8.0 ± 5.33) and prophage composition (6.40% ± 3.08) than the pathogen Melissococcus plutonius or any of the core bacteria. Prophage populations were highly specific to their bacterial host species, suggesting most prophages were acquired recently relative to the divergence of these bacterial groups. Furthermore, functional annotation of the predicted genes encoded within the prophage regions indicates that some prophages in the honey bee gut encode additional benefits to their bacterial hosts, such as genes in carbohydrate metabolism. Collectively, this survey suggests that prophages within the honey bee gut may contribute to the maintenance and stability of the honey bee gut microbiome and potentially modulate specific members of the bacterial community, particularly S. alvi and G. apicola.

Colonization order of bacterial isolates on treefrog embryos impacts microbiome structure in tadpoles.

Priority effects, or impacts of colonization order, may have lasting influence on ecological community composition. The embryonic microbiome is subject to stochasticity in colonization order of bacteria. Stochasticity may be especially impactful for embryos developing in bacteria-rich environments, such as the embryos of many amphibians. To determine if priority effects experienced as embryos impacted bacterial community composition in newly hatched tadpoles, we selectively inoculated the embryos of laboratory-raised hourglass treefrogs, Dendropsophus ebraccatus, with bacteria initially isolated from the skin of wild D. ebraccatus adults over 2 days. First, embryos were inoculated with two bacteria in alternating sequences. Next, we evaluated the outcomes of priority effects in an in vitro co-culture assay absent of host factors. We then performed a second embryo experiment, inoculating embryos with one of three bacteria on the first day and a community of five target bacteria on the second. Through 16S rRNA gene amplicon sequencing, we observed relative abundance shifts in tadpole bacteria communities due to priority effects. Our results suggest that the initial bacterial source pools of embryos shape bacterial communities at later life stages; however, the magnitude of those changes is dependent on the host environment and the identity of bacterial colonists.

Comparative genomics of Lactobacillaceae from the gut of honey bees, Apis mellifera, from the Eastern United States.

Lactobacillaceae are an important family of lactic acid bacteria that play key roles in the gut microbiome of many animal species. In the honey bee (Apis mellifera) gut microbiome, many species of Lactobacillaceae are found, and there is functionally important strain-level variation in the bacteria. In this study, we completed whole-genome sequencing of 3 unique Lactobacillaceae isolates collected from hives in Virginia, USA. Using 107 genomes of known bee-associated Lactobacillaceae and Limosilactobacillus reuteri as an outgroup, the phylogenetics of the 3 isolates was assessed, and these isolates were identified as novel strains of Apilactobacillus kunkeei, Lactobacillus kullabergensis, and Bombilactobacillus mellis. Genome rearrangements, conserved orthologous genes (COG) categories and potential prophage regions were identified across the 3 novel strains. The new A. kunkeei strain was enriched in genes related to replication, recombination and repair, the L. kullabergensis strain was enriched for carbohydrate transport, and the B. mellis strain was enriched in transcription or transcriptional regulation and in some genes with unknown functions. Prophage regions were identified in the A. kunkeei and L. kullabergensis isolates. These new bee-associated strains add to our growing knowledge of the honey bee gut microbiome, and to Lactobacillaceae genomics more broadly.

Body condition, skin bacterial communities and disease status: insights from the first release trial of the limosa harlequin frog, Atelopus limosus.

Many endangered amphibian species survive in captive breeding facilities, but there have been few attempts to reintroduce captive-born individuals to rebuild wild populations. We conducted a soft-release trial of limosa harlequin frogs, Atelopus limosus, which are highly susceptible to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), to understand changes associated with the transition from captivity to the wild. Specifically, we assessed changes in body condition, skin-associated bacterial communities and disease status after release. Frogs were housed individually in field mesocosms and monitored for 27 days. Body condition did not significantly change in the mesocosms, and was similar to, or higher than, that of wild conspecifics at day 27. The skin bacteria of captive-born frogs, based on 16S rRNA gene amplicons, became similar to that of wild conspecifics after 27 days in mesocosms. Prevalence of Bd in wild conspecifics was 13-27%, and 15% of the A. limosus in mesocosms became infected with Bd, but no mortality of infected frogs was observed. We conclude that mesocosms are suitable for systematically and repeatedly monitoring amphibians during release trials, and that body condition, the skin microbiome, and Bd status can all change within one month of placement of captive-born individuals back into the wild.

Antibiotic perturbation of gut bacteria does not significantly alter host responses to ocular disease in a songbird species.

