Lessons from the diet: Captivity and sex shape the gut microbiota in an oviparous lizard (Calotes versicolor)

Abstract Studies have indicated that the abundance and community structure of gut microbiota are altered by diet. In this study, next‐generation sequencing of the 16S rRNA gene amplicon was performed to evaluate variations in the gut microbiota of wild and captive individuals of both sexes of Calotes versicolor. The results showed that there was a significant sex difference in microbial community structure for wild C. versicolor, Bacteroide was the dominant genus in wild females (WF), whereas Ochrobactrum was the dominant genus in wild males (WM). Acinetobacter and Hymenobacter were the dominant genera in WF, while Clostridium was the dominant genus in captive females (CF). The results indicated that differences in diet between wild and captive C. versicolor also resulted in variations in gut microbiota. Thus, it was not surprising that captivity and sex shape the gut microbiota in C. versicolor. In summary, the fundamental information presented about the gut microbiota of both sexes of wild (and captive females) C. versicolor, indicates that the artificial environments are not suitable for the wild C. versicolor.


| INTRODUC TI ON
Gut microbiota plays a critical role in host health and provides fundamental information about host physiology (Hale et al., 2018).
Bacteroidetes, Firmicutes, and Proteobacteria are three of the most important components of the gut microbiota in vertebrate species (Kohl et al., 2017;Ren et al., 2016). Several studies have shown that gut microbiota may play a significant role in vertebrate evolution, as its diversity is correlated with the evolutionary history of these animals. However, vertebrate gut microbial communities are influenced by several other factors, such as host features (age, body size, and sex) and environment (diet and season) (Delsuc et al., 2014;Martin et al., 2010;Zhou, Nelson, et al., 2020). For example, in nonreproductive mice the relative abundance of Lactobacillus spp. was higher in males than in females, whereas the contrary was observed in reproductive mice (Maurice et al., 2015). Female Sceloporus virgatus display significantly lower microbial diversity and richness than males (Martin et al., 2010). Moreover, compared to males, Rhinella marina females display an increased relative abundance of Bacteroides, Comamonas, Flavobacterium, Microvirgula, Parabacteroides, and Pseudomonas species, with a decreased relative abundance of Cetobacterium, Clostridium, Epulopiscium, Plesiomonas, and Vibrio species (Zhou, Nelson, et al., 2020). However, no influences of sex have been observed in the microbial communities of Liolarmus and Phymaturus lizards (Kohl et al., 2017). Thus, the influence of sex on gut microbial communities in wildlife species is complex and largely unknown.
Recent studies have shown that captivity plays an important role in endangered species conservation by maintaining the breeding population, particularly for lizards; however, captivity has been shown to significantly alter the gut microbial community of lizards (Jiang et al., 2017;Kohl et al., 2017;Tang et al., 2020;Zhou, Zhao, et al., 2020), amphibians (Bataille et al., 2016;Tong et al., 2019), and other taxa (Chi et al., 2019;Hale et al., 2018;Martínez-Mota et al., 2020;Oliveira et al., 2020). For instance, a study by Kohl et al. (2017) found that captive species exhibited less Firmicutes and Actinobacteria compared to wild species, and Bacteroidetes was only present in captive species. Furthermore, some studies have reported a significant difference between the alpha diversity of wild and captive animal gut microbiotas (Kohl et al., 2017;Ren et al., 2016), whereas other studies have indicated a general loss of microbial diversity as a result of captivity .
Microbiome characterization and monitoring tools are being developed and are recommended for wild species conservation (Oliveira et al., 2020;Redford et al., 2012), particularly endangered species such as the giant panda (Ailuropoda melanoleuca) (Zhu et al., 2011), the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) (Wan et al., 2016), and the Chinese crocodile lizard (Shinisaurus crocodilurus) (Jiang et al., 2017;Tang et al., 2020). Considering of the sex of individuals in captivity is important of conservation activities, particularly for health breeding programs. However, the important of gut microbiota to conservation efforts for lizards of both sexes in captive and wild environments is largely unknown. Therefore, the main goal of this study was to characterize the gut microbiota of the lizard Calotes versicolor to address the follow- ing Diptera, Coleoptera, Lepidoptera, and Orthoptera (Qiu et al., 2001).
Adult lizards do not display sexually dimorphism in snout-vent length (Qiu et al., 2001), but females have relatively narrow heads compared to males (Shanbhag & Parsad, 1993). Hindgut samples from C. versicolor tend to have more Firmicutes and Bacteroidetes, and less Proteobacteria than those of the small intestine (Zhang et al., 2021).

