## 6 Discussion
We performed i-means analysis on the MiSeq dataset in order to divide the data into three groups because it has been previously reported that there are three human enterotypes based on gut microbiota at the phylum level, predominantly Firmicutes, Bacteroidetes, or Actinobacteria [31]. Previous studies have proposed that Proteobacteria represent the prominent microbial phyla in the fish gut [34]. The present study revealed the fish gut microbiota to be predominantly composed of Firmicutes, Actinobacteria, or Proteobacteria, rather than Bacteroidetes as in human gut enterotypes (Figures S3 and S4). Bacteroidetes were in low-to-high abundance in individual fish gut microbiomes (Figure S3A), but the composition ratio had an impact on group separation because the importance value calculated by i-means was lower than Proteobacteria, Firmicutes, and Actinobacteria (Figure S3), which may indicate that Proteobacteria and Bacteroidetes play similar roles in the gut microbiota of fish and humans, respectively. In marine biofilms, a considerable amount of carbohydrate metabolism is carried out by Bacteroidetes and Gammaproteobacteria, including Proteobacteria; thus, Proteobacteria in the fish gut might decompose consumed carbohydrates [35]. Our analysis revealed that Bacterial Class 3 was associated with a high abundance of Proteobacteria and a low abundance of Bacteroides, and the high-intensity NMR signal that was annotated as acetate might be derived from carbohydrate degradation (Figure 3B). Bacterial Class 1 was found to have fewer association rules by our proposed scheme than Bacterial Classes 2 and 3. The associations were a higher proportion of Firmicutes, while food category 4 included crustaceans eaters and was found to share ecology category 2 with Bacterial Class 3. Crustacea use chitin and/or chitosan to form their exoskeletons [36], and it has been reported that many species of bacteria, including bacillus which are Firmicutes, express enzymes with chitinase activity [37]. Fish with a Bacterial Class 1 gut microbiome may, therefore, digest chitin in their feed via enzymes originating from their intestinal Firmicutes. This suggestion indicates that food influences the intestinal microbiome [38; 39]. We performed i-means analysis on NMR datasets in order to divide the data into four groups. This revealed that the intestinal contents of fish have many variables, but the high importance value of dividing the NMR signal clusters calculated by i-means was only TMAO, which suggested that the number of high impact factors (Gini impurity) was less than bacterial factors (Figure S3). The most important variable for dividing the NMR dataset was annotation as a TMAO signal. The group with the highest TMAO proportion was NMR Class 4 (Figure S4B). However, this group did not exhibit associations with other NMR classes, bacterial classes, or qualitative information. It is known that TMAO is involved in osmoregulation in the muscle of marine creatures [40; 41]. TMAO indicated association rules to four habitat factors: brackish water, coast, offshore, and deep sea (not fresh) water (Figure 3D). Freshwater fish have been reported to have lower TMAO concentrations in their muscle tissue than seawater fish [42; 43; 44]. Our scheme could be used to identify the characteristics of organisms using qualitative and non-numerical information.