Bacterial communities in and on wild hosts are increasingly appreciated for their importance in host health. Through both direct and indirect interactions, bacteria lining vertebrate gut mucosa provide hosts protection against infectious pathogens, sometimes even in distal body regions through immune regulation. In house finches (Haemorhous mexicanus), the bacterial pathogen Mycoplasma gallisepticum (MG) causes conjunctivitis, with ocular inflammation mediated by pro- and anti-inflammatory cytokines and infection triggering MG-specific antibodies. Here, we tested the role of gut bacteria in host responses to MG by using oral antibiotics to perturb bacteria in the gut of captive house finches prior to experimental inoculation with MG. We found no clear support for an impact of gut bacterial disruption on conjunctival pathology, MG load, or plasma antibody levels. However, there was a non-significant trend for birds with intact gut communities to have greater conjunctival pathology, suggesting a possible impact of gut bacteria on pro-inflammatory cytokine stimulation. Using 16S bacterial rRNA amplicon sequencing, we found dramatic differences in cloacal bacterial community composition between captive, wild-caught house finches in our experiment and free-living finches from the same population, with lower bacterial richness and core communities composed of fewer genera in captive finches. We hypothesize that captivity may have affected the strength of results in this experiment, necessitating further study with this consideration. The abundance of anthropogenic impacts on wildlife and their bacterial communities, alongside the emergence and spread of infectious diseases, highlights the importance of studies addressing the role of commensal bacteria in health and disease, and the consequences of gut bacterial shifts on wild hosts.

Host preferences inhibit transmission from potential superspreader host species.

Host species that are particularly abundant, infectious and/or infected tend to contribute disproportionately to symbiont (parasite or mutualist) maintenance in multi-host systems. Therefore, in a facultative multi-host system where two host species had high densities, high symbiont infestation intensities and high infestation prevalence, we expected interspecific transmission rates to be high. Instead, we found that interspecific symbiont transmission rates to caged sentinel hosts were an order of magnitude lower than intraspecific transmission rates in the wild. Using laboratory experiments to decompose transmission rates, we found that opportunities for interspecific transmission were frequent, where interspecific and intraspecific contact rate functions were statistically indistinguishable. However, most interspecific contacts did not lead to transmission events owing to a previously unrecognized transmission barrier: strong host preferences. During laboratory choice experiments, the symbiont preferred staying on or dispersing to its current host species, even though the oligochaete symbiont is a globally distributed host generalist that can survive and reproduce on many snail host species. These surprising results suggest that when managing symbiont transmission, identifying key host species is still important, but it may be equally important to identify and manage transmission barriers that keep potential superspreader host species in check.

Classic Hoarding Cages Increase Gut Bacterial Abundance and Reduce the Individual Immune Response of Honey Bee (Apis mellifera) Workers.

Laboratory experiments have advanced our understanding of honey bee (Apis mellifera) responses to environmental factors, but removal from the hive environment may also impact physiology. To examine whether the laboratory environment alters the honey bee gut bacterial community and immune responses, we compared bacterial community structure (based on amplicon sequence variant relative abundance), total bacterial abundance, and immune enzyme (phenoloxidase and glucose oxidase) activity of cohort honey bee workers kept under laboratory and hive conditions. Workers housed in the laboratory showed differences in the relative abundance of their core gut taxa, an increase in total gut bacterial abundance, and reduced phenoloxidase activity, compared to bees housed in hives.

An experimental test of disease resistance function in the skin-associated bacterial communities of three tropical amphibian species.

Variation in the structure of host-associated microbial communities has been correlated with the occurrence and severity of disease in diverse host taxa, suggesting a key role of the microbiome in pathogen defense. However, whether these correlations are typically a cause or consequence of pathogen exposure remains an open question, and requires experimental approaches to disentangle. In amphibians, infection by the fungal pathogen Batrachochytrium dendrobatidis (Bd) alters the skin microbial community in some host species, whereas in other species, the skin microbial community appears to mediate infection dynamics. In this study, we completed experimental Bd exposures in three species of tropical frogs (Agalychnis callidryas, Dendropsophus ebraccatus,andCraugastor fitzingeri) that were sympatric with Bd at the time of the study. For all three species, we identified key taxa within the skin bacterial communities that were linked to Bd infection dynamics. We also measured higher Bd infection intensities in D. ebraccatus and C. fitzingeri that were associated with higher mortality in C. fitzingeri. Our findings indicate that microbially mediated pathogen resistance is a complex trait that can vary within and across host species, and suggest that symbiont communities that have experienced prior selection for defensive microbes may be less likely to be disturbed by pathogen exposure.

Horsenettle (Solanum carolinense) fruit bacterial communities are not variable across fine spatial scales.

