Digestive and respiratory tracts are inhabited by rich bacterial communities that can vary between their different segments. In comparison with other bird taxa with developed caeca, parrots that lack caeca have relatively lower variability in intestinal morphology. Here, based on 16S rRNA metabarcoding, we describe variation in microbiota across different parts of parrot digestive and respiratory tracts both at interspecies and intraspecies levels. In domesticated budgerigar (Melopsittacus undulatus), we describe the bacterial variation across eight selected sections of respiratory and digestive tracts, and three non-destructively collected sample types (faeces, and cloacal and oral swabs). Our results show important microbiota divergence between the upper and lower digestive tract, but similarities between respiratory tract and crop, and also between different intestinal segments. Faecal samples appear to provide a better proxy for intestinal microbiota composition than the cloacal swabs. Oral swabs had a similar bacterial composition as the crop and trachea. For a subset of tissues, we confirmed the same pattern also in six different parrot species. Finally, using the faeces and oral swabs in budgerigars, we revealed high oral, but low faecal microbiota stability during a 3-week period mimicking pre-experiment acclimation. Our findings provide a basis essential for microbiota-related experimental planning and result generalisation in non-poultry birds.

• Keywords
• Budgerigar, Domestic parakeet, Gastrointestinal tract microbiota, Microbiome composition, Psittaciformes, Symbiosis,
• MeSH
• Bacteria genetics   MeSH
• Respiratory System microbiology   MeSH
• Microbiota * MeSH
• Parrots * genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Ribosomal, 16S MeSH

During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity, and survival in vertebrates. Bird eggs are thought to be (1) initially sterile (sterile egg hypothesis) and (2) colonized after oviposition through horizontal trans-shell migration, or (3) initially seeded with bacteria by vertical transfer from mother oviduct. To date, however, little empirical data illuminate the contribution of these mechanisms to gut microbiota formation in avian embryos. We investigated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit (Parus major), using a methodologically advanced procedure combining 16S rRNA gene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that the avian egg is (nearly) sterile, but acquires a slightly richer microbiome during the embryonic development. Of the three potentially pathogenic bacteria targeted by qPCR, only Dietzia was found in E0-egg (yet also in negative controls), E13 gut and female samples, which might indicate possible vertical transfer. Unlike in poultry, we have shown that major bacterial colonization of the gut in passerines does not occur before hatching. We emphasize that protocols that carefully check for environmental contamination are critical in studies with low-bacterial biomass samples.

• Keywords
• egg microbiome, embryo, gastrointestinal tract microbiota, passerine bird, pathogenic bacteria, sterile egg,
• MeSH
• Bacteria genetics   MeSH
• Microbiota * MeSH
• Passeriformes * microbiology   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Gastrointestinal Microbiome * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Ribosomal, 16S MeSH

INTRODUCTION: Decreasing biotic diversity with increasing latitude is an almost universal macroecological pattern documented for a broad range of taxa, however, there have been few studies focused on changes in gut microbiota (GM) across climatic zones. METHODS: Using 16S rRNA amplicon profiling, we analyzed GM variation between temperate (Czechia) and tropical (Cameroon) populations of 99 passerine bird species and assessed GM similarity of temperate species migrating to tropical regions with that of residents/short-distance migrants and tropical residents. Our study also considered the possible influence of diet on GM. RESULTS: We observed no consistent GM diversity differences between tropical and temperate species. In the tropics, GM composition varied substantially between dry and rainy seasons and only a few taxa exhibited consistent differential abundance between tropical and temperate zones, irrespective of migration behavior and seasonal GM changes. During the breeding season, trans-Saharan migrant GM diverged little from species not overwintering in the tropics and did not show higher similarity to tropical passerines than temperate residents/short-distance migrants. Interestingly, GM of two temperate-breeding trans-Saharan migrants sampled in the tropical zone matched that of tropical residents and converged with other temperate species during the breeding season. Diet had a slight effect on GM composition of tropical species, but no effect on GM of temperate hosts. DISCUSSION: Consequently, our results demonstrate extensive passerine GM plasticity, the dominant role of environmental factors in its composition and limited effect of diet.

• Keywords
• climatic zones, faecal microbiome, gastrointestinal tract, metabarcoding, passerine birds,
• Publication type
• Journal Article MeSH

Gut microbiota (GM) often exhibit variation between different host species and co-divergence with hosts' phylogeny. Identifying these patterns is a key for understanding the mechanisms that shaped symbiosis between GM and its hosts. Therefore, both GM-host species specificity and GM-host co-divergence have been investigated by numerous studies. However, most of them neglected a possibility that different groups of bacteria within GM can vary in the tightness of their association with the host. Consequently, unlike most of these studies, we aimed to directly address how the strength of GM-host species specificity and GM-host co-divergence vary across different GM clades. We decomposed GM communities of 52 passerine species (394 individuals), characterized by 16S rRNA amplicon sequence variant (ASV) profiles, into monophyletic Binned Taxonomic units (BTUs). Subsequently, we analyzed strength of host species specificity and correlation with host phylogeny separately for resulting BTUs. We found that most BTUs exhibited significant host-species specificity in their composition. Notably, BTUs exhibiting high host-species specificity comprised bacterial taxa known to impact host's physiology and immune system. However, BTUs rarely displayed significant co-divergence with host phylogeny, suggesting that passerine GM evolution is not shaped primarily through a shared evolutionary history between the host and its gut microbes.

