Scallop visceral mass and mantle are generally considered as inedible portions of aquatic products. However, they are also rich in protein, amino acids, fatty acids and other nutrient substances (Li et al., 2014; Wu et al., 2016; Xing et al., 2011), which is a potential source for the feed supplement of precious marine products. In this case, we turned our attention towards the effects of dietary scallop visceral mass and mantle on growth, amino acid and fatty acid profiles, immune responses and intestinal microbiota of sea cucumber (A. japonicus). Growth performance is one of the most important indices to evaluate the effects of sea cucumber aquaculture. Our results show that the dietary supplementation of scallop viscera mass could promote the SGR of sea cucumber, which was increased by 356% on day 20 compared with that in the CK group. This indicates a beneficial effect of dietary supplementation of scallop visceral mass on the growth of juvenile A. japonicas. Furthermore, scallop viscera was also found to improve the nutrition composition of sea cucumber. Fatty acids, especially highly unsaturated fatty acids (HUFAs), have been reported to play important roles in physiology and reproductive processes of both plants and animals (Bergé and Barnathan, 2005; Liu et al., 2007). Tissue fatty acid composition is an important index of the metabolism and growth of sea cucumber (Dalsgaard et al., 2003). In this case, the results show that dietary supplementation of scallop visceral significantly increased the contents of ω-3 fatty acids including 20:3ω3, EPA, DHA, as well as ω-3/ω-6 ratio. ω-3 fatty acid is essential to the growth and reproduction of sea cucumber. Study in Parastichopus californicus indicated that high level of EPA and DHA in diet resulted in higher fecundity of female broodstock and better survival rate of descendant larvae (Whitefield et al., 2018). Higher ω-3 fatty acid content in A. japonicas tissue indicates the beneficial effect of scallop visceral mass on the health of sea cucumber. On the other hand, ω-3 PUFAs such as EPA and DHA bring more nutritional benefits to human health, and a diet with high ratio of ω-3/ω-6 fatty acid is more desirable in reducing the risk of many chronic diseases, such as cardiovascular disease, cancer and inflammatory (Simopoulos, 2008). Therefore, higher ω-3 fatty content and ω-3/ω-6 ratio also suggest higher commercial value of A. japonicas.
The increase of tissue ω-3 fatty acids in the SV group likely results from the extremely high content of total lipid as well as ω-3 fatty acids in the scallop visceral mass (Table. 1). In addition, the protein contents of scallop visceral mass and mantle approach to that of fish powder (about 70%) (Yu et al., 2015) and far exceed algae (Table 1). Therefore, scallop visceral mass and mantle, as aquatic product waste, could be used as the protein source substituting fish powder in sea cucumber diet, and increase the ω-3 fatty acid content of sea cucumber tissue at the same time.
Previous studies on sea cucumber culture have indicated that dietary supplementation of probiotics and biologically active substances could regulate intestinal microbiota and immunity (Wei et al., 2015; Yang et al., 2019; Yang et al., 2015). Hence, we further investigated the effect of dietary supplementation of scallop visceral mass and mantle on the immune related gene expressions and intestine microbial community of A. japonicas. Sea cucumbers are invertebrates that lack adaptive immune responses, and their defense mechanisms mainly rely on the nonspecific immune system of pattern recognition receptors (PRRs) and signal transduction (Xue et al., 2015). Toll-like receptors (TLRs) are well-characterized among the various types of PRRs in A. japonicus, which could specifically recognize conserved molecular structures and activate immune system (Kongchum et al., 2011). TLRs can recruit adaptor molecules MyD88 and TRAF6 for signal transduction to activate nuclear factor-kappa B (NF-κB) and mitogen activated protein kinases (MAPKs) pathways (Akira and Takeda, 2004; Lu et al., 2013a; Lu et al., 2013b). Here, it is found that the expression of some genes related to the Toll-like receptor signal transduction in nonspecific immune system was up-regulated by dietary supplementation of scallop visceral mass and mantle, including AjToll, MyD88 and TRAF6 (two key adaptor molecules), p50, p105 and rel (three NF-κB proteins), MKK36 and p38 (two MAPK proteins). These results suggest a promising effect of dietary scallop visceral mass and mantle on the enhancement of immune defense in sea cucumber. Additionally, it is shown that the benefit for the immune system resulting from dietary scallop visceral mass and mantle have time effect. For SM group, the up-regulation of these immune related genes showed the most remarkable effect on day 15, while SV group exhibited the best immunostimulatory activity on day 30, suggesting different action modes of scallop visceral mass and mantle on immunity regulation of the sea cucumber.
Furthermore, dietary supplementation of scallop viscera and mantle are also found to manipulate the diversity of microbial community in intestine of A. japonicas. Dietary scallop viscera markedly increased the specific OUT of intestinal microbe during the whole process (Fig. 5), suggesting higher microbial diversity and more potential functions of microbial community. The microbial diversity index and the number of biomarkers were significantly increased by dietary supplementation of scallop viscera and mantle (Fig. 6, 7), especially on day 30, when the abundance of Bifidobacteriaceae, Streptomycetaceae, Clostridiaceae, Lachnospiraceae, Monoglobaceae and Rhizobiales in group SV and SM markedly increased compared with the CK group.
Family Bifidobacteriaceae, Streptomycetaceae, Clostridiaceae in the gut of A. japonicas in this case are dominantly contributed by genus Bifidobacterium, Streptomyces and Clostridium, respectively. Bifidobacterium is one of the best known probiotic bacteria, exhibiting antagonistic activities against microbial pathogens, immunomodulatory, antimutagenic and anticarcinogenic activities, and the effects of prevention and cure of pathogen induced diarrheas (Divya et al., 2012; Kim et al., 2012; Servin, 2004; Zorriehzahra et al., 2016). Streptomyces could produce many kinds of metabolites which have been used as antibiotics and drug primers with strong inhibition of bacteria and pathogens (Bi and Yu, 2016; Bibb, 2013). Streptomyces has been used in aquaculture and demonstrated the potential of bioremediation and improving animal growth and water quality (Babu et al., 2018; Das et al., 2006). Clostridium is one of the richest bacterial cluster in the intestine of human and animals. Clostridium species have been reported to attenuate inflammation and maintain the intestinal health via their cellular components and metabolites including butyrate, secondary bile acids and indolepropionic acid (Guo et al., 2020). It has been used in aquaculture and exhibited beneficial effects of promoting the growth performance, immune response, and digestive enzyme activities (Wang et al., 2019). Rhizobiales has been found in the intestine of zebrafish and shark and is associated with nitrogen fixation (Sapountzis et al., 2015; Stoll et al., 2007). The existence of Rhizobiales could alleviate nitrogen limitation through nitrogen fixation, producing bacterial nifH protein and enhance the growth of colony (Sapountzis et al., 2015).
It is shown that supplementation of scallop visceral mass and mantle increased the microbial diversity and the abundance of beneficial microbes, which might facilitate to establish a heathier microbial ecosystem in the intestine of A. japonicus. Existing studies show that the dietary supplementation of probiotics might improve the innate immunity of sea cucumber (Lu et al., 2021; Sonnenburg et al., 2006; Wang et al., 2021). The enhancement of immune response of A. japonicus probably is also associated with the optimization of microbial community with dietary supplementation of scallop visceral mass and mantle (Supplementary Fig. S3).