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.