| Gut microbiota analyses
DNA was extracted from all samples using the cetyltrimethylammonium bromide (CTAB)/sodium dodecyl sulfate (SDS) method.
Universal primers were employed to amplify the V3-V4 regions of the bacterial 16S rRNA genes that contained Illumina sequences at the 5′-end of forward primers harboring 7-12 bp barcodes.

| Statistical analyses
Variations in alpha diversity were analyzed using Tukey's HSD test.
Nonmetric multidimensional scaling (NMDS) based on the Bray-Curtis distance was constructed to determine the variations in beta-diversity.
To obtain the unique genus, features that occurred in ≥75% of the replicates in each group were retained. The linear discriminant analysis (LDA) effect size (LEfSe) method was employed to identify the variations in microbial communities based on LDA sources (Segata et al., 2011). To explore the functional profiles of gut microbiota between F I G U R E 1 Composition of the gut microbiota of each group at the phylum, family and genus levels F I G U R E 2 The non-metric multidimensional scaling (NMDS) of the gut microbiota composition. The variation explanation is indicated on each axis, respectively WM and WF, or between WF and CF, all OTUs were assigned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways by Tax4Fun (Aßhauer et al., 2015). The differences in gene function analyses were identified in STAMP (v2.1.3) (Parks et al., 2014), and Welch's t-test was used for the comparisons between the two groups.
The relationships between various microbial communities were analyzed using Spearman's correlation coefficients. Partial Mantel tests were performed to evaluate relationships between the relative abundance of microbiota and gene functions. All analyses were conducted using the linKET package (Huang, 2021) in R version 4.0.4 (R Core Team, 2021).

| RE SULTS
In all lizards, Firmicutes, Proteobacteria, Bacteroidetes, and Verrucomicrobia, were the four dominant phyla identified (mean relative abundance >1%, Figure 1a). Actinobacteria only was a dominant phylum in WM, but not in WF (Figure 1a). Richness, Chao1, and ACE indices were lower in WF than in WM (all adj p < .01), but Shannon, Simpson, and Pielou's E diversity indices showed no differences (all adj p > .05) between WM and WF ( Figure S1). There were including 172 (for sex) and 169 (for wild vs captivity) KEGG metabolic pathways were selected between WF and WM, and WF and CF, respectively, associated with metabolism (68.60% and 68.05%), genetic information processing (10.47% and 10.65%), cellular processes (3.49% and 5.33%), environmental information processing (4.07% and 3.55%), organismal systems (2.91% and 3.01%), and human diseases (9.30% and 8.28%). There was significant difference in organismal systems between WF and WM (adj p = .038), but no significant differences were detected between WF and CF (adj p > .20). Functional

