Study On The Food Composition Of Black-necked Crane(Grus Nigricollis) And The Adaptability Of Its Gut Microbiota During The Stable Period Of Overwintering In Caohai

Gut microbiota play an important role in maintaining the health of the body.Many factors affected the structure of gut microbiota,such as internal and external factors.Food is the main limiting factor for animal survival,and also the most important factor affecting the host gut microbiota.The black-necked Crane is a vulnerable species in the world,and which is a national protected animal.And its food is the key factor for the long-term survival.To introduce protection for the black-necked Crane,study the composition of gut microbiota is very important.However,in recent years,the research on the gut microbiota of black necked Crane has not done in-depth research on its function,and the research on food composition still used the direct observation method and fecal microscopic analysis method.In this study,Caohai National Nature Reserve is one of the largest wintering areas of birds in Weining county,China,was selected as the research area.During the stable wintering period,the fresh feces of the black necked Crane were collected by non-invasive sampling method,From the result of DNA extraction and species identification,the samples identified as black necked Crane were combined with amplicon and metagenomic sequencing technology to explore the diet of black necked Crane during the stable wintering period and the relationship between the gut microbiota and their function.As the results showed,during the wintering period in Caohai wetland:1)The black necked Crane mainly feeds on Solanaceae(Solanum tuberosum,Solanum lycopersicum and Capsicum annuum),Poaceae(Corn),Cyperaceae(Schoenoplectus tabernaemontani,Cyperus alternifolius),Araceae(Pinellia ternata,Spirodela polyrhiza)and Polygonaceae(Fallopia multiflora,Fagopyrum luojishanense)and other local crops as well as wild plants.It also feeds on a small number of arthropods(Coleoptera,Hemiptera and Diptera)and rodents(Sus scrofa and Rattus norvegicus);2)The more complex the Black necked Crane feeds,the more complex the composition of intestinal microflora,and the higher the richness of intestinal microflora;3)Proteobacteria,Firmicutes and Actinobacteria are the main intestinal microflora of the black necked Crane.Proteobacteria is highly enriched in the intestine,mainly to help the black necked Crane digest and metabolize carbohydrate rich food(corn,etc.);Firmicutes is mainly to help black necked Crane digest and metabolize starch and cellulose rich food(potato and Schoenoplectus,etc.),that is to say,the structure of gut microbiota of black necked Crane is mainly composed of Proteobacteria and Firmicutes,which is to adapt to the diet of black necked Crane with high sugar,high starch and high cellulose;4)Functional prediction analysis showed that the gut microbiota of black necked Crane had abundant metabolic activities,mainly including carbohydrate metabolism and amino acid metabolism,in which starch and sucrose metabolism had the highest abundance,and the related enzymes were glycogen synthase [EC: 2.4.1.11],starch synthase [EC:2.4.1.21] and glycogen phosphorylase [EC: 2.4.1.1],This series of enzymes were related to glycosyltransferases GT2 and GT4,as well as glycoside hydrolases GH23,GH13,GH1,GH3 and GH2 in the intestine of black necked Crane.The bacteria associated with these enzymes were mainly from Proteobacteria and Firmicutes;5)The sequencing results from the amplicon sequencing(bacterial16S r RNA gene,chloroplast rbc L gene,mitochondrial COI gene)were similar to those of the metagenome,however,the species annotation of amplicon sequencing was higher than that of the metagenome sequencing at the genus level,and the number of species information annotation of the metagenome sequencing were more accurate at the species level.This study is helpful to explore the function of intestinal microflora of black necked Crane in wintering period,and provide strong theoretical support for better protection of black necked Crane.The study of food composition of black necked Crane is helpful to further understand the feeding species of black necked Crane in stable wintering period,so as to establish the relationship between the food composition and the structure of gut microbiota,and also provide important theoretical basis and reference value for the research on the feeding habits of other waterbirds.