Habitat environmental factors and gender inuence intestinal microbial diversity of the short-faced moles (Scaptochirus moschata)

The short-faced moles (Scaptochirus moschata) are unique Chinese mammal that live in burrows for life. They have complex ecological adaptation mechanisms to adapt to perennial underground life. Intestinal microbes play an important role in the ecological adaptation of wild animals. The gut microbiota diversity and its function in short-faced moles’ ecological adaptation is a scientic issue worth exploring. In this study, the Illumina HiSeq sequencing platform was used to sequence the V3-V4 hypervariable regions of the 16S rRNA genes of 22 short-faced moles’ intestinal samples to study the composition and functional structure of their intestinal microbiota. The results showed that in the short-faced moles’ intestine, there are four main phyla, Firmicutes, Proteobacteria, Actinobacteria and Bacteroidete. At the family level, Peptostreptococcaceae and Enterobacteriaceae have the highest abundance. At the genus level, Romboutsia is the genus with the highest microbial abundance. According to the KEGG database, the main functions of short-faced mole gut microbes are metabolism, genetic information processing, environmental information processing, and cellular processes. The function of short-faced mole intestinal microbiota is suitable for its long-term burrowing life. No gender difference is found in the composition and function of the short-faced mole intestinal microbiota. There are signicant differences in the composition and functional structure of the short-faced mole gut microbiota between samples collected from different habitats. We conferred that this is related to the different environment factors in which they live, especially to the edaphic factors.


Introduction
Short-faced mole (Scaptochirus moschata) belongs to the Mogera of the Talpidae, is a small mammal that lives in burrows for life, feeds on plant rhizomes and soil insects such as grubs, etc. It has developed morphological and physiological characteristics adapted to burrowing, such as eye degeneration, foreclaw valgus, smell and hearing sensitivity, etc. Short-faced mole is Chinese endemic species, mainly distributed in Henan, Shandong, Inner Mongolia, Ningxia, Shanxi and other provinces in China. Several studies on Talpidae have focused on the phylogenetic and morphological evolution, while the ecological adaptability of Talpidae is less studied.
Intestinal microbes are important symbionts in mammalian gut and occupy important niche (Sherwood et al. 2013; Saxena et al. 2015). They play an important role in maintaining intestinal health and immune function, including in the proliferation of intestinal cells, defense against pathogens, metabolism of secondary products, and digestion of complex compounds (Flint et al. 2012). Intestinal microbes can also affect animal nutrition metabolism and intestinal immunity through intestinal-brain axis ( Therefore, this study intends to study the gut microbial diversity of the short-faced moles, and to investigate the differences of intestinal microbes in moles with different sex and distribution.
Furthermore, through the prediction of microbiome function, we also try to explore the function of gut microbes and their possible contribution in the adaptation to underground life of the short-faced moles.
This study will provide data for the understanding of the physiological and ecological mechanism of short-faced moles to adapt to burrowing life, and provide data for the ecological studies of small burrowing mammals.
1 Materials and Methods

Samples collection
In this study, 22 short-faced moles (SM) were collected using non-damagingly self-made mouse traps in September 2020, including 12 males (named M) and 10 females (named F). The sampling locations were Guanxian (115°E, 36°N; named G; 11) and Huimin (117°E, 37°N; named H; 11). The short-faced moles were captured and placed in the soil of the original habitat, and transported to the laboratory alive.
Subsequently, the gasi ed diethyl ether was put into the transparent sealing device, and then the collected samples were put into the device, and the diethyl ether was continued. During this process, the state of short-faced moles were kept observing. After euthanasia, short-faced moles were dissected and the colon contents were collected aseptically. The collected colon contents samples were stored at -80℃ in the laboratory until DNA extraction. This study was approved by the Medical Ethics Committee of Qufu Normal University (No. 2020018), and during the approval period, the short-faced moles was captured and dissected as required.

DNA extraction, PCR ampli cation and sequencing
We used QIAamp DNA Stool Genomic DNA Extraction Kit (Qiagen, Germany) to extract DNA from intestinal samples according to the instructions. The purity and concentration of the DNA were detected by 1% agarose gel electrophoresis. We used quali ed DNA samples for PCR ampli cation, then diluted the sample to 1 ng/µl using sterile water. PCR ampli cation of the V3-V4 region of 16S rRNA gene was performed with the primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT). The PCR reaction steps were as follows: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 30s (35 cycles); and nally extension at 72°C for 5min.
The concentration of PCR ampli ed products was detected by 2% agarose gel electrophoresis. Then the gel extraction kit (Qiagen, Germany) was used to purify the PCR products. The TruSeq® DNA PCR-Free Sample Preparation Kit was used to construct the library. The constructed library was quantitatively tested by Qubit. After the library was quali ed, the Illumina HiSeq sequencing platform was used for sequencing.

