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.