4 | DISCUSSION
The intestinal microbiota has been found that plays an important role in
sea cucumber health, growth and function
(C. Li et al., 2017;
Yamazaki et al., 2016). The sea cucumberA. japonicus is one of the best model animals to study
host-microbiota interactions during organ regeneration. We have
previously reported that intestinal microbial composition and functional
genes of A. japonicus are associated with intestine regeneration
stages after evisceration and that Rhodobacterales and Flavobacteriaceae
may function as keystone taxa in the intestinal microbial community ofA. japonicus during intestine regeneration
(H. Zhang et al., 2019). However, how the
intestinal microbiota affects intestine regrowth is still unknown. Here,
we present the first description of the effects of intestinal microbial
quantity and microbiome features on developing intestine in A.
japonicus .
We demonstrated that decreased cell counts were observed in the
intestine of faster regenerating A. japonicus individuals, who
contained large fractions of Rhodobacterales and Flavobacteriaceae.
Given the importance of microbial quantity in regulating cell renewal in
the intestine of the faster regenerating individuals, we asked whether
microbial quantity could change the beneficial bacterial community and
then promote the regrowth rate by depleting the intestinal microbiota in
the GF group. We assessed the interactions between microbial quantity
and potential key players with the intestinal regrowth rate by
developing a GF sea cucumber model. We provide evidence that intestinal
microbiota depletion is both necessary and sufficient to increase the
abundances of Flavobacteriaceae and Rhodobacterales and then to promote
the intestine regrowth rate of A. japonicus , which was observed
in the GF samples and the slower and faster regenerating individuals,
respectively.
Previous studies have reported that the gut microbiota influences cell
proliferation in the gut epithelium. Intestinal epithelial cell
proliferation was reduced in GF zebrafish model
(Cheesman et al., 2011;
Rawls, Samuel, & Gordon, 2004), and
secretory cells were less abundant in the gut epithelium of GF animals
(Bates et al., 2006;
Uribe, Alam, Johansson, Midtvedt, &
Theodorsson, 1994). In contrast to previous findings, our data suggest
that depletion of the intestinal microbiota promotes the intestine
regeneration rate of A. japonicus . The intestine regeneration
rate was significantly faster in the GF sea cucumbers than in the CV sea
cucumbers. Notably, the relative abundance of Flavobacteriaceae was
significantly increased in GF samples compared to that in CV samples,
and Flavobacteriaceae was the most abundant family in GF samples. The
possible mechanism for promoting the intestine regeneration rate could
be that the reduced bacterial populations increased the abundance of
potential functional microbes during intestine regeneration since
Flavobacteriaceae and Rhodobacteraceae have been reported to be
potential key players in the intestine of A. japonicus during
intestine regeneration (H. Zhang et al.,
2019).
Flavobacteriaceae present low pathogenicity
(Jooste & Hugo, 1999), and can produce
carotenoids that have antioxidative activities
(Shindo et al., 2007). More importantly,
the marine Flavobacteriaceae usually produce enzymes that degrade agars,
fucoidan, fucose, laminarin, xylan, and carrageenans from micro- or
macroalgae (Sakai, Kimura, & Kato,
2002). Within Flavobacteriaceae, the predominant genus wasFlavobacterium , which presents high levels of resistance to a
wide range of antibiotics (Thomson,
1988). Some Flavobacterium species play a role in mineralizing
various types of organic matter (carbohydrates, amino acids, proteins,
and polysaccharides) in aquatic ecosystems and are able to degrade
various cellulose derivatives, such as carboxymethylcellulose
(Bernardet & Bowman, 2006). The enzymatic
abilities of Flavobacteriaceae may either directly or indirectly
mineralize various types of organic matter from seawater and increase
the production of carbohydrates, which is beneficial to the sea cucumber
during the intestine regeneration process and thus promotes the
intestinal regrowth rate.
Previous studies have shown that Rhodobacteraceae species are frequently
found in the intestine of sea cucumber A. japonicus(F. Gao et al., 2014;
Sha et al., 2016;
Luo Wang et al., 2018;
Yang, Xu, Tian, Dong, & Peng, 2015;
H. Zhang et al., 2019). Rhodobacterales
retaining polyhydroxybutyrate (PHB) metabolism genes, as a PHB producer,
promoted the growth of sea cucumber A. japonicus(Yamazaki et al., 2016). In addition to
our research, Rhodobacteraceae were identified as keystone taxa in the
microbial community associated with Nannochloropsis salina in
aquatic ecosystems (Geng, Tran-Gyamfi,
Lane, Sale, & Yu, 2016). In the present study, the relative abundance
of Rhodobacteraceae was obviously increased in GF samples. Thus, they
might be important for the intestinal regrowth of A. japonicus .
In this study, quantitative analysis revealed that the abundance of the
bacterial 16S rRNA gene was reduced in faster regenerating A.
japonicus individuals during intestine regeneration. Meanwhile, the
relative abundances of Flavobacteriaceae and Rhodobacteraceae in the
faster regenerating individual were higher than those in the slower
regenerating individual. Interestingly, metagenomic analyses suggested
that genes annotated to carbohydrate metabolism were more abundant in
the faster regenerating individual and that genes annotated topha A (acetyl-CoA C-acetyltransferase) were also enriched, which
is essential in PHB synthesis from acetyl-CoA to PHB
(Yamazaki et al., 2016). PHB accumulates
in commonly nutrient-limited bacterial cells
(Jendrossek & Pfeiffer, 2014), and the
bacterial PHB, especially that produced by Rhodobacterales, might also
serve as an energy source for the sea cucumber
(Yamazaki et al., 2016).
Together, our results indicate a direct link between intestinal
bacterial 16S rRNA abundance to Flavobacteriaceae and Rhodobacteraceae
and to the intestine regeneration rate of A. japonicus .
Flavobacteriaceae is related to carbohydrate production, and
Rhodobacteraceae is related to PHB production, which are beneficial to
the regenerating intestine that has an disrupted function. We predict
that depletion of the intestinal microbiota promotes the potential key
players during intestine regeneration and thus promotes the intestinal
regrowth rate of A. japonicus . In the present study, the most
abundant species in GF sea cucumbers wasFlavobacterium_unclassified . The lack of information on the
detection and/or isolation of Flavobacterium in the regenerating
intestine of A. japonicus resulted in delays in discovering
details in the quantity-structure-function relationships. Hereafter,
additional experiments should be conducted to further assess how the
keystone taxa and functional genes respond to changes in the intestinal
microbial quantity and intestinal regeneration and to further study the
ecophysiology and ecogenomics of the key players; then, we could infer
their effects on host animals. Once the specific functions are unveiled,
these bacteria could also be candidates for probiotics in sea cucumber
aquaculture.
Broadly, this study contributes to the understanding of complex
interactions between the components of intestinal ecology, which include
intestinal microbial populations and developing intestine that has
unsound function. By sequencing the intestinal microbiome of regrowth
intestine, we identified microbiota abundance as a key driver of
microbial community alterations, especially beneficial bacterial
members, in the regenerating intestine of A. japonicus . The
results indicated that intestinal microbial quantity and community
structure exert regulatory mechanisms for organ development of the host
and provide new insights into the host-microbiota interaction. Applying
similar molecular techniques to wild and farmed systems around the world
will not only further our understanding of the role of intestinal
microbiomes in host health and development, but may also provide
insights useful for improving intestinal ecology, management, and
conservation of fishery resources.