Intestinal Bacterial Composition and Characteristics of Wintering Mandarin Duck (Aix Galericulata) in Shiqian Mandarin Duck Lake National Wetland Park, Guizhou Province

Introduction

Currently, a large number of studies have been conducted on the relationship between intestinal microorganisms and the host. In the early, studies focused on the composition, diversity, ecological and physiological characteristics of intestinal microorganisms for humans and mammals [1]. However, studies on intestinal microorganisms of birds are only found in economic species of artificial cultivation [2, 3]. With the wide application of high-throughput sequencing technology and the rapid development of bioinformatics analysis technology, the grand plan of intestinal micro biome research has been launched in many countries [4, 5] so that people are no longer limited to research poultry intestinal microorganisms [6], but gradually turn to wild birds intestinal microorganisms [7, 8]. Previous studies have shown that the number and abundance of intestinal microorganisms have been gradually increased from carnivores and omnivores to herbivores [9]. Therefore, mastering composition of intestinal microorganisms is the basis for studying the relationship between their functions and hosts.

Shiqian Mandarin Duck Lake National Wetland Park, Guizhou Province is located in 27 ° 24 '37 "- 27 ° 28' 35 'N, 108 ° 12' 08" 18 '- 108 ° 38' E, elevation of 468m-754m. At 8:00-9:00, 13:00-14:00 and 18:00-19:00 every day of January 31 and February 1, 2018, non-invasive sampling technology was used to collect 14 fresh feces form the wetland park. In order to ensure that all samples came from different individuals, the collection points of two adjacent samples were more than 2m apart. After observing Mandarin Duck feeding or resting on shore about 30minutes by 10x60 binoculars, we arrived at activity sites in time, using disposable sterile gloves and cotton swabs to quickly obtain about 3g fresh feces and putting them into 5ml sterile centrifuge tube containing anhydrous ethanol. At the same time, recording sample number and information (Table 1) and putting them into portable ice box in time. Then, back to the laboratory within 24h and save in ultra-low temperature freezer - 75℃.

Mandarin Duck (Aix galericulata) belongs to omnivorous birds, for our country Ⅱ class protected animals. At present, most studies on Mandarin Duck focus on macro aspects, such as reproduction and overwintering ecology [10, 11]. However, the study of intestinal microbial composition and characteristics is still blank. In addition, Mandarin Ducks are main protected objects of Shiqian Mandarin Duck Lake National Wetland Park. Therefore, the analysis of intestinal bacterial composition and characteristics of overwintering Mandarin Ducks not only has certain reference value for understanding their physiological function and health, but also has important significance for the protection of their population in Wetland Parks.

Based on this, high-throughput sequencing of bacterial 16S rRNA gene and modern molecular biological information analysis technology are used to study intestinal bacteria of wintering Mandarin Duck. The aim of the study are: (1): To understand their composition and dominant groups to enrich the content of its micro ecology research; (2): Characteristics of dominant bacteria are analyzed to provide basic data for the study of the relationship between intestinal bacteria diversity, host's dietary changes and physiological functions.

Discussion and Conclusions

Detected OTUs were not fully identified, and identification rate at Species level was only 22.34%. This was due to the limited selection of bacterial identification database, which limited the amount of biological information of sequences to some extent. It might also be that a few novel or unknown microorganisms that coexist with the gut of Mandarin Ducks. The V3-V4 variable region of 16S rRNA gene obtained sequence fragments within 500bp, which is far less than complete length of bacteria 16S rRNA gene. Moreover, most of sequences could only be identified at genus level (the identification rate of bacteria genus was 68.03%). Therefore, in future studies, sequencing depth of target fragment should be increased to obtain more biological information.

Firmicutes, Proteobacteria, Fusobacteria and Bacteroidetes are play leading roles (93.69%) in intestinal bacteria composition of Mandarin Ducks. This is consistent with the dominant bacteria of 75 intestinal bacterial phyla being identified by humans [16]. They are ubiquitous and predominant in intestinal tract of humans and animals, which is related to their metabolic function. Under the condition of long-term evolution and symbiosis with the host, a relatively stable symbiosis mechanism is formed, which then dominates composition and structure of the host intestinal bacteria.

