The Effect of Host, Habitat and Fasting Time on the Gut Microbiota of Amphibian

Wild animals entering captivity experience radical lifestyle changes resulting in microbiota alterations, in large part due to differences in diet. However, little is known about how external environmental factors inuences the gut microbiota and the interaction of the environment-host-microbe interactions in host fasting. The gut microbiota in the early stage (amA and dyA groups) and late stage of hibernation in Rana amurensis and R. dybowskii of entering captivity (amL and dyL groups) and wild environments (amS and dyS groups) was determined, and the effects of host, environmental factors and fasting time on the gut microbiota were investigated via high-throughput Illumina sequencing. The Shannon index differed signicantly between the amL and dyL groups and between the amA and amS groups. The PD index differed signicantly between the dyL and dyS groups. Eight core OTUs were widely distributed between species, habitats and fasting times and were dominant in abundance. Captive and wild environments, host species, and fasting time signicantly affected the composition and structure of the gut microbiota. Akaike information criterion (AIC)-based model results suggested that the environment and host were the variables that needed to be included in redundancy analysis (RDA) to explain the variance in taxa. The pairwise distances between the early and late stages of hibernation of were greater in R. amurensis and R. dybowskii entering captivity than in wild. The average of OTUs shared by early and late stages of hibernation of captive frogs was signicantly lower than the average of wild frogs. These results can reveal the impact of environmental changes on the gut microbiota, thereby revealing the important interactions between environment-host-microbes, and helping to protect vertebrate hosts.


Introduction
Gut microbial diversity is important for the nutrition, physiology, and pathology of host organisms Compared with terrestrial amniotic vertebrates, amphibians inhabit more complex ecological environments, and their gut microbiota has greater diversity and complexity (Bletz et al. 2016). At present, the gut microbiota of mammals is a popular topic in microbial ecology (Nelson 2015), but little is known about the gut microbiota of amphibians, the intermediate clade between shes and reptiles (Jiménez & Sommer 2016).
Among the factors that may change the gut microbiota, host genotype has a signi cant effect on its composition (Nap in & Schmid-Hempel 2018). Genetic factors can determine the physiological structure of the host gut, the function of the gut barrier and host immune function, which interact directly with the gut microbiota and participate in the production and stability of the gut microecological balance ( Fasting affects the gastrointestinal (GI) tract and the gut microbiota . Previous studies have shown signi cant differences in the gut microbiota between hibernation fasting frogs and frogs that are eating (Weng, Yang & Wang 2016;Wiebler et al. 2018). Amphibians (such as R. amurensis and R. dybowskii) living in the temperate zone experience changes in the annual cycle of feeding or fasting and hibernation, and they hibernate and fast for more than 6 months (Tattersall & Ultsch 2008). There may be large differences in the immune system in terms of intestinal nutrients, intestinal structure and host in the early and late stages of hibernation fasting, which may affect the gut microbiota. Although some studies have investigated seasonal and fasting effects on the amphibian gut microbiota (Kohl et al. 2014), the difference between the effects of long-term fasting and short-term fasting is unclear.
In this study, the gut microbiota in the early and late stages of hibernation in two frog species in two different hibernation habitats (a natural hibernation pool and a captive hibernation pool) was investigated, and the effects of host, environmental factors and hibernation fasting time on the gut microbiota were investigated. We hypothesized that (1) Wild animals entering captivity experience lifestyle changes resulting in microbiome alterations; (2) there would be signi cant differences in the gut microbiota diversity between short-term fasting and long-term fasting; and (3) there were signi cant differences between the gut microbiota of the two species in the same habitat, i.e., the host had a signi cant effect on the gut microbiota.

Sample collection
There were two main experimental groups. The gut microbiota in the early and late stages of hibernation (amA, dyA, amS, and dyS groups) in R. amurensis and R. dybowskii in captive and wild environments (amL, dyL, amS, and dyS groups) were obtained and studied, and the effects of host, environmental factors and hibernation fasting on the gut microbiota were investigated (Table 1). All frog samples were brought to a laboratory at Northeast Agricultural University for immediate pro ling of the intestinal microbial composition. Gut content samples were collected from brown frog intestinal contents within 20 min after euthanasia. A mixture of ether and alcohol was used to anaesthetize the frogs, after which cervical dislocation was performed (Tong et al. 2020). Then, the digestive tract was carefully isolated from the body, and lower GI tract contents were collected. To avoid crosscontamination, each sample was collected using a fresh pair of sterile tweezers. The contents of each intestine were emptied into a sterile vial and immediately stored at -80 °C.