Sequencing data preprocessing
The Raw Tags obtained by sequencing were ltered strictly by quality control to obtain high-quality clean tags. After detect and remove chimeric sequences, the nal effective tags were obtained for further analysis.

OTUs classi cation and species abundance analysis
In order to study the species composition of the gut microbes in each sample, the effective tags contained from 22 samples were clustered to OTUs (Operational Taxonomic Units) with 97% identity.
Then the OTUs were compared to SILVA's SSUrRNA database for species annotation (set threshold of 0.8) by using Mothur software (Version 1.35.1). R software (v2.15.3) was used to generate Rarefaction curve and Rank Abundance curve. The end of the Rarefaction curve is gradually attened, which proves the rationality of the sequencing amount, and the results obtained by sequencing can effectively re ect the abundance of microorganisms contained from the sample. We analyzed the OTUs distribution to study the species abundance of the gut microbes of each sample, and constructed a histogram of the relative abundance of OTUs at each classi cation levels (phylum, class, order, family, and genus).

Diversity analysis and signi cant test
In order to analyze the diversity of the microbial community intragroup, we performed alpha diversity analysis by using Mothur software (Version 1.35.1) to re ect the abundance and diversity of the microbial community of each sample, including the observed-species (OTUs) abundance, chao1, Shannon, Simpson, and ACE indices (Li et al. 2013). Wilcoxon rank sum test was performed by using R software to evaluate the difference among the alpha diversity indices of different groups. R software was also used for beta diversity analysis based on Unweighted & Weighted Unifrac distances to compare the We used R vegan software package to perform Bray-Curtis distance-based Anosim (Chapman et al. 1999), MRPP (O Reilly et al. 1980) and ADONIS (Stat et al. 2013) analysis to assess whether the difference between groups was signi cantly higher than the difference within groups. Mothur software was used for AMOVA (Roewer et al. 1996) based on weighted and unweighted Unifrac distance to compare microbial diversity between different groups. LEfSe (Segata et al. 2011 ) was used to analyze the difference of abundance on each classi cation level (phyla, class, order, family, and genus) between groups, and to nd the biomarkers that have a signi cant contribution to the difference between groups. Unweighted Pair-group Method with Arithmetic Means (UPGMA) trees based on Unweighted & Weighted Unifrac distance were constructed to assess the similarity of the intestinal microbial diversity between different samples.

Function prediction of short-faced moles intestinal microbiota
We perform Tax4Fun function prediction by the Neighbor-Joining test based on minimum 16S rRNA gene sequence similarity. We extracted the 16S rRNA gene sequence of the prokaryotic whole genome from the KEGG database and used the BLASTN algorithm to compare it to the SILVA SSU Ref NR database (BLAST bitscore > 1500) to establish a correlation matrix. The KEGG database prokaryotic genome function information annotated by UProC and PAUDA methods was corresponding to the SILVA database to realize the function annotation of the SILVA database. Sequencing samples were clustered with the SILVA database sequence as the reference sequence OTUs, thus the functional annotation information were obtained (Aßhauer et al. 2015).

Sequencing data processing and OTU analysis
A total of 2085117 raw PE reads are obtained from 22 short-faced moles intestinal samples. After quality control and chimera ltering, a total of 1367088 effective tags can be used for subsequent analysis with an average length of 411.91bp. By clustered with 97% consistency, an average of 283 OTUs is obtained for each sample. According to the ower gure, it can be seen that there are only 17 OTUs shared by 22 samples, and the speci city between samples is relatively large (Fig. 1). According to the rarefaction curve and rank abundance curve, the sample size of this experiment is su cient and can be used for subsequent analysis. Furthermore, the rarefaction curve ( Fig. S1a) indicates that the intestinal microbial species abundance of SMGF06 is signi cantly higher than other samples. The rank abundance curve ( Fig. S1b) also shows that the species abundance of SMGF06 samples is higher than that of other samples, but there is no obvious difference in species evenness.

