The relict gull (Larus relictus) is a medium-sized gull that breeds in scattered locations in drought lakes in the Russian Far East, eastern Mongolia, and Kazakhstan. The largest breeding group is called the Ordos subpopulation, which is distributed in northern China in Shaanxi and Inner Mongolia (1). It is listed as vulnerable in the International Union for Conservation of Nature Red List and is a national first-class protected bird in China. There are approximately 10,000–19,999 relict gulls worldwide (2).

The Ordos subpopulation of relict gulls migrates over the Inner Mongolia Plateau and reaches the coast of the Bohai Sea for winter. Mu Us Desert (KX) and Bojiang Haizi (BJ) in Ordos City, Inner Mongolia Autonomous Region, China are both located on the Inner Mongolia Plateau and are important breeding grounds and stopover sites for relict gulls (3, 4). The relict gulls mainly live around sandy lakes and feed on sand flora and lake fauna. The habitats of the two breeding sites are different. Previous studies highlighted that habitat and dietary nutrition are both potential influencing factors of the animal gut microbial community (5–8), thus, the gut microbiota may also change accordingly.

The gut microbiome is a collection of all microbial cells and related genetic substances present in the host gastrointestinal tract. The intestinal microbiota is numerous and diverse and is closely related to various physiological functions of the host's body. Some types of gut microbiota are associated with metabolic diseases, such as obesity (9) and diabetes (10), and the occurrence and progression of gastrointestinal diseases (11–13). Others participate in functional processes that are critical for homeostasis (14, 15). In summary, the composition and diversification of the gut microbiota are closely associated with the health of host. To date, studies on the gut microbiota of avian species have focused on the influence of particular bacteria or bacterial pathogens on the host (16, 17). The rapid development of high-throughput 16 S rRNA gene sequencing technology has enriched our knowledge of gut microbiota diversity. It is widely used for quick and efficient characterization of the gut microbial communities of many organisms, such as humans (18), cows (19), pigs (20), chickens (21), and a selected number of avian species (22–25).

Characterization of the avian gut microbial community contributes to the knowledge of avian microbiology and will likely advance efforts to protect the living environments of wild birds. Moreover, the identification of pathogenic bacteria harbored by wild birds helps to prevent the spread of related diseases. At present, neither there is any related research on relict gull gut microbiota nor that of different breeding grounds. In the present study, fecal samples of relict gulls from two different breeding grounds were collected. Then, the bacterial microbiota composition and differences in relict gulls across the different sites during the breeding period were determined. Analysis of the bacterial microbiota composition and its relation to the breeding environment has great significance for the physiological ecology and population management of relict gulls. The findings of the present study can provide insight into the gut microbial composition of relict gulls and provide a theoretical basis for their protection, such as disease prevention and control.

The relict gull is a vulnerable species that relies heavily on dry lakes in Asia (30). The Ordos subpopulation of relict gulls migrates over the Inner Mongolia Plateau and reaches the coast of the Bohai Sea for winter (3, 31). Ordos Wetland, including the KX and BJ, are both vital stopover sites and breeding grounds. The habitats of the two breeding sites are different. Previous studies have highlighted that habitat and dietary nutrition are potential influencing factors of the animal gut microbial community (5–8). At the same time, the gut microbiota plays important roles in energy intake and metabolism, immune homeostasis maintenance, and the reproductive health of the host (32). Therefore, it is important to know the composition of the intestinal bacterial community to aid assessment of the breeding environment and determine whether sufficient food is available to the species living there. However, to date, there have been no studies on the intestinal microbiota of relict gulls. In the present study, 16S rRNA gene high-throughput sequencing technology was used to detect differences in the bacterial microbiota of relict gull (Larus relictus) in different places.

In terms of alpha diversity, no difference in the abundance and diversity of OTUs was observed between the relict gulls of the KX and BJ (BJ). Beta diversity was represented by NMDS based on unweighted UniFrac distance and demonstrated that there were significant differences in the gut microbiota composition between the two groups. This indicates that different habitat environments affected the gut microbial communities.

At the phylum level, 15 phyla were identified in the 13 fecal samples, and Firmicutes, Proteobacteria, Fusobacteria, Tenericutes, Bacteroidetes, and Chloroflexi showed an average relative abundance above 1%. Firmicutes and Proteobacteria were the most abundant microbiota in the relict gulls at both sites. This is consistent with the results of previous studies on other avian species (27, 33). These phyla exist in multiple avian species, indicating that they are important functional bacteria in the gastrointestinal tract of relict gulls.

The presence of these phyla in the gut microbiota of multiple avian species indicates that these bacteria might play important functional roles in the gastrointestinal tract of relict gulls, such as digestion, absorption of nutrients, and immune response. Firmicutes and Proteobacteria are both key metabolic phyla in the gut and can break down compound sugars, fatty acids, and polysaccharides in foods, generating energy for animals to absorb and accumulate (34–36). Fusobacteria play important roles in the production of butyrate, which in turn accelerates fat accumulation in the body and strengthens immunity (37). Tenericutes are widespread in the environment and are opportunistic pathogenic bacteria (33). Furthermore, Bacteroidetes are considered to be related to chronic intestinal inflammation (38). Chloroflexi is abundant and ubiquitous freshwater microbes (39). These results show that relict gull breeding in the two sites may be at risk of gastrointestinal disease. Local government departments should take certain measures to protect the health of the breeding relict gulls.

