A First Insight into the Structural and Functional Comparison of Environmental Microbiota in Freshwater Turtle Chinemys reevesii at Different Growth Stages under Pond and Greenhouse Cultivation

Abstract

The microbial community structure of water is an important indicator for evaluating the water quality of the aquaculture environment. In this study, the investigation and comparison of the bacterial communities of pond cultivation (PC) and greenhouse cultivation (GC) between hatchling, juvenile, and adult growth stages of C. reevesii were performed. In addition, the V4 regions of the 16S rRNA gene were sequenced. The Chao1 richness estimator of the PC group was significantly higher than that of the GC group. The beta diversity showed that the microbiotas of the two groups were isolated from each other. The dominant phyla were Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes in the PC group and Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, Chloroflexi, and Actinobacteria in the GC group. Both the numbers and the types of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations differed between the PC and GC groups. The prediction of bacterial phenotype implied that the GC environment is more likely to deteriorate, and turtles are more susceptible to pathogens than those of the PC environment. In addition, a total of nine potential pathogenic bacteria were identified and the correlation of environmental factors analyses showed significant differences of bacterial species between the PC and GC groups, while the potential pathogenic bacteria showed significant correlation with the stocking density, temperature, pH, orthophosphate (PO₄-P), and dissolved oxygen (DO) in both the PC and GC groups. Noticeably, this is the first report to describe the different microbiota characteristics of the different cultivation environments in the different growth stages of C. reevesii, which will provide valuable data for water quality adjustment, disease prevention, and the healthy breeding of turtles.

Keywords: Chinemys reevesii; different growth stages; environmental microbiota; greenhouse cultivation; pond cultivation.

Figure 1

Alpha- and beta-diversity, analysis of similarities (ANOSIM) group similarity, NDMS, and group distance analyses of different growth stages of freshwater turtle C. reevesii in six groups. (A): Chao1, (B): Shannon, (C): Simpson, (D): Bray–Curtis, (E): binary Jaccard, (F): ANOSIM group similarity, (G): NDMS analysis, (H): group distance. The red boxes represent the maximum group distances and the green boxes represent the minimum group distances.

Figure 2

The bacterial community, microbiota structure and OTUs in phylum and genus levels of the pond cultivation (PC) and greenhouse cultivation (GC) groups. (A): microbial community structure; (B,C): Venn diagram of PC and GC groups in phylum level; (D,E): Venn diagram of PC and GC groups in genus levels; (F): Size of each OUTs in HTPC and HTGC, JTPC and JTGC, ATPC and ATGC.

Figure 3

Bacterial and Archaea, LEfSe analysis and random forest model of different classifications annotation of the freshwater turtle C. reevesii in six groups. (A–F): HTPC group, JTPC group, and ATPC group, HTGC group, JTGC group, and ATGC group. The circles represent different classification levels from inside to outside, and the size of the fan represents the relative proportion of different OTU annotation results. The group level was constituted by the average level of relative abundance of each species in each group (n = 3 or 4); (G): LEfSe cladogram, the circle radiating from the inside to the outside represents the classification level from the phylum to the genus (the innermost yellow circle is the Kingdom). Each small circle at a different classification level represents a classification at their levels, and the diameter of the circle represents the relative abundance; (H–M): random forest model maps of PC and GC in phylum and genus level based on MDA algorithm.

Figure 4

Gene function prediction (top30) (A) and top10 in gene function predictive expression p value (B–K) of all six groups based on the COG database.

Figure 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation information and prediction of bacterial phenotype of PC and GC groups. (A): KEGG pathway annotation, the red and green boxes represent the high abundance of PC and GC environments. The black boxes represent the high abundance of the HTPC group, the blue boxes represent the high abundance of the JTPC group, and the red boxes represent the high abundance of the HTGC group. The green boxes represent the high abundance of the JTGC group, and the gray boxes represent the high abundance of the ATGC group. (B): Gram Positive; (C): Gram Negative; (D): Biofilm Forming; (E): Pathogenic Potential; (F): Mobile Elements; (G): Aerobic; (H): Anaerobic; (I): Facultative anaerobic; (J): Oxidative Stress Tolerant. The red boxes represent the highest relative abundance and the green boxes represent the lowest relative abundance.

Figure 6

Correlation analysis of environmental factors of PC and GC groups. (A,B) Redundancy analysis (RDAs) of PC and GC groups based on OTUs of 16S rRNA genes. (C): RDA analysis of PC and GC groups based on potential pathogenic bacteria. The blue arrows above represent different environmental factors, and the green arrows represent different phylum of microbiota (top10) and different genus of potential pathogenic bacteria. The arrow length of the environmental factor represents the correlation magnitude between the environmental factors and the samples, and longer arrows represent greater influence on the distribution of the samples and microbiota. The angle between the lines of the arrows represents the correlation. The acute angle indicates that the two environmental factors are positively correlated, and the obtuse angle is negatively correlated. The value in the (D) represent the abundance of potential pathogenic bacteria in PC and GC groups.