Gut microbiota composition of short-faced moles
Except for unclassi ed microbial sequences and a little percentage of archaea, the short-faced moles intestinal microbiota include 46 bacterial phyla, 60 classes, 138 orders, 270 families and 688 genera.

Comparison of intestinal microbial diversity between groups
In order to test the similarity of microbial diversity in different samples from the same sampling location, we performed Anosim analysis based on the Bray-Curtis distance. The result showed that SMH vs SMG R = 0.06965, P = 0.06, which proved that the difference between groups was greater than the difference within groups (Fig. S4). The Anosim analysis of the groups SMF and SMM showed that R = 0.0009, P = 0.451, proving that the difference between groups is also greater than the difference within the group (Fig.  S5). MRPP analysis and AMOVA analysis were also performed to further verify this result. The MRPP results showed that SMG vs SMH A = 0.01714, P = 0.036, and SMF vs SMM A = 0.0004643, P = 0.405. The AMOVA analysis also showed that the difference both between the location groups and the gender groups was greater than that within the group, but the difference was all not signi cant (SMG vs SMH: weighted Unifrac AMOVA analysis, P = 0.396; unweighted, P = 0.118. SMF vs SMM: weighted Unifrac AMOVA analysis, P = 0.306; unweighted, P = 0.31).
Then we performed wilcoxon test both on the alpha diversity indices of the groups SMH and SMG, and on the alpha diversity indices of groups SMM and SMF, the results showed that there was no signi cant difference in alpha diversity indices between the groups SMH and the SMG (Table 1), and between the groups SMH and SMG (Table 2). Based on the weighted and unweighted Unifrac distance, we conducted a beta diversity test between different sampling locations. Beta analysis based on weighted Unifrac distance showed no signi cant difference between the groups SMG and SMH (P = 0.51), while beta diversity test based on the unweighted Unifrac distance showed extremely signi cant differences (P = 0.0033) (Fig. 3). Similarly, we also conducted a beta diversity test on the gut microbiota of different genders. The results showed that there was no signi cant difference between the groups SMM and SMF (weighted Unifrac beta analysis, P = 0.7346; unweighted Unifrac beta analysis, P = 0.4176).
In order to further nd out the differences between groups, we conducted wilcoxon tests between the groups on OTU abundance at each classi cation level (phylum, class, order, family, genus). Then LEfSe analysis was performed to nd the biomarkers that contribute most to the signi cant differences between the two groups. There are a total of 6 phyla,  (Fig. 4a). The biomarker between group SMM and group SMF are phylum Bacteroidetes; class Bacteroidia; family Ruminococcaceae (SMF 1.04%; SMM 6.09%) (SMF < SMM); order Enterobacteriales, family Enterobacteriaceae (SMF 19.50%; SMM 12.40%); genus Unidenti ed-Enterobacteriaceae and Escherichia-coli (SMF > SMM) (Fig. 4b).
In addition, by UPGMA clustering, we analyzed the similarity between different samples based on microbial diversity to determine whether different samples have aggregation associated with sampling site or sex. The results show that there is neither sampling-related aggregation nor gender-related aggregation (Fig. 5).

Prediction of intestinal microbial function
According There is signi cant difference in the function abundance of carbohydrate metabolism and enzyme families between groups SMG and SMH by Wilcoxon test (P < 0.05). The second most important function is the genetic information processing (~ 21.38%), including translation (~ 9.59%), replication and repair (~ 8.44%), etc. There is signi cant difference in the function abundance of translation between the groups SMG and SMH (P < 0.01). The other important function is environmental information processing (~ 14.38%), including membrane transport (~ 10.90%), signal transduction (~ 3.35%), etc. The function of intestinal microbes of short-faced moles is also associated with several diseases, such as infectious diseases, drug resistance and cancer, etc. Groups SMG and SMH have signi cant differences in the function process of cancer (P < 0.05) (Fig. 6). At KEGG pathway level 3, the function of short-faced moles intestinal microbiota is mainly annotated to transporters, two component system, DNA repair and recombination proteins, purine metabolism, transfer RNA biogenesis, ABC transporters, pyrimidine metabolism, amino acid related enzymes, quorum sensing, peptidases, etc (Fig. 7).

