microorganisms Article Taxonomic and Functional Characteristics of the Gill and Gastrointestinal Microbiota and Its Correlation with Intestinal Metabolites in NEW GIFT Strain of Farmed Adult Nile Tilapia (Oreochromis niloticus) Zhenbing Wu 1,2 , Qianqian Zhang 1,3, Yaoyao Lin 1,2, Jingwen Hao 1,2, Shuyi Wang 1,2, Jingyong Zhang 1,3 and Aihua Li 1,3,* 1 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; [email protected] (Z.W.); [email protected] (Q.Z.); [email protected] (Y.L.); [email protected] (J.H.); [email protected] (S.W.); [email protected] (J.Z.) 2 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 3 Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, Wuhan 430072, China * Correspondence: [email protected]; Tel.: +86-27-68780053 Abstract: The gill and gastrointestinal tract are primary entry routes for pathogens. The symbiotic microbiota are essential to the health, nutrition and disease of fish. Though the intestinal microbiota of Nile tilapia (Oreochromis niloticus) has been extensively studied, information on the mucosa-associated microbiota of this species, especially the gill and gastrointestinal mucosa-associated microbiota, is Citation: Wu, Z.; Zhang, Q.; Lin, Y.; lacking. This study aimed to characterize the gill and gastrointestinal mucosa- and digesta-associated Hao, J.; Wang, S.; Zhang, J.; Li, A. microbiota, as well as the intestinal metabolite profiles in the New Genetically Improved Farmed Taxonomic and Functional Characteristics of the Gill and Tilapia (NEW GIFT) strain of farmed adult Nile tilapia by high-throughput sequencing and gas chro- Gastrointestinal Microbiota and Its matography/mass spectrometry metabolomics. The diversity, structure, composition, and predicted Correlation with Intestinal function of gastrointestinal microbiota were significantly different across gastrointestinal regions and Metabolites in NEW GIFT Strain of sample types (Welch t-test; p < 0.05). By comparing the mucosa- and digesta-associated microbiota, Farmed Adult Nile Tilapia linear discriminant analysis (LDA) effect size (LEfSe) analysis revealed that Pelomonas, Ralstonia (Oreochromis niloticus). Microorganisms pickettii, Comamonadaceae, and Staphylococcus were significantly enriched in the mucosa-associated 2021, 9, 617. https://doi.org/ microbiota, whereas many bacterial taxa were significantly enriched in the digesta-associated mi- 10.3390/microorganisms9030617 crobiota, including Chitinophagaceae, Cetobacterium, Candidatus Competibacter, Methyloparacoccus, and chloroplast (LDA score > 3.5). Furthermore, Undibacterium, Escherichia-Shigella, Paeniclostridium, Academic Editor: and Cetobacterium were dominant in the intestinal contents and mucosae, whereas Sphingomonas Łukasz M. Grze´skowiak aquatilis and Roseomonas gilardii were commonly found in the gill and stomach mucosae. The Phylo- Received: 1 February 2021 genetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) analysis Accepted: 16 March 2021 revealed that the predictive function of digesta-associated microbiota significantly differed from that Published: 17 March 2021 of mucosa-associated microbiota (R = 0.8152, p = 0.0001). In addition, our results showed a signif- icant interdependence between specific intestinal microbes and metabolites. Notably, the relative Publisher’s Note: MDPI stays neutral abundance values of several potentially beneficial microbes, including Undibacterium, Crenothrix, with regard to jurisdictional claims in and Cetobacterium, were positively correlated with most intestinal metabolites, whereas the relative published maps and institutional affil- abundance values of some potential opportunistic pathogens, including Acinetobacter, Mycobac- iations. terium, Escherichia-Shigella, Paeniclostridium, Aeromonas, and Clostridium sensu stricto 1, were negatively correlated with most intestinal metabolites. This study revealed the characteristics of gill and gastroin- testinal mucosa-associated and digesta-associated microbiota of farmed Nile tilapia and identified a close correlation between intestinal microbes and metabolites. The results serve as a basis for Copyright: © 2021 by the authors. the effective application of targeted probiotics or prebiotics in the diet to regulate the nutrition and Licensee MDPI, Basel, Switzerland. health of farmed tilapia. This article is an open access article distributed under the terms and Keywords: Nile tilapia; gill; gastrointestinal tract; microbiota; microbial function; high-throughput conditions of the Creative Commons sequencing; metabolomics Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Microorganisms 2021, 9, 617. https://doi.org/10.3390/microorganisms9030617 