• Keywords
• 16S rRNA, co‐divergence, gut microbiota, metabarcoding, passerines,
• Publication type
• Journal Article MeSH

Quality and quantity of food items consumed has a crucial effect on phenotypes. In addition to direct effects mediated by nutrient resources, an individual's diet can also affect the phenotype indirectly by altering its gut microbiota, a potent modulator of physiological, immunity and cognitive functions. However, most of our knowledge of diet-microbiota interactions is based on mammalian species, whereas little is still known about these effects in other vertebrates. We developed a metabarcoding procedure based on cytochrome c oxidase I high-throughput amplicon sequencing and applied it to describe diet composition in breeding colonies of an insectivorous bird, the barn swallow (Hirundo rustica). To identify putative diet-microbiota associations, we integrated the resulting diet profiles with an existing dataset for faecal microbiota in the same individual. Consistent with previous studies based on macroscopic analysis of diet composition, we found that Diptera, Hemiptera, Coleoptera and Hymenoptera were the dominant dietary components in our population. We revealed pronounced variation in diet consumed during the breeding season, along with significant differences between nearby breeding colonies. In addition, we found no difference in diet composition between adults and juveniles. Finally, our data revealed a correlation between diet and faecal microbiota composition, even after statistical control for environmental factors affecting both diet and microbiota variation. Our study suggests that variation in diet induce slight but significant microbiota changes in a non-mammalian host relying on a narrow spectrum of items consumed.

• MeSH
• Diet MeSH
• Feces MeSH
• Microbiota * MeSH
• Mammals MeSH
• Gastrointestinal Microbiome * genetics   MeSH
• Swallows * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The composition of gut bacterial communities is strongly influenced by the host diet in many animal taxa. For birds, the effect of diet on the microbiomes has been documented through diet manipulation studies. However, for wild birds, most studies have drawn on literature-based information to decipher the dietary effects, thereby, overlooking individual variation in dietary intake. Here we examine how naturally consumed diets influence the composition of the crop and cloacal microbiomes of twenty-one tropical bird species, using visual and metabarcoding-based identification of consumed diets and bacterial 16S rRNA microbiome sequencing. We show that diet intakes vary markedly between individuals of the same species and that literature-based dietary guilds grossly underestimate intraspecific diet variability. Furthermore, despite an effect of literature-based dietary guild assignment of host taxa, the composition of natural diets does not align with crop and cloacal microbiome similarity. However, host-taxon specific gut bacterial lineages are positively correlated with specific diet items, indicating that certain microbes associate with different diet components in specific avian hosts. Consequently, microbiome composition is not congruent with the overall consumed diet composition of species, but specific components of a consumed diet lead to host-specific effects on gut bacterial taxa.

• MeSH
• Diet * MeSH
• Microbiota physiology   MeSH
• Eating physiology   MeSH
• Birds classification   microbiology   physiology   MeSH
• Gastrointestinal Microbiome physiology   MeSH
• Tropical Climate * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Animal hosts have evolved intricate associations with microbial symbionts, where both depend on each other for particular functions. In many cases, these associations lead to phylosymbiosis, where phylogenetically related species harbour compositionally more similar microbiomes than distantly related species. However, evidence for phylosymbiosis is either weak or lacking in gut microbiomes of flying vertebrates, particularly in birds. To shed more light on this phenomenon, we compared cloacal microbiomes of 37 tropical passerine bird species from New Guinea using 16S rRNA bacterial gene sequencing. We show a lack of phylosymbiosis and document highly variable microbiomes. Furthermore, we find that gut bacterial community compositions are species-specific and tend to be shaped by host diet but not sampling locality, potentially driven by the similarities in habitats used by individual species. We further show that flight-associated gut modifications, coupled with individual dietary differences, shape gut microbiome structure and variation, contributing to the lack of phylosymbiosis. These patterns indicate that the stability of symbiosis may depend on microbial functional diversity rather than taxonomic composition. Furthermore, the more variable and fluid host-microbe associations suggest probable disparities in the potential for coevolution between bird host species and microbial symbionts.

