Discussion
The strict nocturnal activity rhythm of T. roborowskii (Song et al., 2009) and their special burrow construction not only avoid high temperatures in the daytime but also create a relatively constant living temperature. These behaviors are effective mechanisms for adapting to harsh and hot desert environments (Song et al., 2017). It is clear that seasonal dietary shifts are also a way to cope with the drought environment and enrich the food composition of T. roborowskii , which may be the main reason for the significantly higher gut microbial abundance and diversity ofT. roborowskii in autumn relative to the spring. Studies on insectivorous and carnivorous bats have shown that diet variation significantly affects the gut microbial community structure (Gong et al., 2021). Therefore, improvement of gut microbial diversity and changes in community structure in T. roborowskii may be the main factors in coping with seasonal dietary shifts. Kohl et al. (2016) fed Liolaemus ruibali a diet ratio of 50% insects + 50% plants or 10% insects + 90% plants and found that lizards fed a rich, plant-based diet showed a higher level of gut microbial diversity. This is consistent with our results; that is, the intake of caper fruit significantly increased the diversity of gut microbes, and this change may be beneficial to the achievement of gut function and the stabilization of microbiota inT. roborowskii (Soriano et al., 2018).
At the phylum level, the contents of Firmicutes, Bacteroidetes, Proteobacteria, and Verrucobacterium in the gut microbes ofT. roborowskii in spring and autumn accounted for more than 91% of the core flora. Studies have shown that Firmicutes, Bacteroidetes, and Proteobacteria are important members of the gut microbiota in most vertebrates (Zhao et al., 2018; Hale et al., 2019; Wang et al., 2018; Kohl et al., 2013). As a nocturnal desert gecko, the leopard gecko (Euplepharis macularus ) possesses a gut microbial composition that is highly similar to that of T. roborowskii and remains stable after 28 d of fasting, suggesting that animals living in extreme desert environments have a high degree of control over gut microbes (Kohl et al., 2014). Other lizard species, such as a toad-headed lizard (Phrynocephalus vlangalii ) (Zhang et al., 2018), S. crocodilurus (Jiang et al., 2017), T. septentrinalis (Zhou et al., 2020), and S. occidentalis (Moeller et al., 2020), are also dominated by Firmicutes and Bacteroidetes, which indicates that different lizard taxa have similar gut microbial composition at higher taxonomic levels.
However, the gut microbial composition ofT. roborowskii is unique at both the family and genus levels. S. crocodilurus lives in warm, moist habitats and feeds mainly on earthworms and loaches. The main bacterial families are Pasteurellaceae, Deinococcaceae, and Comamonadaceae (Jiang et al., 2017). There were significant differences compared with T. roborowskii . A study of T. septentrionali s from eastern China showed that this insect-eating lizard has a composition similar to that of gut microbes of T. roborowskii at the family level, but the abundance differs greatly among families (Zhou et al., 2020). P. vlangalii living at high altitudes appears to have a more similar gut microbial composition to that of T. roborowskii (Zhang et al., 2018), which may be caused by similar food sources and arid environments. It can be seen that some factors such as diet (Campos et al., 2018) and environmental factors (Kartzinel et al., 2019) can significantly affect the composition of lizards’ gut microbes.
Individuals with a high ratio of Firmicutes to Bacteroidetes have a higher ability to obtain energy from food (Clarke et al., 2012). Our results showed that T. roborowskii has more than 65% abundance of Firmicutes and Bacteroidetes in different seasons. Firmicutes are responsible for the metabolic transformation of carbohydrates and proteins (Flint et al., 2015), and can produce large amounts of energy-rich short-chain fatty acids that are associated with digestive efficiency (Turnbaugh et al., 2009). Bacteroidetes have the capacity to ferment amino acids and carbohydrates and are involved in bile acid, polysaccharide, and steroid metabolism (Rios-Covian et al., 2017). They work together to promote fat accumulation (Turnbaugh et al., 2006). The relative stability of the core flora may be favorable for maintaining a high level of nutrient absorption capacity during the active season. On one hand, this meets the direct cost of summer breeding input (Speakman, 2008), and on the other hand, it ensures energy accumulation before hibernation.