| DISCUSS ION
Our results revealed that the core microbiota of C. versicolor consisted of Firmicutes, Proteobacteria, Bacteroidetes, and Verrucomicrobia at the phylum level, which is consistent with the results of previous studies (Jiang et al., 2017;Kohl et al., 2017;Tang et al., 2020;Zhou, Zhao, et al., 2020). Furthermore, our results showed that sex does influence gut microbial communities, with WF having significantly lower gut microbial diversity and richness compared to WM.
Previous studies have detected sex-related differences in the gut microbiota of S. virgatus (Martin et al., 2010), and R. marina (Zhou, Nelson, et al., 2020). Sexual dimorphism may be related to differences in the spatial and temporal niches (Butler, 2007), with male lizards having higher a perch than females for defending territories and remaining visible to potential mates (Logan et al., 2021). However, the snout-vent length of C. versicolor is not sexually dimorphism (Qiu et al., 2001). A previous study showed that sex hormones intercept changes in gut microbiota via gonadectomy and testosterone hormone replacement in mice (Org et al., 2016). Hormonal changes and sex differences strongly affect bile acid profiles (Org et al., 2016), which respond to high-fat/high-sugar diets, which in turn affect gut microbiota (Islam et al., 2011;Li & Chiang, 2015). Moreover, the relatively narrow heads of female C. versicolor may be related to the selection of a small-sized diet. The food niche overlap between sexes is 0.522 (Qiu et al., 2001). The main diet of adult females includes Orthoptera (Acrididae, 9.0%), Coleoptera (Chrysomelidae 14.7% and Scarabaeoidae 9.0%), and Diptera (Platypezidae 34.6%), whereas that of adult males includes Orthoptera (Acrididae 11.6%), Coleoptera (Chrysomelidae 24.5%), and Lepidoptera (Nymphalidae 11.1% and Papilionidae 17.6%) (Qiu et al., 2001). In the present study, we found that Bacteroides was the most dominant genus in WF, with Previous studies have shown that captivity influences gut microbiota (Tang et al., 2020;Zhou, Zhao, et al., 2020). Our results showed that captivity was related to a loss of Firmicutes and Proteobacteria, and the introduction of Bacteroidetes and Verrucomicrobia to gut microbiota. Firmicutes play an important role in fiber and cellulose degradation by breaking down cellulose into volatile fatty acids, which can be used by the host. The higher abundance of Firmicutes in wild lizards probably leads to improved digestion and absorption of nutrients. Captive lizards were fed with an artificial fodder composed of Tenebrio molitor and Gryllulus chinensis, which had a relatively high-fat content, but a relatively low fiber content. Moreover, two discriminative features (Acinetobacter and Hymenobacter) were identified in CF, while another (Clostridium) was identified in WF. Clostridium was positively correlated with the serum levels of total cholesterol, low-density lipoprotein cholesterol, and triacylglycerols (Guo et al., 2018). The LEfSe showed that Moraxellaceae, Rhodospirillaceae, Acinetobacter, and Hymenobacter were discriminative features in the WF group. The relationships between bacterial and KEGG pathways indicated that the presence of Moraxellaceae, Rhodospirillaceae, Acinetobacter, and Hymenobacter had a significant effect on the metabolism of cofactors and vitamins, transport and catabolism, pentose phosphate pathway, and epithelial cell signaling in the H. pylori infection. Therefore, the results of this study indicated that a simple diet in captivity directly influences the gut microbial communities of C. versicolor.

| CON CLUS IONS
In conclusion, the bacterial phyla Firmicutes, Proteobacteria, Bacteroidetes, and Verrucomicrobia dominated the core microbiota of F I G U R E 5 Functionally predicted KEGG pathways differing in (a) between wild males and wild females and (b) between wild females and captive females of Calotes versicolor. The bar plot shows mean proportions of differential level 3 of KEGG pathways predicted using Tax4Fun. The difference in proportions between the groups is shown with 95% confidence intervals. Only p value < .05 (Welch's t-test, FDR adjusted) are shown and composition C. versicolor. Our results led to the following conclusions: (1) WF having significantly lower microbial diversity and richness compared to WM; (2) captivity is related to a loss of Firmicutes and Proteobacteria, but also to the introduction of Bacteroidetes and Verrucomicrobia; (3) metabolic functions were differentially determined by the bacterial variations. The types and size of food items in the diet were F I G U R E 6 Relationships between bacterial and KEGG pathway by sex (a) and captive (b). Pairwise comparisons of bacterial were displayed with a color gradient denoting Spearman's correlation coefficient. Bacterial and KEGG community composition was related to each bacterium by Mantel test significantly different for WM and WF, as well as CF and WF. It was not surprising we found that captivity and sex influence the gut microbiota in C. versicolor. The relationship between bacterial and KEGG pathways indicated that the artificial environments used here are not suitable for wild C. versicolor.

ACK N OWLED G M ENTS
The authors wish to thank the members of Hainan Key Laboratory of Herpetological Research for assistance in the field and laboratory, and the anonymous reviewers and the editor of the journal for their valuable comments. The authors would like to thank Editage (www. edita ge.cn) for English language editing.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.