• Keywords
• diet, gut retention time, microbial heterogeneity, passeriformes, phylosymbiosis,
• MeSH
• Diet MeSH
• Phylogeny MeSH
• Passeriformes * MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Gastrointestinal Microbiome * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• New Guinea MeSH
• Names of Substances
• RNA, Ribosomal, 16S MeSH

BACKGROUND: It has been proposed that divergence in the gut microbiota composition between incipient species could contribute to their reproductive isolation. Nevertheless, empirical evidence for the role of gut microbiota in speciation is scarce. Moreover, it is still largely unknown to what extent closely related species in the early stages of speciation differ in their gut microbiota composition, especially in non-mammalian taxa, and which factors drive the divergence. Here we analysed the gut microbiota in two closely related passerine species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia). The ranges of these two species overlap in a secondary contact zone, where both species occasionally hybridize and where interspecific competition has resulted in habitat use differentiation. RESULTS: We analysed the gut microbiota from the proximal, middle and distal part of the small intestine in both sympatric and allopatric populations of the two nightingale species using sequencing of bacterial 16S rRNA. We found small but significant differences in the microbiota composition among the three gut sections. However, the gut microbiota composition in the two nightingale species did not differ significantly between either sympatric or allopatric populations. Most of the observed variation in the gut microbiota composition was explained by inter-individual differences. CONCLUSIONS: To our knowledge, this is the first attempt to assess the potential role of the gut microbiota in bird speciation. Our results suggest that neither habitat use, nor geographical distance, nor species identity have strong influence on the nightingale gut microbiota composition. This suggests that changes in the gut microbiota composition are unlikely to contribute to reproductive isolation in these passerine birds.

• Keywords
• Diet, Gut microbiome, Habitat use, Luscinia, Passerines, Reproductive isolation,
• MeSH
• Ecosystem MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Gastrointestinal Microbiome * MeSH
• Sympatry MeSH
• Songbirds * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Ribosomal, 16S MeSH

Animal-associated microbiota is expected to impose crucial effects on the host's fitness-related performance, including reproduction. Most research to date has focused on interactions between the host with its gut microbiota; however, there remain considerable gaps in knowledge regarding microbial consortia in other organs, including interspecific divergence, temporal stability, variation drivers, and their effects on the host. To fill these gaps, we examined oral and vaginal microbiota composition in four free-living mouse species of the genus Apodemus, each varying in the degree of female promiscuity. To assess temporal stability and microbiota resistance to environmental change, we exposed one of the species, Apodemus uralensis, to standardized captive conditions and analyzed longitudinal changes in its microbiota structure. Our results revealed the existence of a "core" oral microbiota that was not only shared among all four species but also persisted almost unchanged in captivity. On the other hand, vaginal microbiota appears to be more plastic in captive conditions and less species-specific in comparison with oral microbiota. This study is amongst the first to describe oral microbiota dynamics. Furthermore, the vaginal microbiota results are especially surprising in light of the well-known role of stable vaginal microbiota as a defense against pathogens. The results indicate the existence of diverse mechanisms that shape each microbiota. On the other hand, our data provides somewhat ambiguous support for the systematic effect of phylogeny and social system on both oral and vaginal microbiota structures.

• MeSH
• Bacteria classification   genetics   isolation & purification   MeSH
• Phylogeny MeSH
• Microbiota MeSH
• Mice MeSH
• Organ Specificity MeSH
• Sequence Analysis, DNA methods   MeSH
• Mouth microbiology   MeSH
• Vagina microbiology   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Comprehensive studies of wild bird microbiomes are often limited by difficulties of sample acquisition. However, widely used non-invasive cloacal swab methods and under-explored museum specimens preserved in alcohol provide promising avenues to increase our understanding of wild bird microbiomes, provided that they accurately portray natural microbial community compositions. To investigate this assertion, we used 16S rRNA amplicon sequencing of Great tit (Parus major) gut microbiomes to compare 1) microbial communities obtained from dissected digestive tract regions and cloacal swabs, and 2) microbial communities obtained from freshly dissected gut regions and from samples preserved in alcohol for 2 weeks or 2 months, respectively. RESULTS: We found no significant differences in alpha diversities in communities of different gut regions and cloacal swabs (except in OTU richness between the dissected cloacal region and the cloacal swabs), or between fresh and alcohol preserved samples. However, we did find significant differences in beta diversity and community composition of cloacal swab samples compared to different gut regions. Despite these community-level differences, swab samples qualitatively captured the majority of the bacterial diversity throughout the gut better than any single compartment. Bacterial community compositions of alcohol-preserved specimens did not differ significantly from freshly dissected samples, although some low-abundant taxa were lost in the alcohol preserved specimens. CONCLUSIONS: Our findings suggest that cloacal swabs, similar to non-invasive fecal sampling, qualitatively depict the gut microbiota composition without having to collect birds to extract the full digestive tract. The satisfactory depiction of gut microbial communities in alcohol preserved samples opens up for the possibility of using an enormous resource readily available through museum collections to characterize bird gut microbiomes. The use of extensive museum specimen collections of birds for microbial gut analyses would allow for investigations of temporal patterns of wild bird gut microbiomes, including the potential effects of climate change and anthropogenic impacts. Overall, the utilization of cloacal swabs and museum alcohol specimens can positively impact bird gut microbiome research to help increase our understanding of the role and evolution of wild bird hosts and gut microbial communities.

• Keywords
• Digestive tract microbiota, Microbiomes, Museum alcohol bird collections, Non-invasive sampling, Passeriformes,
• Publication type
• Journal Article MeSH