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S13568-021-01252-2.Pdf Chen et al. AMB Expr (2021) 11:93 https://doi.org/10.1186/s13568-021-01252-2 ORIGINAL ARTICLE Open Access Habitat environmental factors infuence intestinal microbial diversity of the short-faced moles (Scaptochirus moschata) Lei Chen*, Di Xu, Jing Zhu, Shen Wang, Mi Liu, Mengyao Sun, Geyang Wang, Lingyu Song, Xiaoyu Liu and Tianyu Xie Abstract 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 scientifc 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’ intes- tinal 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 func- tions 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 diference is found in the composition and function of the short-faced mole intestinal microbiota. There are signifcant diferences in the composition and functional structure of the short-faced mole gut microbiota between samples collected from diferent habitats. We conferred that this is related to the diferent envi- ronment factors in which they live, especially to the edaphic factors. Keywords: Short-faced moles, Gut microbiome, Gender, Sampling locations, Edaphic factor Introduction Several studies on Talpidae have focused on the phyloge- Short-faced mole (Scaptochirus moschata) belongs to the netic and morphological evolution, while the ecological Mogera of the Talpidae, is a small mammal that lives in adaptability of Talpidae is less studied. burrows for life, feeds on plant rhizomes and soil insects Intestinal microbes are important symbionts in mam- such as grubs, etc. It has developed morphological and malian gut and occupy important niche (Willey et al. physiological characteristics adapted to burrowing, such 2014; Saxena and Sharma 2016). Tey play an important as eye degeneration, foreclaw valgus, smell and hear- role in maintaining intestinal health and immune func- ing sensitivity, etc. Short-faced mole is Chinese endemic tion, including in the proliferation of intestinal cells, species, mainly distributed in Henan, Shandong, Inner defense against pathogens, metabolism of secondary Mongolia, Ningxia, Shanxi and other provinces in China. products, and digestion of complex compounds (Flint et al. 2012). Intestinal microbes can also afect animal nutrition metabolism and intestinal immunity through *Correspondence: [email protected] College of Life Science, Qufu Normal University, Jingxuan West Street intestinal-brain axis (Schoster et al. 2017; Li et al. 2018; No.57, Qufu, Shandong Province 273165, China Wang et al. 2007), which is of great signifcance to animal © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Chen et al. AMB Expr (2021) 11:93 Page 2 of 12 survival and environmental adaptation (Wu et al. 2016; samples according to the instructions. Te concentration Zhu et al. 2011; Chen et al. 2017). Tere are many fac- of the DNA was detected by NanoDrop™ OneC (Termo tors that afect gut microbes, including the genetic back- Fisher Scientifc, USA). We used qualifed DNA samples ground of the host, the diet, age, gender, social class of for PCR amplifcation, then diluted the sample to 1 ng/ the host, and the climate of the habitat. Soil, as an essen- μl using sterile water. PCR amplifcation of the V3-V4 tial part of terrestrial mammal habitat, also plays an region of 16S rRNA gene was performed with the prim- important role in the composition and diversity of animal ers 341F (CCT AYG GGRBGCASCAG) and 806R (GGA gut microbes (Zhao et al. 2018; Wang et al. 2019). CTA CNNGGG TAT CTAAT). Te PCR reaction steps Short-faced moles live in an underground hypoxic were as follows: pre-denaturation at 98 °C for 1 min; environment all year round. Te physiological adaptation denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, mechanisms are important and representative in the eco- extension at 72 °C for 30 s (35 cycles); and fnal extension logical research of burrowing mammals. Te functional at 72 °C for 5 min. contribution of intestinal microbes in the burrowing life Te concentration of PCR amplifed products was of the short-faced moles is an important feld in the study detected by 2% agarose gel electrophoresis. Ten the of the physiological ecology of the burrowing mammals. gel extraction kit (Qiagen, Germany) was used to purify Terefore, this study intends to study the gut microbial the PCR products. Te TruSeq® DNA PCR-Free Sam- diversity of the short-faced moles, and to investigate the ple Preparation Kit was used to construct the library. diferences of intestinal microbes in moles with diferent Te constructed library was quantitatively tested by sex and distribution. Furthermore, through the predic- Qubit. After the library was qualifed, the Illumina HiSeq tion of microbiome function, we also try to explore the sequencing platform was used for sequencing. function of gut microbes and their possible contribution in the adaptation to underground life of the short-faced moles. Tis study will provide data for the understanding Sequencing data preprocessing of the physiological and ecological mechanism of short- According to the quality control process of Qiime faced moles to adapt to burrowing life, and provide data (V1.9.1) (Caporaso et al. 2010), the raw tags obtained for the ecological studies of small burrowing mammals. by sequencing were fltered strictly by quality control to obtain high-quality clean tags. Truncate Raw Tags from Materials and methods the frst low-quality base site where the number of con- Samples collection secutive low-quality values (the default quality threshold In this study, 22 short-faced moles (SM) were collected is < = 19) has reached the set length (the default length is using non-damagingly self-made mouse traps in Sep- 3). Ten flter out the Tags whose continuous high-qual- tember 2020, including 12 males (named M) and 10 ity base length is less than 75% of the Tag length. After females (named F). Te sampling locations were Guanx- detect and remove chimeric sequences, the fnal efective ian (115°E, 36°N; named G; 11) and Huimin (117°E, 37°N; tags were obtained for further analysis. named H; 11). Te short-faced moles were captured and placed in the soil of the original habitat, and transported to the laboratory alive. Subsequently, the gasifed diethyl OTUs classifcation and species abundance analysis ether was put into the transparent sealing device, and To detect the species richness in each sample, the efec- then the collected samples were put into the device, and tive tags in all samples is classifed as operational taxo- the diethyl ether was continued. During this process, nomic units (OTUs) based on 97% sequence similarity. the state of short-faced moles was kept observing. After Ten the OTUs were compared to SILVA’s SSUrRNA euthanasia, short-faced moles were dissected and the database for species annotation (set threshold of 0.8) colon contents were collected aseptically. Te collected by using Mothur software (Version 1.35.1). R software colon contents samples were stored at – 80℃ in the labo- (v2.15.3) was used to generate Rarefaction curve and ratory until DNA extraction. Rank Abundance curve. Te end of the Rarefaction curve Tis study was approved by the Medical Ethics Com- is gradually fattened, which proves the rationality of the mittee of Qufu Normal University (No. 2020018), and sequencing amount, and the results obtained by sequenc- during the approval period, the short-faced moles was ing can efectively refect the abundance of microorgan- captured and dissected as required. isms contained from the sample. We analyzed the OTUs distribution to study the species abundance of the gut DNA extraction, PCR amplifcation and sequencing microbes of each sample, and constructed a histogram of We used QIAamp DNA Stool Genomic DNA Extraction the relative abundance of OTUs at each classifcation lev- Kit (Qiagen, Germany) to extract DNA from intestinal els (phylum, class, order, family, and genus). Chen et al. AMB Expr (2021) 11:93 Page 3 of 12 Diversity analysis and signifcant test Results To analyze the diversity of the microbial community Sequencing data processing and OTU analysis intragroup, we performed alpha diversity analysis by A total of 2,085,117 raw paired-end reads are obtained using Mothur software (Version 1.35.1) to refect the from 22 short-faced moles intestinal samples.
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