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.