Fruit house microbial communities that are unique from the rest of the plant. While symbiotic microbial communities complete important functions for their hosts, the fruit microbiome is often understudied compared to other plant organs. Fruits are reproductive tissues that house, protect, and facilitate the dispersal of seeds, and thus they are directly tied to plant fitness. Fruit microbial communities may, therefore, also impact plant fitness. In this study, we assessed how bacterial communities associated with fruit of Solanum carolinense, a native herbaceous perennial weed, vary at fine spatial scales (<0.5 km). A majority of the studies conducted on plant microbial communities have been done at large spatial scales and have observed microbial community variation across these large spatial scales. However, both the environment and pollinators play a role in shaping plant microbial communities and likely have impacts on the plant microbiome at fine scales. We collected fruit samples from eight sampling locations, ranging from 2 to 450 m apart, and assessed the fruit bacterial communities using 16S rRNA gene amplicon sequencing. Overall, we found no differences in observed richness or microbial community composition among sampling locations. Bacterial community structure of fruits collected near one another were not more different than those that were farther apart at the scales we examined. These fine spatial scales are important to obligate out-crossing plant species such as S. carolinense because they are ecologically relevant to pollinators. Thus, our results could imply that pollinators serve to homogenize fruit bacterial communities across these smaller scales.

Experimental test of microbiome protection across pathogen doses reveals importance of resident microbiome composition.

The commensal microbes inhabiting a host tissue can interact with invading pathogens and host physiology in ways that alter pathogen growth and disease manifestation. Prior work in house finches (Haemorhous mexicanus) found that resident ocular microbiomes were protective against conjunctival infection and disease caused by a relatively high dose of Mycoplasma gallisepticum. Here, we used wild-caught house finches to experimentally examine whether protective effects of the resident ocular microbiome vary with the dose of invading pathogen. We hypothesized that commensal protection would be strongest at low M. gallisepticum inoculation doses because the resident microbiome would be less disrupted by invading pathogen. Our five M. gallisepticum dose treatments were fully factorial with an antibiotic treatment to perturb resident microbes just prior to M. gallisepticum inoculation. Unexpectedly, we found no indication of protective effects of the resident microbiome at any pathogen inoculation dose, which was inconsistent with the prior work. The ocular bacterial communities at the beginning of our experiment differed significantly from those previously reported in local wild-caught house finches, likely causing this discrepancy. These variable results underscore that microbiome-based protection in natural systems can be context dependent, and natural variation in community composition may alter the function of resident microbiomes in free-living animals.

Assessing age, breeding stage, and mating activity as drivers of variation in the reproductive microbiome of female tree swallows.

Sexually transmitted microbes are hypothesized to influence the evolution of reproductive strategies. Though frequently discussed in this context, our understanding of the reproductive microbiome is quite nascent. Indeed, testing this hypothesis first requires establishing a baseline understanding of the temporal dynamics of the reproductive microbiome and of how individual variation in reproductive behavior and age influence the assembly and maintenance of the reproductive microbiome as a whole. Here, we ask how mating activity, breeding stage, and age influence the reproductive microbiome. We use observational and experimental approaches to explain variation in the cloacal microbiome of free-living, female tree swallows (Tachycineta bicolor). Using microsatellite-based parentage analyses, we determined the number of sires per brood (a proxy for female mating activity). We experimentally increased female sexual activity by administering exogenous 17ß-estradiol. Lastly, we used bacterial 16S rRNA amplicon sequencing to characterize the cloacal microbiome. Neither the number of sires per brood nor the increased sexual activity of females significantly influenced female cloacal microbiome richness or community structure. Female age, however, was positively correlated with cloacal microbiome richness and influenced overall community structure. A hypothesis to explain these patterns is that the effect of sexual activity and the number of mates on variation in the cloacal microbiome manifests over an individual's lifetime. Additionally, we found that cloacal microbiome alpha diversity (Shannon Index, Faith's phylogenetic distance) decreased and community structure shifted between breeding stages. This is one of few studies to document within-individual changes and age-related differences in the cloacal microbiome across successive breeding stages. More broadly, our results contribute to our understanding of the role that host life history and behavior play in shaping the cloacal microbiomes of wild birds.

Morphological and molecular characterization of Quinqueserialis (Digenea: Notocotylidae) species diversity in North America.

Estimates of trematode diversity are inaccurate due to unrecognized cryptic species and phenotypic plasticity within species. Integrative taxonomy (genetics, morphology and host use) increases the clarity of species delineation and improves knowledge of parasite biology. In this study, we used this approach to resolve taxonomic issues and test hypotheses of cryptic species in a genus of trematode, Quinqueserialis. Specimens from throughout North America were field collected from hosts and obtained from museums. We found three morphologically distinct groups and successfully sequenced specimens from two of these groups. DNA sequencing at the 28S and CO1 gene regions revealed that two of the three groups were genetically distinct. One genetic group included two morphological clusters demonstrating host-induced phenotypic plasticity within Quinqueserialis quinqueserialis. The other unique genetic group is a novel species, Quinqueserialis kinsellai n. sp., which is described herein. Our study illustrates the importance of integrating multiple sources of evidence when investigating trematode diversity to account for the influence of cryptic species or phenotypic plasticity. However, further sampling is needed to understand Quinqueserialis spp. diversity as some species have no genetic information associated with them.

