fmars-08-705332 July 13, 2021 Time: 16:51 # 1 ORIGINAL RESEARCH published: 19 July 2021 doi: 10.3389/fmars.2021.705332 Dynamic Alterations of the Distal Intestinal Microbiota, Transcriptome, and Metabolome of Hybrid Grouper by b -Conglycinin With Reconciliations by Sodium Butyrate in Feed Bin Yin1,2,3, Hongyu Liu1,2,3*, Beiping Tan1,2,3*, Xiaohui Dong1,2,3, Shuyan Chi1,2,3, Qihui Yang1,2,3 and Shuang Zhang1,2,3 1 Laboratory of Aquatic Animal Nutrition and Feed, Fisheries College, Guangdong Ocean University, Zhanjiang, China, Edited by: 2 Aquatic Animals Precision Nutrition and High Efficiency Feed Engineering Research Centre of Guangdong Province, Ikram Belghit, Zhanjiang, China, 3 Key Laboratory of Aquatic, Livestock and Poultry Feed Science and Technology in South China, Ministry Norwegian Institute of Marine of Agriculture, Zhanjiang, China Research (IMR), Norway Reviewed by: Different doses of b-conglycinin produce different regulations on the intestinal health Fotini Kokou, Wageningen University and Research, of aquatic animals, affecting the absorption of nutrients, indirectly changing water Netherlands quality. Sodium butyrate (NaB) can effectively alleviate the negative effects caused Seyyed Morteza Hoseini, Iranian Fisheries Science Research by high-dose b-conglycinin. We investigated the positive response to low-dose Institute (IFSRI), Iran (1.5%, bL) and negative response to high-dose (6.0%, bH) b-conglycinin and *Correspondence: supplementation with NaB (6.0% b-conglycinin + 0.13% NaB, bHNaB) in terms of water Hongyu Liu pollutants, microbiota, transcriptome, and metabolome in hybrid grouper (Epinephelus [email protected] Beiping Tan fuscoguttatus × E. lanceolatus ). The ammonia nitrogen, nitrite, total nitrogen, and [email protected] total phosphorus♀ contents were♂ significantly higher in the water from bH than from FMb, bL, and bHNaB. Supplementing with NaB significantly reduced the ammonia Specialty section: This article was submitted to nitrogen, nitrite, total nitrogen, and total phosphorus contents. Low-dose b-conglycinin Aquatic Physiology, increased the relative abundance of Pelagibacterium, Pediococcus, Staphylococcus, a section of the journal Frontiers in Marine Science and Lactobacillus and promoted the “ribosome,” “peroxisome proliferator-activated Received: 05 May 2021 receptor (PPAR) signaling” and “histidine metabolism.” High-dose b-conglycinin Accepted: 23 June 2021 increased the relative abundance of pathogenic bacteria Ralstonia and Photobacterium Published: 19 July 2021 and inhibited the “cell cycle” “PPAR signaling” and “starch and proline metabolism.” NaB Citation: supplementation at high-dose b-conglycinin reduced the Ralstonia and Photobacterium Yin B, Liu H, Tan B, Dong X, Chi S, Yang Q and Zhang S (2021) abundance and promoted the “cell cycle,” “linoleic acid metabolism,” and “ABC Dynamic Alterations of the Distal transporters.” Overall, these results reveal differences in the effects of high- and low- Intestinal Microbiota, Transcriptome, and Metabolome of Hybrid Grouper dose b-conglycinin, as well as NaB supplementation, on the utilization of proteins, by β -Conglycinin With carbohydrates, and lipids and on substance transport and signaling among distal Reconciliations by Sodium Butyrate intestinal cells of hybrid grouper. A total of 15 differential metabolite biomarkers were in Feed. Front. Mar. Sci. 8:705332. doi: 10.3389/fmars.2021.705332 identified: FMb vs. bL contained 10-methylimidazole acetic acid, N-acetyl histamine, Frontiers in Marine Science| www.frontiersin.org 1 July 2021| Volume 8| Article 705332 fmars-08-705332 July 13, 2021 Time: 16:51 # 2 Yin et al. b-Conglycinin and Sodium-Butyrate on Grouper urocanic acid, creatinine, glutathione, taurine, nervonic acid, stearic acid, docosanoic acid, and D-serine; FMb vs. bH contained 4-L-fucose, sucrose, a,a-trehalose, and quercetin; and bH vs. bHNaB contained 4-N-acetyl histamine, urocanic acid, creatinine, and S-adenosylhomocysteine, respectively. Our study provides new insights into the regulation of intestinal health by b-conglycinin in aquatic animals and the protective mechanism of NaB. Keywords: hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus ), b-conglycinin, sodium butyrate, intestinal microbiota, transcriptome, metabolome ♀ ♂ INTRODUCTION It is accepted that b-conglycinin (7S) of soybean meal is a major factor in the induction of intestinal injury. b-Conglycinin, The hybrid grouper (Epinephelus fuscoguttatus one of the major antigen proteins in soybean, often causes allergic × E. lanceolatus ) is an economically important fish along♀ reactions in young animals, causing inflammatory damage to the southern coast♂ of China, with a fast growth rate and strong intestinal epithelial cells accompanied by lipid peroxidation