Firmicutes is ubiquitous and species-rich in the intestinal tract of vertebrates, which could involve in carbohydrate metabolism and nutrient absorption widely ，has the most abundance in intestinal bacteria of Mandarin Ducks, among them, Lactobacillus is the most dominant. In Lactobacillus, many bacteria are probiotics that could coexist with their hosts. They have the function of enhancing the antibody-mediated immune response and inducing intestinal T cells to express cytokines [17, 18]. Therefore, Lactobacillus could decompose sugars in food timely to provide energy for wintering Mandarin Ducks to survive, and also play a important role in maintaining the stability of the intestinal environment.

As the second dominant bacteria phylum of Mandarin Ducks, Proteobacteria is also the most diverse type in all bacteria, which had a variety of physiological functions, among them a small part is probiotics, which could obtain a large amount of carbon sources in food [19]. Pseudomonas is the most dominant in Proteobacteria, probably to better adapt to or cope with the complex feeding characteristics of Mandarin Ducks. Because, Mandarin Ducks still eat very complex foods in overwintering, ranging from high-protein meat, a variety of insects, aquatic plants to terrestrial plants [20]. However, many bacteria groups of Proteobacteria could regulate metabolism flexibly and tolerate different nutritional levels of foods [21], and some also had the function of degrading plant cellulose [22]. Therefore, compared with other bacteria kinds, they are more competitive or have strong broad-spectrum properties and coexist with other bacteria, and enriched in the intestinal tract of Mandarin Ducks.

Fusobacteria is associated with host nutrient absorption and synthesis of short-chain fatty acids [23], but many of them are pathogenic bacteria [24]. Studies have shown that various bacteria of Fusobacterium had pathogenic to humans and animals [25]. In this study, only Fusobacterium is found in Mandarin Ducks intestinal Fusobacterium, and its abundance is the highest, accounting for 13.95% of the total bacterial genera. Although no obvious health problems were found in Mandarin Ducks during years of observation and sampling by the reserve administrators, some pathogenic bacteria of Fusobacterium also might be latent in the intestinal tract of Mandarin Ducks or inhibited or lost under coexistence with other bacteria.

Bacteroidetes is able to involve in metabolism of steroids and bile acids, degradation polysaccharides and fermentation carbohydrates, etc [26]. Among them, Bacteroides is the most dominant, which were able to participate in metabolism of plant fiber and polysaccharide [27], and help Mandarin Ducks obtain nutrients in food. However, when the normal micro- ecological balance was broken, they also caused endogenous infection [28]. Results of intestinal bacterial composition of Mandarin Duck verified the conclusion that dominant intestinal bacteria of Anseriformes birds were very similar to that of other birds in phylum level, and intestinal bacteria of most birds were mainly concentrated in Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes, Actinobacteria and Tenericutes, such as Anseriformes and Sphenisciformes, Galliformes, Gruiformes [7, 29, 30, 31].

Compared with Anser, Proteobacteria (23.71%) and Cyanobacteria (0.30%) in intestines of wintering Mandarin Ducks are much lower than that of Anser indicus (64.69% and 8.48%) [32], while Fusobacteria (14.05%) is much higher (that of Anser indicus 0.56%) [33]. This apparent abundance difference is largely due to different genetic factors (host itself) and host's diet [34]. Richness and evenness of intestinal bacteria of Mandarin Ducks is Fang ZY, et al. Intestinal Bacterial Composition and Characteristics of Wintering Mandarin Duck (Aix Galericulata) in Shiqian Mandarin Duck Lake National Wetland Park, Guizhou Province. Int J Zoo Animal Biol 2019, 2(3): 000158.

significant differences form different samples, which is probably influenced by changes in food resources.

The relative abundance of Proteobacteria (9.18%) in artificial feeding sites is much lower than that in other foraging habitats (including reservoirs, shoals and farmland) (average 31.96%). Moreover, the bacterial groups of the feeding point samples were clustered into a separate group, while the bacteria of the non-feeding point samples were clustered into three groups. Such results were likely to be caused by food resources changes in different foraging habitats, because, intestinal microorganisms were highly sensitive to food changes or feeding types of the host [35]. However, specific differences or effects need to be further studied on the relationship between diet and intestinal bacteria of Mandarin Ducks. Foundation project: Natural Science Foundation Project of Guizhou province (Guizhou Forestry Science and Technology Cooperation No. 11 [2017] ); National Natural Science Foundation of China (No. 31801990); Guizhou Science and Technology Cooperation Platform Talent Project (No. 5781 [2018]); School-county Cooperative Project.