Discussion
Short-faced mole is a small mammal that lives in underground caves all year round. Report on most aspects of short-faced mole remains scarce. In this study, we attempt to discuss the in uence of gender and distribution area on the intestinal microbial diversity of the short-faced moles, and to nd out whether there are signi cant differences in the gut microbes of short-faced moles of different genders or in different distribution locations. Actinobacteria and Bacteroidete are also dominant phyla in the short-faced moles intestinal microbiota. Relevant studies have shown that an increase in protein and fat in animal foods will increase Actinobacteria abundance in intestinal microbiota (Guan et al. 2016). And carnivorous animals have high abundance of Actinobacteria in their gut may be associated with high fat and high protein feeding habits.
In the present study, there is a high abundance of Actinobacteria in short-faced moles intestinal microbiota (second to Proteobacteria). We speculate that it may be related to high percentage of insect larvae in its food. Microbes of Bacteroidetes can promote digestion. , the content of Bacteroidetes in the short-faced moles' intestinal microbiota is low (only 3.00%), which is similar to the results of the study on the intestinal microbes of grey geese (Anser anser) ). According to the researchers, a low Bacteroidetes abundance of grey geese is due to dietary reasons, or other gut microbiota perform biopolymer degradation functions ). Therefore, we speculate that the low content Bacteroidetes in short-faced moles' intestinal microbiota may be related to their food composition. And we speculate that the dietary polysaccharides in the short-faced moles' diet take only a small percentage.
The beta diversity test results based on unweighted Unifrac distance showed that there are extremely signi cant differences between groups from different sampling locations. However, the beta diversity test results based on the weighted Unifrac distance showed no signi cant difference between the two groups. Study on the intestinal microbial community of free-range and captive cheetahs (Acinonyx jubatus) found that based on the weighted Unifrac distance analysis results have higher resolution than the results based on the unweighted Unifrac distance. Then they indicated that the difference between captive and free-range cheetahs is attributed more to the different abundance of several microbes than to the existence of the unique OTUs (Wasimuddin et al. 2017). So we consider that the differences between different regional groups may also be caused by the differences in the abundance of some OTUs.
Previous studies have found that the composition and diversity of Arctic sediment is similar to that of local mammalian intestinal microbial ora ). Researchers also found that the living environment of baboons (Papio), especially the geological history of soil and the content of exchangeable sodium, signi cantly affect the intestinal microbial ora (Grieneisen et al. 2019 ). In the present study, one of the sampling site (Huimin) is mainly a plain formed by sediment deposition of the Yellow River, and it is about 1,000 meters away from the Yellow River. The other sampling site (Guanxian) is the alluvial plain of the Yellow River. Because of the diversion of the ancient Yellow River, it is more than 8000 meters away from the Yellow River channel. Though examining the physical and chemical properties of the soil in the mole burrows of two sampling sites, we found that the amount of organic matter and cation exchange in soil of Huimin is lower than that in Guanxian. There are signi cant difference in cation exchange capacity (cmol/kg), exchangeable sodium (cmol (Na + ) / kg) and organic matter (g/kg) (SMG > SMH) in the soil between Huimin and Guanxian (Tab. S1). Therefore, we believe that soil factors will have a certain impact on the intestinal microbes of short-faced moles.
UPGMA analysis found that the gut microbiota did not cluster associated with sampling locations. There are still large differences in gut microbes between different individuals in the same sampling location, which may be related to the difference in the microenvironment between individuals. Therefore, environmental factors in the microenvironment may have a more signi cant impact on the gut microbes of short-faced moles, which have a long-term burrowing underground life with poor migration ability. In addition, due to the number of samples from the same sampling location is still small, whether the microenvironment has more in uence on the diversity of intestinal microbiota or the distribution area has more in uence on intestinal microbiota steel needs further study.
Many studies have reported that intestinal microbial diversity in humans and many wild mammals is related to gender. Studies on the intestinal microbes of dholes (Cuon alpinus) found that the abundance of Bacteroidetes in the gut microbes of female samples was higher than that of males ). Studies on golden takin (Budorcas taxicolor bedfordi) also found that the gut microbial diversity was affected by gender ). In the present study, the results showed that there was no signi cant difference in the diversity and abundance of intestinal microbial communities in different gender groups by alpha diversity and beta diversity analysis. However, when compared the abundance of short-faced moles intestinal microbial abundance of different gender groups at different classi cation levels, we found that there were signi cant differences between male and female groups in 5 phyla, 2 classes, 1 orders, 4 families and 23 genera. By LEfSe analysis, we found the biomarkers of signi cant difference between group SMM and group SMF, included family Ruminococcaceae (SMF < SMM) and family Enterobacteriaceae (SMF > SMM).