Seasonal changes and the unexpected impact of environmental disturbance on skin bacteria of individual amphibians in a natural habitat.

Amphibians host diverse skin bacteria that have a role in pathogen defense, but these skin communities could change over time and impact this function. Here, we monitored individual Eastern red-spotted newts (Notophthalmus viridescens; N = 17) for 2 years in a field pond enclosure and assessed the effects of season and disturbance on skin bacterial community dynamics. We created disturbances by adding additional pond substrate to the enclosure at two timepoints. We planned to sample the skin bacterial community and metabolite profiles of each newt every 6 weeks; we ultimately sampled eight individuals at least six times. We used 16S rRNA gene amplicon sequencing to characterize the bacterial communities and HPLC-MS for metabolite profiling. We found that disturbance had a dramatic effect on skin bacterial communities and metabolite profiles, while season had an effect only using select metrics. There were seven core bacterial taxa (97% OTUs) that were found on all newts in all seasons, pre- and post-disturbance. Lastly, there was a correlation between bacterial and metabolite profiles post-disturbance, which was not observed pre-disturbance. This longitudinal study suggests that environmental disturbances can have lasting effects on skin bacterial communities that overwhelm seasonal changes, although the core bacteria remain relatively consistent over time.

Spatial scale and structure of complex life cycle trematode parasite communities in streams.

By considering the role of site-level factors and dispersal, metacommunity concepts have advanced our understanding of the processes that structure ecological communities. In dendritic systems, like streams and rivers, these processes may be impacted by network connectivity and unidirectional current. Streams and rivers are central to the dispersal of many pathogens, including parasites with complex, multi-host life cycles. Patterns in parasite distribution and diversity are often driven by host dispersal. We conducted two studies at different spatial scales (within and across stream networks) to investigate the importance of local and regional processes that structure trematode (parasitic flatworms) communities in streams. First, we examined trematode communities in first-intermediate host snails (Elimia proxima) in a survey of Appalachian headwater streams within the Upper New River Basin to assess regional turnover in community structure. We analyzed trematode communities based on both morphotype (visual identification) and haplotype (molecular identification), as cryptic diversity in larval trematodes could mask important community-level variation. Second, we examined communities at multiple sites (headwaters and main stem) within a stream network to assess potential roles of network position and downstream drift. Across stream networks, we found a broad scale spatial pattern in morphotype- and haplotype-defined communities due to regional turnover in the dominant parasite type. This pattern was correlated with elevation, but not with any other environmental factors. Additionally, we found evidence of multiple species within morphotypes, and greater genetic diversity in parasites with hosts limited to in-stream dispersal. Within network parasite prevalence, for at least some parasite taxa, was related to several site-level factors (elevation, snail density and stream depth), and total prevalence decreased from headwaters to main stem. Variation in the distribution and diversity of parasites at the regional scale may reflect differences in the abilities of hosts to disperse across the landscape. Within a stream network, species-environment relationships may counter the effects of downstream dispersal on community structure.

Cloacal bacterial communities of tree swallows (Tachycineta bicolor): Similarity within a population, but not between pair-bonded social partners.

Host-associated microbial communities can influence the overall health of their animal hosts, and many factors, including behavior and physiology, can impact the formation of these complex communities. Bacteria within these communities can be transmitted socially between individuals via indirect (e.g., shared environments) or direct (e.g., physical contact) pathways. Limited research has been done to investigate how social interactions that occur in the context of mating shape host-associated microbial communities. To gain a better understanding of these interactions and, more specifically, to assess how mating behavior shapes an animal's microbiome, we studied the cloacal bacterial communities of a socially monogamous yet genetically polygynous songbird, the North American tree swallow (Tachycineta bicolor). We address two questions: (1) do the cloacal bacterial communities differ between female and male tree swallows within a population? and (2) do pair-bonded social partners exhibit more similar cloacal bacterial communities than expected by chance? To answer these questions, we sampled the cloacal microbiome of adults during the breeding season and then used culture-independent, 16S rRNA gene amplicon sequencing to assess bacterial communities. Overall, we found that the cloacal bacterial communities of females and males were similar, and that the communities of pair-bonded social partners were not more similar than expected by chance. Our results suggest that social monogamy does not correlate with an increased similarity in cloacal bacterial community diversity or structure. As social partners were not assessed at the same time, it is possible that breeding stage differences masked social effects on bacterial community diversity and structure. Further, given that tree swallows exhibit high variation in rates of extra-pair activity, considering extra-pair activity when assessing cloacal microbial communities may be important for understanding how these bacterial communities are shaped. Further insight into how bacterial communities are shaped will ultimately shed light on potential tradeoffs associated with alternative behavioral strategies and socially-transmitted microbes.