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 617 2 of 24 1. Introduction The microbiota of fish, like other vertebrates, play critical roles in host health and metabolism [1]. Fish microbiota include resident communities (autochthonous or associated with mucosa) and transient communities (allochthonous or associated with digesta) [2]. Most infections start at or affect the fish mucosal epithelia. The three major infection routes in fish are the gills, the skin, and the gastrointestinal tract [3], which are the first points of contact for pathogens. The mucosal surfaces of these organs represent an important barrier that supports and regulates a wide variety of microbial communities [2]. These diverse and balanced mucosa-associated microbial communities are essential for fish health [4], but the mechanism underlying their interactions with the host remains unclear. Characterizing the microbiota in healthy fish is an essential first step to elucidating the impacts of microbial manipulation in aquaculture systems. The evaluation of the bacte- rial community attached to the gills, which is the respiratory organ, has a long history [5]. However, except for several recent studies [6–10], this topic has received little attention. The gastrointestinal tract is a complex ecosystem that harbors diverse microbial commu- nities that can increase digestion efficiency and the use of nutrients, boost the immune system, and prevent the attachment and proliferation of opportunistic pathogens [11]. The establishment of balanced gastrointestinal microbiota is important in fish health and digestive function [11]. However, information regarding the functional roles that these microbes play in host metabolism is limited. The New Genetically Improved Farmed Tilapia (NEW GIFT) strain of Nile tilapia is the most commonly farmed tilapia in China and Southeast Asia [12]. The great impact and success of NEW GIFT is evident in developing countries, in which it has helped improve food and income security [12]. However, intensive aquaculture of tilapia has caused various disease problems, especially bacterial diseases [13], which has ultimately led to considerable mortality and consequent economic losses. Recently, insights into the farmed fish microbiome have opened up tantalizing new prospects for optimizing health and productivity in aquaculture systems, such as an increase in the application of probiotics [14]. Host-associated probiotics have a greater chance of competing with resident microorganisms, thus more effectively colonizing the host [15]. Therefore, the use of host- associated probiotics for disease prevention has become a consensus in aquaculture [15], and related studies have been conducted in tilapia [16–18]. To better screen host-associated probiotics, the gill and gastrointestinal mucosa-specific microbiota of farmed tilapia need to be further studied. To date, knowledge of the fish normal microbiome has mostly focused on the char- acteristics of microbial diversity, and little information on the functional capacity of the fish microbiome is available [19]. In the present study, we used 16S rRNA gene high-throughput sequencing and non-targeted gas chromatography/mass spectrometry (GC/MS) metabolomics technology to characterize the taxonomic diversity and functional potential of a farmed tilapia microbiome. The objectives of this study were as follows: (1) to characterize the structure and diver- sity of the gill and gastrointestinal digesta- and mucosa-associated microbiota in the NEW GIFT strain of farmed adult Nile tilapia, (2) to compare the predicted function of symbiotic microbiota at different sites in NEW GIFT Nile tilapia, and (3) to explore the correlation between intestinal microbiota and metabolites in NEW GIFT Nile tilapia under commercial aquaculture conditions. Results provide basic support for the nutritional health and disease prevention of farmed tilapia in aquaculture. 2. Materials and Methods 2.1. Sample Collection and Preparation Nile tilapia (NEW GIFT strain of Oreochromis niloticus) was sourced from the same batch of artificially incubated seeds and raised in flow-through aquaculture systems at Yingshan County, Huanggang City, Hubei Province, China. The experimental water was from a warm spring in the local mountain, and the water quality was fresh and Microorganisms 2021, 9, 617 3 of 24 unpolluted. After oxygenation and purification treatment in the aeration tank, all water quality parameters were within the normal range for tilapia. The water temperature was kept at about 28.5 ◦C, which was within the optimal temperature range for tilapia farming [20,21]. The fish were fed with the commercial diet at a daily rate of 3%