Gut microbiota is composed of many bacteria, fungi, viruses, and other organisms in the gastrointestinal system. The composition of gut microbiota in animals varies greatly in their early life but becomes stable in adulthood (Lim et al., 2015; Yin et al., 2020). Many large-scale studies have revealed effects of gut microbiota on the well-being of the host (Wan et al., 2020; Kuang et al., 2022). Host factors such as phylogeny, physiology, behavior, and dietary habit have also been shown to affect the composition of gut microbiota (Benson et al., 2010; Kurilshikov et al., 2021).

A recent large-scale study of the gut bacteria of bats, amphibians, and birds showed that bats and birds share similar profiles of gut bacteria (Song et al., 2020), suggesting that an innate ability, such as flying, may have similar effects on gut bacteria in animals of different phylogenies. In contrast, other studies disclosed a link between specific microorganisms and their hosts (Brucker and Bordenstein, 2012; Easson and Thacker, 2014; Sanders et al., 2014; Brooks et al., 2016). Such relationship is referred to as phylosymbiosis that predicts host clades harbor distinguishable microbial communities (Brucker and Bordenstein, 2012) and is common in some animals, such as sponges (Easson and Thacker, 2014), Nasonia (Brucker and Bordenstein, 2012), ants, and apes (Sanders et al., 2014). These findings suggest a relationship between the composition of gut microbiota and host evolutionary history, behaviors, and ecological factors (Zepeda Mendoza et al., 2018; Lutz et al., 2019).

Bat is the second largest order of mammals and have a long evolutionary history, distinct physiology, diverse dietary habits, echolocations, and flying ability (Adams and Pedersen, 2013). Similar to carnivores, the gut microbiota of bats are dominated by Firmicutes and Proteobacteria (Sun et al., 2020). It has been shown that the compositions of gut bacteria of bats vary with their diet, sex, age, mass, geographic location, physiology, reproduction, and symbiotic parasites (Phillips et al., 2012; Carrillo-Araujo et al., 2015; Lutz et al., 2019). Although the effects of host factors on gut bacteria have been described (Phillips et al., 2012; Carrillo-Araujo et al., 2015; Lutz et al., 2019), the mechanisms of such effects are unknown. We hypothesized that host phylogeny and dietary habits could affect the diversity of gut microbiota and therefore conducted this study to compare the compositions of gut microbiota in bats of different phylogenies and those of insect-eating (insectivores) bats, fruit-eating (frugivores) bat, and bats that feed on both insects and fish, aiming to uncover the impact of various host factors on the gut microbiota of bats.

The compositions of gut microbiota vary among different species of animals and may also vary under different physiological and behavioral conditions. As bats are highly differentiated animals with various dietary preferences and some bat species such as M. pilosus are in transition from insect-eating to fish-eating, they are ideal for investigation of the effects of diets on gut microbiota. In this study, we found that the gut microbiota profile of the one frugivorous bat (R. leschenaultii) examined was greatly different from that of insectivorous bats and bats that feed on both insects and fish, but those of the latter two dietary types of bats were similar. However, the abundance of 62 different species of bacteria was found to vary among different species of bats. These observations suggest that the difference in the profiles of gut microbiota in bats of different phylogenies was higher than those with different dietary habits as previously described (Lutz et al., 2019). We also found a much less difference in gut microbiota among bats of different phylogenies at the genus (R² = 0.54, p = 0.06) level than at the family level (R² = 0.049, p = 0.743) by PCA analysis. A possible reason for such is that closely related bats have similar behaviors, physiologies, and environmental factors and therefore have similar gut microbial compositions (Archie, 2019).

Many of the physiological or behavioral characteristics of bats have a unique evolutionary history (Brualla et al., 2023). For example, the basic immune systems of bats (e.g., innate and adaptive immune systems) are more tolerant than those of other mammals (Randolph and Barreiro, 2018). A study of bat rabies virus found that bat antibodies failed to penetrate the blood-brain barrier and therefore were unable to defend the infection in the central nervous system (Roy et al., 2007). However, bats are known to carry many potentially lethal viruses, such as filoviruses, henipaviruses, lyssaviruses, paramyxoviruses, and coronaviruses (Karunarathna et al., 2020; Sun et al., 2020). Such symbiosis of various viruses may affect their physiologies, behaviors, and adaption to the virulent gut ecology. Bats also harbor many bacterial pathogens such as various species of Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Pseudomonas, and Serratia (Daniel et al., 2013; Sun et al., 2020). We postulate that the symbiosis of various viruses and pathogenic bacteria is a survival strategy for bats to evade predators and that the host-microbe interactions may be a driving force for the evolution of bats to survive in various ecologic conditions. Dietary habits and other behaviors may also affect animal evolution. For instance, the bamboo diet promotes the adaptive development of strong teeth and paws in giant panda (Vallittu et al., 2021). Another example is that the flying ability of birds or bats is endowed with light bone, high degree of skull healing, shoulder strap, sternum with keel protuberance, and degradation of posterior tibia and fibula (Chang et al., 2019).