Ecological and statistical analysis
The paired-end sequences were merged into a single 434-bp-long sequence using FLASH ( Alpha diversity (phylogenetic diversity (PD), Shannon, and Sobs indices) was analysed using mothur (Hadizadeh et al. 2017). Relative abundance differences between groups of bacterial taxa were compared using the Wilcoxon rank sum test. Only differences with corrected P values < 0.05 are presented. Ordination plots were constructed with Bray-Curtis distances and unweighted UniFrac distances using the R vegan package. These matrices were further analysed in the R vegan package with nonmetric multidimensional scaling (NMDS) and permutational multivariate analysis of variance (PERMANOVA) (Mcardle & Anderson 2001) to statistically analyse the comparisons outlined above. Furthermore, host species, habitat, and fasting time effects on the gut microbiota were evaluated using two-way PERMANOVA (Zhang & Li 2018).
A member of the core gut microbiota of the frogs was assigned if it was found in 90% of the groups and represented > 0.1% of the reads. Differential microbial taxa between the two frog species in two different hibernating habitats and fasting times were identi ed using the linear discriminant analysis (LDA) effect size (LEfSe) technique (Zhong, Yan & Shangguan 2015), which considers both statistical signi cance and biological relevance. Differences between populations were analysed using one-way analysis of variance (ANOVA) at a signi cance level of P < 0.05.
The Venn analysis method performs pairwise analysis on the shared OTUs of each sample in the early hibernation (amA and dyA) and each sample in the late hibernation (amL, dyL, amS and dyS groups). The chi-square test was used to assess differences in the proportions of shared OTUs and total OTUs of the groups. P < 0.05 was considered as a signi cant difference. As a measure of similarities between the gut and skin bacterial communities over time, the pair-wise distance of Bray-Curtis dissimilarities and weighted UniFrac distances of the gut microbiota between the early and late stages of hibernation of captive and wild were compared via the Wilcoxon rank-sum test. Only differences with p values of < 0.05 are presented.
Since the longest gradient of all axes determined by detrended correspondence analysis (DCA) was < 4, redundancy analysis (RDA) was used to link the changes in the microbial community with environmental variables (Janczyk et al. 2010). All variables were standardized by the "scale" function before analysis.
Since there may be multicollinearity among the three impact factors, the variance in ation factor (VIF < 3) for each variable was estimated using the R package. Akaike's information criterion (AIC) was applied to select useful impact factors in an AIC-based model for vector tting. Permutation tests with marginal effects and 1,000 permutations were applied to estimate the signi cance of the AIC-based model and the VIF-based model. The smaller the AIC value was, the better the model t. All of the analyses were performed using R software (version 3.5.3), and the vegan package was used for RDA and nonparametric multivariate ANOVA.

Overview of alpha diversity and the core microbiota
Rana amurensis and R. dybowskii were sampled for sequencing, resulting in 2,496,447 high-quality sequences with a mean of 43,042 sequences per sample. A total of 2,607 OTUs were obtained with an average length of 442.74 bp per read. The average number of OTUs per sample was 325.78, with a range of 72 (sample dyL02) to 1,140 (sample dyS02). Rarefaction analysis showed that the sequenced samples mostly reached the plateau phase, particularly the amL and amA samples (Fig. S1).
There were no signi cant differences in the Sob index between groups (Wilcoxon rank sum test, P > 0.05, Fig. 1a). The Shannon index differed signi cantly between the amL and dyL groups and between the amA and amS groups (Wilcoxon rank sum test, P < 0.05; Fig. 1b). The PD index differed signi cantly between the dyL and dyS groups (Wilcoxon rank sum test, P < 0.05) (Fig. 1c).
With respect to the core microbiota of all frogs, there were 8 core OTUs (found in > 90% of all frogs) ( Fig. 1d and 2). Twenty and 7 species-speci c core OTUs were identi ed in R. amurensis and R. dybowskii, respectively (Figs. 2). Most notable were Arthrobacter, Chryseobacterium, Erysipelatoclostridium, Hafnia-Obesumbacterium and Pseudomonas, as they were widely distributed between species, habitats and fasting times and were dominant in abundance ( Fig. 1d and 2).

