Gut microbiota contributes to the growth of fast-growing transgenic common carp (Cyprinus carpio L.)

Abstract

Gut microbiota has shown tight and coordinated connection with various functions of its host such as metabolism, immunity, energy utilization, and health maintenance. To gain insight into whether gut microbes affect the metabolism of fish, we employed fast-growing transgenic common carp (Cyprinus carpio L.) to study the connections between its large body feature and gut microbes. Metagenome-based fingerprinting and high-throughput sequencing on bacterial 16S rRNA genes indicated that fish gut was dominated by Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes, which displayed significant differences between transgenic fish and wild-type controls. Analyses to study the association of gut microbes with the fish metabolism discovered three major phyla having significant relationships with the host metabolic factors. Biochemical and histological analyses indicated transgenic fish had increased carbohydrate but decreased lipid metabolisms. Additionally, transgenic fish has a significantly lower Bacteroidetes:Firmicutes ratio than that of wild-type controls, which is similar to mammals between obese and lean individuals. These findings suggest that gut microbiotas are associated with the growth of fast growing transgenic fish, and the relative abundance of Firmicutes over Bacteroidetes could be one of the factors contributing to its fast growth. Since the large body size of transgenic fish displays a proportional body growth, which is unlike obesity in human, the results together with the findings from others also suggest that the link between obesity and gut microbiota is likely more complex than a simple Bacteroidetes:Firmicutes ratio change.

Figure 1. UPGMA clustering over Sørensen similarity of gut microbiota composition between transgenic fish and wild-type controls across different developmental stages.

The similarity matrix was calculated using the binary data, and clustering was performed using the unweighted pair-group method with arithmetic average (UPGMA). T_i and C_i indicate the i^th replication of transgenic fish and wild-type control, respectively; T_fi, T_mi, and T_hi represent the i^th foregut, midgut, and hindgut samples from transgenic fish, respectively, and C_fi, C_mi, and C_hi from the corresponding part of controls; T_s and T_w represent the sediment and water samples collected from the pond where transgenic fish were reared, and C_s and C_w represent controls; F indicates food sample.

Figure 2. Comparison of similarities and differences for gut microbiota composition between transgenic fish and wild-type controls across different developmental stages.

(a) Comparison of the average Sørensen index obtained from DGGE patterns of 16S rRNA genes between transgenic fish and wild-type controls or within each group of transgenic fish and wild-type controls. (b) Comparison of Raup and Crick similarity index (S _RC) obtained from DGGE patterns of 16S rRNA genes between transgenic fish and wild-type controls or within each group of transgenic fish and wild-type controls. Dashed lines indicate significant cutoff for difference (low line) and similarity (upper line). Error bars represent the standard error of the mean.

Figure 3. Principal Component Analysis (PCA plot with UniFrac scaled axis) of individual samples with different diet treatments.

For each sample code, the first letter T represents transgenic fish, and C wild-type controls; the middle letter(s) indicates the diet treatments (C: control diet, HP: high protein diet, HC: high carbohydrate diet, HL: high lipid diet), and the last numbers represent replicate samples.

Figure 4. Comparison of relative Bacteroidetes and Firmicutes abundance at different developmental stages.

Real-time quantitative PCR (Q-PCR) was used to quantify the abundance of gut Firmicutes and Bacteroidetes based on the 16S rRNA genes (V3 region). (a) Relative abundance of Firmicutes and Bacteroidetes in transgenic fish. (b) Relative abundance of Firmicutes and Bacteroidetes in wild-type controls.

Figure 5. Triplot of the redundancy analysis (RDA) showing significant relationship between metabolic-related factors (response variables) and microbial groups (explanatory variables).

First and second ordination axes were plotted, representing 22.6% and 7.9% of the variability in the data set, respectively. P-values obtained by Monte Carlo test were reported. For each sample code, the first letter T represents transgenic fish, and C wild-type controls; the middle letter(s) indicates the diet treatments (C: control diet, HP: high protein diet, HC: high carbohydrate diet, HL: high lipid diet), and the last numbers represent replicated samples. GLU represents glucose, AST aspartate aminotransferase, and HDL high-density lipoprotein.

Figure 6. Histological analyses of liver tissues. Liver tissues from 4 different diet treatments were formaldehyde-fixed, followed by staining with Hematoxylin and eosin.

The results (75×) from transgenic fish are shown in the upper panels, and wild-type controls with corresponding diet treatments in the lower panels. Arrows in upper panels indicate lipid droplet (blue) and those in lower panels (green) show glycogen deposits.