Different from most terrestrial mammals, the gut microbiota of bats with different dietary habits have few Bacteroidetes, but similar to carnivores, bat gut microbiota is enriched in Firmicutes and Proteobacteria (Sun et al., 2020). A previous study of bat gut bacteria showed that species of Gammaproteobacteria and Firmicutes were dominant in non-breeding adult bats; this observation is also consistent with ours in this study. We also did not observe a significant correlation between dietary habits and gut microbiota in bats that feed on insets or both insects and fish as that reported by other investigators (Phillips et al., 2012; Carrillo-Araujo et al., 2015; Lutz et al., 2019). We noticed that Aeromonas species were much more abundant in bats that feed on both insects and fish than those with insect diet. As Aeromonas is often detected in fish (Hanninen et al., 1997), it may alter the composition of gut microbiota in bats that feed on fish.

As observed in previous studies (Zepeda Mendoza et al., 2018; Ingala et al., 2021), we found that the predominant metabolic pathways were Transporters for insectivorous bats; Transporters and Carbon metabolism for bats that feed on both insects and fish; and Transporters and Biosynthesis of amino acids for frugivorous bat. A recent study showed that the numbers of genes involved in 37 metabolic pathways were different between herbivorous and animalivorous bats (Ingala et al., 2021). However, we found only 3 metabolic pathways involved in stilbenoid, diarylheptanoid and gingerol biosynthesis that were different in the number of genes between insectivorous bats and bats (M. pilosus) that feed on both insects and fish. A probable reason for this discrepancy is that we examined a very small dietary shift, i.e., from insect to both insect and fish diet. As M. pilosus bats have a high viral load, the enrichment of genes in these 3 metabolic pathways may be a symbiotic strategy with the viruses. The finding that the number of genes involved in photosynthesis of antenna proteins varied between IB and IFB (p = 0.036) suggests that photosynthesis also plays a role in gut microbial variation among bats with different dietary habits as previously postulated (Jin et al., 2021).

By investigating the effects of host speciation and dietary habits on gut microbiota, this study disclosed the effect of host–microbe interactions on bat evolution. A major weakness in this study is in bat sampling. To avoid the influence of geographic variation on gut microbiota, bats used in this study were captured from the same cave. As a result, we only captured bats from 3 families (Pteropodidae, Rhinolophidae, Vespertilionidae), and the number of bats is very limited with only one bat in most species. Such problem may introduce deviations in the comparison of microbial diversity. Furthermore, a previous study surveyed the variations in gut microbiota among 9 bat families, and nearly half of the families showed significant differences in gut bacterial composition (Lutz et al., 2019). We investigated 3 bat families and detected no significant difference in gut microbiota among them, indicating that investigations with more families are warranted in future studies. Although we have investigated the effects of host speciation and diets on the composition of gut microbiota, there may be other host factors that also play a role in the formation of gut microbiota in bats. We also noticed that more than 50% of gut microbial genes in each sample were annotated as “function unknown.” It has been postulated that these genes may be involved in echolocation, longevity, flying, and immune system (Zhang et al., 2013; Jebb et al., 2020).

In this study, we found a very weak correlation between dietary habits and gut microbiota in bats that feed on insects or both insects and fish. However, the compositions of gut microbiota were found to vary among different species of bats, suggesting that host speciation plays a major role in the formation of gut microbiota. We also found differences in the number of genes related to certain metabolic pathways among bats with different dietary habits, suggesting the effect of diet on physiological functions of bats.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, PRJNA954561.

The animal study was reviewed and approved by the Animal Ethics Committee of Institute of Zoology, Guangdong Academy of Sciences (No. GIABR20200810).

MG and LZ designed the research. MG performed the research. JW, YZ, and XH collected samples and extracted fecal DNA. MG, SX, JQ, PL, CZ, and CH provided analytic tools. MG, SX, JD, and JQ analyzed data. MG and LZ wrote the paper. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the GDAS Special Project of Science and Technology Development (2021GDASYL-20210103052), the Guangdong Provincial Science and Technology Program (2021B1212110003 and 2021B1212050021), and the National Natural Science Foundation of China (32172435).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor DJ declared a shared parent affiliation with some of the authors MG, JW, YZ, XH, PL, JD, CZ, and LZ at the time of review.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.