The habitat environment has an important effect on gut microbial communities. Kiritimatiellaeota was present in relict gulls in BJ but not in KX. Kiritimatiellaeota mediates arginine biosynthesis and regulates fatty acid synthesis. It is often found in plateau animals (40) and is a strategy for plateau animals to use low-fat food to provide energy in order to adapt to extreme environments. BJ has an altitude of 1,367–1,412 m, which is higher than that of the KX (1,300–1,400 m), but the difference is not significant. The annual average temperature of BJ is 5.2°C, which is lower than that in the KX, but is not significantly different. Thus, we speculate that the presence of Kiritimatiellaeota may be a consequence of the difference in microbial communities between the two sites. However, the dominant plant species in these two sites have not yet been identified, thus, the plants consumed by the relict gull Ordos subpopulation at the two study sites are unknown. Hence, further study is required to establish the consumption behaviors of the relict gull Ordos subpopulation.

Additionally, together Firmicutes and Proteobacteria were the dominant bacterial phyla at both sites. However, Firmicutes were the more dominant gut microbiota of relict gulls in the KX, whereas the dominant phylum in BJ was Proteobacteria. The results of LEfSe analysis indicate that at the phylum level, the relative abundance of Bacteroidales was significantly higher in BJ than in KX. Previous studies have reported that an increased Firmicutes/Bacteroidetes ratio is related to obesity in humans (41, 42) and strengthens the capacity to harvest energy from their diet (43). In this study, relict gulls in the KX had a higher Firmicutes/Bacteroidetes ratio than those in BJ (BJ), indicating that relict gulls in the KX had an enhanced capacity to promote food energy intake and maintain metabolic equilibrium.

In previous studies, abundant bacterial genera were considered to have derived from the food sources and the wider environment. For example, Catellicoccus was highly abundant in various gulls' fecal samples (44–46), indicating that it may be a symbiont or associated with their special diets and lifestyle. Cetobacterium is a bacterium that is commonly found in the intestinal tract of fish (47), Candidatus_Arthromitus is a bacterium genus that is commonly found in insect intestines (48). Critically, fish and insects are likely important to the diet of relict gulls. Therefore, these bacteria may have colonized the gull's intestines through the food consumed. Uncultured_bacterium_f_Enterobacteriaceae, Enterococcus, Paeniclostridium, Mycoplasma, Plesiomonas, and Proteus are all dominant bacterial genera in the soil and water environment (49–53), indicating that environmental factors have an important influence on the gut microbiota of relict gulls.

At the same time, two genera of probiotics were found in the dominant bacteria Lactobacillus and Sporosarcina. Lactobacillus and Sporosarcina have been previously reported to be beneficial bacteria in the intestine, capable of anti-inflammatory activities and protection against pathogens (54, 55). Therefore, the presence of these two types of bacteria can help relict gulls to resist disease. However, most of the highly abundant bacteria found were potentially pathogenic bacteria, such as Paeniclostridium, Clostridium_sensu_stricto_1, Enterococcus, Mycoplasma, Proteus, and Plesiomonas, which have been associated with diseases in multiple species (49–51, 53, 56, 57). Through functional prediction, these bacteria were found to be related to human diseases. This result proves that the relict gulls in KX and BJ carry pathogenic microorganisms that threaten the health of humans and other animals. Therefore, certain measures should be taken to prevent the spread of avian zoonotic diseases.

In summary, in the current study, high-throughput sequencing was performed to analyze fecal samples of relict gull (Larus relictus) breeding in the KX and BJ in Inner Mongolia, China. The analysis revealed that the gut microbiota differed among different breeding sites. We propose that the most abundant bacterial genus had a significant relationship with the diet and living environment. Moreover, the presence of some bacterial genera may be due to adaptation to the plateau environment in which relict gulls live. This would enable the relict gulls to effectively use local resources and accumulate energy. Simultaneously, a variety of highly abundant pathogenic bacteria were found, suggesting that these individuals are at risk and may spread diseases. Certain measures should be taken to protect this species and to prevent the spread of avian zoonotic diseases. This study provides a theoretical basis for relict gull protection and related disease prevention and control.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA788023.

The animal study was reviewed and approved by the current study was performed in accordance with the suggestions on animal care and ethics in China. Non-invasive techniques were used to collect fecal samples. The Animal Ethics and Welfare Committee of Baotou Teachers College approved the implementation of the project, and the management authorities of KX and BJ of Ordos City in Inner Mongolia approved the sampling of relict gull fecal samples.

LG and LL designed the research. LG, YL, and LL collected the samples. CD analyzed the data. LG wrote the manuscript. LG, LL, CD, and YL revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was funded by the Inner Mongolia Natural Science Foundation (No. 2021BS08013), the Baotou Teachers College High-level Research Achievement Cultivation Project (No. BSYKJ2021-ZQ03), and the High-level Talents Introduced Scientific Research Startup Fund Project of Baotou Teacher's College (No. BTTCRCQD2020-003).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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