In this study, we nd that the main functions of the intestinal microbiome of short-faced moles are metabolism, genetic information processing and environmental information processing, etc. By metagenome sequencing and function annotation of three wild giant panda intestinal samples, researchers identi ed homologous sequences with genomic genes encoding cellulase, glucosidase, xylanase, and xylanase. They believe that giant panda intestinal microbes play an important role in the metabolism of lactic acid, amino acids, xenobiotics, nucleotides, polysaccharides, vitamins and lipids in giant pandas (Zhu et al. 2011). A study on the Siberian tiger (Panthera tigris) suggests that the intestinal microbial gene function of the Siberian tiger is mainly related to carbohydrate metabolism subsystem and protein metabolism, respectively. Compared with breast-fed Siberian tiger cubs, the metabolism, translation, replication and repair of carbohydrates, amino acid metabolism, membrane transport and cofactor and vitamin metabolism in the intestinal microbial community of goat breast-fed Siberian tiger cubs decreased (He et al. 2018). Study on the intestinal microbial function of other small mammals, such as shrews (Tupaia belangeri), it is also found that most of the gene function of intestinal microbes are related to metabolism, and are more involved in amino acid metabolism and carbohydrate metabolism. We nd that sugar metabolism (10.81%), amino acid metabolism (9.14%), energy metabolism (4.45%) and nucleotide metabolism (3.86%) account for a large proportion of the main metabolic function of short-faced moles intestinal microbiota. We speculate that this may because that, on one hand, it is bene cial to enhance the digestion and absorption ability of plant cellulose, and on the other hand, it can provide su cient energy to adapt to the low oxygen and lightless living environment, and meet the nutritional and energy needs of reproduction.
At the second level of KEGG pathway, the annotated gene function of short-faced moles intestinal microbiota includes membrane transport, protein translation and other functions besides sugar metabolism, amino acid metabolism and other basic metabolism. Some studies have shown that compared with captive cheetahs (Acinonyx jubatus), the intestinal ora functions of wild cheetahs mainly focus on environmental adaptation, membrane transport, immune system and translation. In order to adapt to environmental requirements such as food supply, competition, territory and family scope, wild cheetahs have enhanced these functions of intestinal ora. A study also found that immune-related genes are enriched in gut microbes in camels (Camelus bactrianus) about two years old (He et al. 2019). Moreover, gastrointestinal diseases caused by intestinal microbiota dysbiosis can limit the growth of captive musk deers ). The gene function of short-faced moles intestinal micro ora annotation shows that intestinal microbes also play an important role in intestinal immunity, which may also be suitable for the life habits of long-term excavation.
In summary, the present study uses high-throughput sequencing technology to study the abundance, diversity and function of short-faced moles gut microbes, and the differences between gender groups and sampling location groups, which will provide data for the study of nutritional ecology of short-faced moles and other small burrowing mammals. In addition to sampling location and gender, other factors may also affect the diversity and function of mammalian gut microbiota. In future research, the intestinal microbial diversity will be further associated with a variety of ecological factors, and the interaction between the diversity of intestinal microbes and environmental factors will be studied in depth, and the sample size will be increased to draw more reliable conclusions. From the perspective of the mutual bene cial symbiosis of gut microbes and their hosts, it will open up a new window to clarify the ecological adaptation mechanism of lifelong burrowing mammals.

Declarations
Ethics approval and consent to participate The capture and dissection of animals and the collection of intestinal samples involved in this study were approved by the Medical Ethics Committee of Qufu Normal University, and all procedures were carried out in accordance with the approval plan of the Medical Ethics Committee of Qufu Normal University (Approval number 2020018).

Consent for publication
The written informed consent forms were taken from the volunteers.

Declarations
Competing interests The authors declare that they have no con ict of interest. Authors' contributions LC conceived, designed, performed the experiments, ML analyzed the data; DX wrote the paper; JZ, SW, MS contributed materials; SW, GW, XL, TX, LS modify the manuscript. All authors read and approved the nal manuscript.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Documentapproved.pdf TableS1.docx