Effects of captive and wild environments and host species on the gut microbiota
The effects of host and habitat on the gut microbiota were assessed using two frog species that hibernated in different habitats, and the results showed that the overall composition and structure of the gut microbiota were strongly associated with the effects of host species (two-way PERMANOVA: Bray-Curtis, P = 0.001; unweighted UniFrac, P = 0.001) and habitat (two-way PERMANOVA: Bray-Curtis, P = 0.003; unweighted UniFrac, P = 0.003), yet no signi cant effects were detected for their interactions (twoway PERMANOVA: Bray-Curtis, P = 0.079; unweighted UniFrac, P = 0.130) ( Table 2; Fig. 3a and b). The gut microbiota differed between host species. The composition and structure of the gut microbiota of the same host (R. amurensis and R. dybowskii) differed signi cantly between the captive and wild environments (R. amurensis, Adonis: Bray-Curtis, R 2 = 0.091, P = 0.022; unweighted UniFrac, R 2 = 0.088, P = 0.037; R. dybowskii, Adonis: Bray-Curtis, R 2 = 0.094, P = 0.014; unweighted UniFrac, R 2 = 0.109, P = 0.015) ( Fig. 3a and b; Table S1).
The LEfSe results revealed signi cant differences in the gut bacterial taxa, mainly in the phyla Actinobacteria (dyL group), Bacteroidetes (amS group) and Spirochaetes (amL group) (LDA > 4, p < 0.05; Fig. 5a). The LEfSe results at the genus level indicated that 5 of the 745 genera present in the dataset were differentially abundant in the two species between different habitats. Flavobacterium, unclassi ed_f__Comamonadaceae and unclassi ed_f__Flavobacteriaceae were most abundant in the dyL group, Acinetobacter was most abundant in the amS group, and Deefgea was most abundant in the dyS group (LDA > 4, P < 0.05; Fig. S4).

The gut microbiota of frog entering captivity environments occurred more signi cant changes
The gut microbiota differed with fasting time in wild environments. In R. amurensis, the composition and structure of the gut microbiota differed signi cantly between the early and late stages of hibernation (Adonis: Bray-Curtis, R 2 = 0.179, P = 0.001; unweighted UniFrac, R 2 = 0.145, P = 0.014; Fig. 3a and b). In R. dybowskii, the composition of the gut microbiota differed signi cantly between the early and late stages of hibernation according to Bray-Curtis distances (Adonis: Bray-Curtis, R 2 = 0.101, P = 0.032; Fig. 3a) but not unweighted UniFrac distances (Adonis: unweighted UniFrac, R 2 = 0.090, P = 0.084; Fig. 3b).
The Bray-Curtis dissimilarities and unweighted UniFrac distances between the early and late stages of hibernation were greater in captive environments than in wild environments ( Fig. 3a and b). Based on Bray-Curtis and unweighted UniFrac distance and comparison of the pairwise distance of the gut microbiota between the early and late stages of hibernation of captive and wild, the pairwise distances of the early and late stages of hibernation between captive and wild were signi cant (Wilcoxon rank-sum test, P < 0.05; Fig. 5a).
We used Venn analysis to pairwise analysed the shared OTUs in each sample in the early of hibernation (amA and dyA) and each sample in the late stages of hibernation (amL, dyL, amS, and dyS groups). The average of the shared OTUs in amA and amL groups was 78.64, and that of the shared OTUs in amA and amS groups was 116.90. There were signi cant differences between the two groups in the average of the shared OTUs (ANOVA, F = 119.425, df = 1, P < 0.01; Fig. 5b). The average of the shared OTUs in dyA and dyL groups was 90. 16, and that of the shared OTUs in dyA and dyS groups was 115.10. There were signi cant differences between the two groups in the average of the shared OTUs (ANOVA, F = 10.818, df = 1, P < 0.01; Fig. 5b).
3.4 The best variables for predicting microbiota structure as selected by the RDA model Constrained ordination (RDA) was used to explore the potential relationships between microbial communities and the environment, fasting time, and host species. Three impact factors with VIF values below 3 were included in the RDA model, including host (VIF: 1.001), fasting (VIF: 2.5385), and habitat (VIF: 2.386). The variation partitioning analysis (VPA) results showed that 5.98% of the variance in gut microbiota structure could be explained by the host (3.94%), fasting (1.43%), and habitat (1.33%) (Fig. 6a). However, the AIC-based model results suggested that host and habitat were the variables that needed to be included in the RDA to explain the variance in taxa (AIC: -25.778, P < 0.05; Fig. 6b).

Captive and wild environments
In the present study, the intestinal microbiota of fasting amphibians entering captivity environments occurred more signi cant changes. Vertebrates may selectively lter particular microbial members from the exogenous species pool to function as gut residents. However The present study selected animals during the fasting period, which better re ected the effect of the host's environment (captive or natural habitat) on the gut microbiota.
In this study, the density of frogs was 30/m 3 , while in natural habitats, the density of frogs is much lower than that in cultured habitats (She & Liu 2009). During high-density farming, farmed animal excreta can lead to water quality deterioration (Tovar et al. 2000). For example, studies have found that the pH of cultured water decreases with an increase in culture density and time, and the amounts of ammonia, nitrite and nitrate nitrogen increase. The concentrations of ammonia, nitrite and nitrate nitrogen in highdensity cultures are also higher than those in low-density cultures. When the aquatic environment changes, the intestinal microenvironment of aquatic animals changes (Rudi et al. 2018). This shows that a high culture density affects animal growth performance and immunity but also signi cantly changes culture water quality, and water quality changes may also affect the intestinal microbiota of cultured aquatic animals.
Space is not a single factor, and its internal complexity (shelter or cover provision) constitutes a spatial element, such that excessive exposure can increase the rigid, tense and fearful behaviours of animals (Michaels, Antwis & Preziosi 2014). Environmental enrichment can limit ghting and cannibalism and can optimize the general health, fecundity, and welfare of captive amphibians (Chum et al. 2013). Both species studied here hibernate mostly in the soil, under rocks, and around roots at the bottom of ponds (She & Liu 2009). In this study, no hiding places were set up for the hibernating frogs, R. dybowskii individuals did not gather together, and both species hibernated in dimly lit areas. Both species are sensitive to exotic stimuli in the summer, and during hibernation, low temperatures make them unresponsive to outside stimuli (Vo & Gridi-Papp 2017). Therefore, due to low temperatures, the effects of space on the physiology, behaviour and intestinal ora of frogs may be limited.

