3.2 | Comparative metagenome analysis of the intestinal microbiome in the regenerating intestine of faster and slower regenerating Apostichopus japonicus
Metagenome sequencing using the Illumina HiSeq platform was performed on the fastest and the slowest regenerating specimens (except specimens containing insufficient intestinal DNA) as representatives of faster and slower regenerating individuals used in the above analyses. To detect the effects of the intestinal microbiota on host regeneration, 3 time points [the beginning (10 d), middle (14 d) and end (21 d) of intestinal regeneration] were chosen. From the fastest and the slowest regenerating individuals, across the 3 stages, 138,957,538 reads (20.8 Gb) and 129,641,842 reads (19.4 Gb) were obtained in total, respectively. After quality filtering and removing host sequences, 33,764 and 34,187 reads from the fastest and the slowest regenerating individual, respectively, were used for MG-RAST annotation.
The metagenomic data of the faster and slower regenerating individuals from different regeneration stages (10 d, 14 d and 21 d) were compared with data in the KEGG database. The relative abundances at different functional levels were calculated, and a total of 300 functional genes were annotated to the metabolic pathways. The KEGG functional profiles also showed similarities in intestinal microbial functions in faster and slower regenerating individuals. The pathways of human diseases and metabolism contained the largest number of annotated genes, followed by pathways relevant to the organism system (Figure S2). However, there were still differences in some KEGG functional categories between the faster and slower regenerating individuals (Figure S3). The analysis demonstrated that 20 functional features in the subsystem category (level 2) were more abundant in one of the samples (Figure 3). In detail, the genes for immune system, carbohydrate metabolism, aging, and infectious diseases were more abundant in the faster regenerating individual than in the slower regenerating individual in the 10 d stage, and the genes for development, xenobiotic biodegradation and metabolism, drug resistance, and membrane transport were more abundant in the faster regenerating individual than in the slower regenerating individual in the 14 d stage. The genes annotated to energy metabolism and lipid metabolism were significantly more abundant in the faster regenerating individual than in the slower regenerating individual in the 21 d stage. Regarding the slower regenerating individual, the more abundant genes were annotated in cell growth and death, signal transduction, digestive system, and glycan biosynthesis and metabolism during intestine regeneration.
The genes were also annotated with eggNOG OGs to explore the difference in microbial functions between the faster and slower regeneratingA. japonicus individuals during intestine regeneration. The functional features of the subsystem category at different levels were analyzed. The genes annotated to ‘replication, recombination and repair’, ‘cytoskeleton’ and ‘amino acid transport and metabolism’ were most abundant, followed by genes annotated to ‘posttranslational modification, protein turnover, chaperones’, ‘cytoskeleton’ and ‘signal transduction mechanisms’ (Figure S4). The gene abundance of eggNOG functions also changed between faster and slower regenerating A. japonicus individuals, but no significant differences were observed.