environmental tolerance. In 2017, groupers were officially of cell membranes, negatively affecting the function of tight included in the construction of China’s National Marine Fish junction protein structures and increasing intestinal epithelial Industry Technology System. As a typical carnivorous fish, it permeability (Zhao et al., 2014). The unbalanced amino acid usually requires up to about 50% of protein in its feed (Jiang in soybean meal may lead to increased excretion of nitrogen et al., 2016). Fishmeal is a high quality protein source and it and phosphorus by fish (Tantikitti et al., 2005), which is not has been traditionally used as the main protein source in the conducive to the healthy and sustainable development of the aquafeed industry. However, due to the high demand for fishmeal aquaculture industry. As a widely used additive in livestock and leading to a high price and lack of sources, the farming industry poultry animals, sodium butyrate (NaB) can not only act as a had to find dietary alternative ingredients. The high nitrogen food attractant, but also improve the tight junctions of intestinal and phosphorus content in the fishmeal feeds is not completely epithelium (Huang et al., 2015) and alleviate inflammation by absorbed by hybrid grouper and is most likely to be excreted inhibiting nuclear factor kappa B (NF-kB) (Albino et al., 2012), along with the feces, leading to serious contamination of the and it also has a positive regulatory effect on the host’s intestinal farmed water. Currently, the use of soybean meal to replace fish microbiota (Zou et al., 2019). The active ingredient in NaB is meal can relieve the pressure of fish meal shortage and greatly butyric acid, which is a short-chain, volatile fatty acid (O’Hara promote the sustainable development of aquaculture industry et al., 2018). In aquatic animals, NaB can stimulate the growth and (Yang et al., 2011; Smith et al., 2017; Miao et al., 2018). However, proliferation of intestinal mucosa and inhibit the proliferation owing to low tolerance to soybean meal, excessive intake of of intestinal pathogenic microorganisms, thus, promoting the soybean meal can lead to varying degrees of intestinal injury growth of fish and enhancing intestinal digestion and antioxidant (Zhang et al., 2019). capacity (Tian et al., 2017; Jesus et al., 2018; Fu et al., 2019). The intestines of aquatic animals are responsible for the Nevertheless, the physiological mechanisms of intestinal injury dual functions of digestion and absorption. The mucosa of the and inflammation in hybrid grouper caused by b-conglycinin intestinal tract is the main interface between the internal and and the protective effects of NaB on the intestinal tract are not external environments of the organism. The intestinal tract is well understood. the main site of nutrient exchange and infection or initiation In a previous study, we demonstrated that ingestion of high of infection by many pathogens (Jiang et al., 2019) and is levels of soybean meal by hybrid grouper resulted in intestinal highly susceptible to the effects of feed ingredients and living injury (Zhang et al., 2019). The inhibitory effects of b-conglycinin environmental conditions (Duan et al., 2017). Thus, it is essential on the growth of fish is usually thought to be due to intestinal to maintain the health and stability of the intestinal tracts of inflammation (Zhang et al., 2013); nevertheless, the reports of aquatic animals. The presence of a large number of microbiota whether b-conglycinin can negatively affect other physiological in the intestinal tract is the result of long-term evolution, and functions in fish are rare. In this study, we investigated the they are closely related to the immune function and nutritional effects of b-conglycinin and the protective mechanism of NaB on requirements of the organism (Yang et al., 2018). Intestinal transcription, intestinal microbiota, and metabolites in the distal microbiota can participate in the mediation of multiple metabolic intestine of hybrid grouper. Then, transcriptomic, intestinal pathways in the host, interacting with host metabolism and microbiota, and metabolomic means were combined to model signal transduction to form a physiologically connected gut- the associations among host metabolism, intestinal microbiota, immune-inflammation axis (Matsumoto et al., 2012). Similarly, and aquaculture water pollutants at the system biology level. This intestinal health status could also influence the composition of will help us understand the metabolic processes of the microflora microorganisms. Therefore, the relationship between intestinal in the host and their effects on host transcription, as well as their flora and metabolite associations and host intestinal health interactions with each