The special frugivory strategy chosen by T. roborowskii in the long-term evolutionary process promotes seasonal changes in its gut microbes. For example, the abundance of Proteobacteria in the autumn group was significantly higher than in the spring group (P <0.05), which seems to be an effective way to cope with this shift. Relevant studies have shown that Proteobacteria are related to a variety of metabolic pathways and can secrete a large number of enzymatic substances related to polysaccharide and protein metabolism, which can effectively decompose polysaccharide and vitamin production (Abdul Rahman et al., 2016; Colston & Jackson, 2016), thus satisfying the combination of Capparis spinosa fruit and insect food and providing the possibility for the accumulation of energy materials before hibernation. The relative abundances of Enterobacteriaceae and Pseudomonas in the autumn group were significantly higher than those in the spring group, which may also be the result of adaptation to fruit food with higher sugar content. Related studies have shown that some Enterobacteriaceae species possess sucrose-specific phosphotransferase systems and sugar transporters with different functions and structures (Le Bouguénec & Schouler, 2011). Carbohydrate metabolism is also widely recognized as a nutritional basis for γ-proteobacteria to colonize the gut and maintain strains (Chang et al., 2004).
Lachnospira and Ruminococcaceae were the most abundant Firmicutes species in the T. roborowskii gut. Most studies have confirmed that both have abundant genes related to polysaccharide degradation and that their ability to utilize dietary polysaccharides is effective (Vacca et al., 2020). The high abundance of ABC transporters is the basis for the utilization of complex plant materials and the transport of various degradation products (Biddle et al., 2013), which can use lactic acid and acetate to produce short-chain fatty acids such as butyrate through the butyryl-CoA: acetate CoA-transferase pathway (Flint et al., 2015). The significant increase in Lachnospiriaceae (Roseburia) in autumn may be an adaptation to the abundant heteropolysaccharides in caper fruits (Bai et al., 2007), and may improve the efficiency of nutrient metabolism.
Akkermansiaceae are widely present in the gut of hibernating animals (Tang et al., 2019), and are considered probiotics with functions such as promoting intestinal mucosal barrier repair and regulating intestinal flora metabolism (Belzer & De Vos, 2012). The relative abundance ofAkkermansia muciniphila was significantly higher in the spring group than in the autumn group. We speculate that the period whenT. roborowskii was collected was still in the transitional period of enterotype transition; therefore, these individuals still had a higher abundance of Verrucobacterium. Akkermansiaceae can produce short-chain fatty acids such as acetate and propionate by degrading intestinal mucin (Feng et al., 2018). The production of fatty acids can maintain the immune state of the intestine and create an anaerobic environment required for the growth of strictly anaerobic symbiotic microorganisms, thus establishing a mutually beneficial relationship with the host (Shealy et al., 2021). Most Enterobacteriaceae and Pseudomonas are facultative anaerobes, and the destruction of the anaerobic environment leads to their rapid proliferation (Rivera-Chávez et al., 2016). Therefore, the high abundance of Verrucomicrobia in spring limited Proteobacteria; however, the abundance of Verrucomicrobia decreased in autumn, and some aerobic Proteobacteria dominated.
Analysis based on PCA and PLS-DA showed significant differences in fecal metabolites among different seasons, which may be caused by differences in seasonal diets (Li et al., 2020). Plant-derived chemicals such as lauric acid and cinnamic acid were significantly enriched in the autumn group because T. roborowskii had limited ability to digest caper seeds (Yang et al., 2021), and some plant-derived components were retained.
Deoxycholic acid glycine conjugate and 3-oxocholic acid are secondary bile acids. Bile acids participate in cholesterol metabolism through the enterohepatic circulation. This plays a crucial role in the digestion and absorption of components, regulates carbohydrate metabolism, and effectively regulates metabolic homeostasis (Bao et al., 2016).
From the differential metabolite KEGG enrichment network map, it can be seen that the biosynthetic pathways of pantothenate and CoA are significantly associated with a variety of metabolites. Pantothenate and D-4’-phosphopantothenate are precursors of pantothenate and CoA. CoA is involved in various biochemical reactions, including the tricarboxylic acid cycle, fatty acid synthesis and oxidation, and amino acid metabolism (Choudhary et al., 2014; Ma et al., 2020). The high CoA biosynthesis in autumn suggests that T. roborowskii has a vigorous metabolic level, which is consistent with our speculation. L-piperic acid, 5-aminopentanoicacid, and D-1-piperideine-2-carboxylic acid were higher in the spring group, demonstrating that lysine was degraded through the pipecolic acid pathway. Related studies have shown that ʟ-lysine can improve the absorption and utilization of food proteins (Hallen et al., 2013). This appears to explain the adjustment of the Turpan wonder geckos in spring to cope with a protein-rich, insect-based diet.
Correlation analysis between metabolite clusters and gut microbes revealed an interesting phenomenon: Verrucomicrobia and Tenericutes were more abundant in spring, whereas Proteobacteria and Elusimicrobia were more abundant in autumn. These have the same correlation with certain metabolite clusters, which means that the gut microbiota in different seasons performs specific metabolic functions, so the gut microbiota ofT. roborowskii shows obvious seasonal patterns with changes in the seasonal diet.