Amphibian host species
In this study, we selected two frog species with a close genetic relationship that were distributed in the same region to clearly study differences in the gut microbiota of two closely related species. The effect of food on gut microbiota is extremely signi cant (Jiménez & Sommer 2016;Rowland et al. 2018). In this study, the two frog species were hibernating and fasting, so the effect of food on the gut microbiota was excluded. In addition, the presence of the same conditions, i.e., low temperature, similar water qualities, the same nutritional status among hosts and similar physiological mechanisms of hibernation, minimized the effects of other factors, such as food, temperature, and physiology (Tong et al. 2019).
Therefore, our study isolated the effects of the endogenous intestinal environment determined by the genetic differences and immune characteristics of host species.
Our study shows that the main inhabitants of the two frog species are Proteobacteria, Bacteroidetes, and Firmicutes, and these frogs have the same core gut microbiota compositions, which indicates that the intestinal habitats of closely related species are highly similar. This is consistent with results from previous studies (Weng, Yang & Wang 2016;Wiebler et al. 2018). In addition, similarities in the gut microbiota often re ect the phylogeny of the hosts, indicating the coevolution of each host and their gut microbiota (Hale et al. 2018;Muletz Wolz et al. 2018). However, even for these hosts with similar genetic relationships, there were signi cant differences in their gut microbial diversity in the same hibernation habitat. The beta diversity of the gut microbiota of the two frog species indicated that their microbial communities were signi cantly different, and the proportion of the core micro ora present in R. dybowskii (7/2,211) was smaller than that in R. amurensis (20/1,752 (Ley et al. 2008), resulting in "directed selection" of a speci c gut microbiota by the host. Low-abundance species will be at a competitive disadvantage or even eliminated. Host selection of microbial groups can be achieved through a variety of mechanisms that, because they show different behaviours or requirements, are not necessarily "active" (Yan et al. 2016). However, when the micro ora is bene cial to the host, a change in the host-selected microbiota is particularly interesting, usually because the host can coordinate the assembly of the micro ora, thereby maximizing its bene ts (Smith et al. 2015). From the point of view of community ecology, the composition of the gut microbiota is not a random combination, and some microbial species have relationships with some hosts, indicating that the host may be "actively" involved in the community assembly process by selecting from all available microbial species libraries ( ltering) (Smith et al. 2015).
In other words, members of microbial communities are recruited together rather than being randomly

Hibernation fasting
This study investigated the gut microbiota of two frog species at different fasting times and the effects of fasting time and host on the gut microbiota, and the results of two-way PERMANOVA showed that hibernation fasting time had a signi cant effect on the composition and structure of the gut microbiota. The microbiota in the late stage of hibernation had more time to become permanently established in the gut because frogs in the early and late stages of hibernation were sampled 20 and 180 days after they started to fast, respectively. Previous studies have also shown that hibernation is associated with changes in the diversity of the gut microbiota; however, in those studies, the gut microbiotas of frogs that were hibernation fasting and frogs that were eating and not hibernating were compared (Weng,  There were signi cant differences in the gut microbiota between the early and late stages of hibernation, which may also be related to host nutrition, the structure and function of the digestive organs and immunosuppression in addition to fasting time. Hibernating frogs must acquire adequate energy in the

Conclusions
In summary, wild frogs entering captivity experience radical lifestyle changes resulting in microbiome alterations. Eight core OTUs were widely distributed between species, habitats and fasting times and were dominant in abundance. Captive and wild environments, host species, and fasting time signi cantly affected the composition and structure of the gut microbiota. The host and habitat factors were the best variables for predicting microbiome composition. Studies of the gut microbiota of frogs that hibernate in two habitats can shed light on the effects of environmental changes on the gut microbiota, which may reveal important environment-host-microbe interactions and help inform the conservation of vertebrate hosts.