Fish and Shellfish Immunology 90 (2019) 141–149 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article The microbiota profile and transcriptome analysis of immune response during metamorphosis stages in orange spotted grouper (Epinephelus T coioides) Joan Tang Xiao Joea,b, Pinwen Peter Chiouc, Chia-Yu Kuoa,b, Jacqueline Ho Jia Linc, ∗ Jen-Leih Wud, Ming-Wei Luc,e, a Doctoral Degree Program in Marine Biotechnology, The College of Life Sciences, National Taiwan Ocean University, Keelung, Taiwan b Doctoral Degree Program in Marine Biotechnology, Academia Sinica, Taipei, Taiwan c Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan d Laboratory of Marine Molecular Biology and Biotechnology, Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan e Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan ARTICLE INFO ABSTRACT Keywords: Metamorphosis is a transformation process in larval development associated with changes in morphological and Epinephelus coioides physiological features, including the immune system. The gastrointestinal tract harbors a plethora of bacteria, Metamorphosis which might affect the digestion and absorption of nutrients, immunity, and gut-brain crosstalk in the host. In 16s rRNA metagenomics this study, we have performed metagenomic and transcriptomic analyses on the intestines of grouper at the pre-, Intestinal microbiota mid- and post-metamorphosis stages. The sequencing data of 16S rRNA gene showed drastic changes in the Immune system microbial communities at different developmental stages. The transcriptomic data revealed that the leukocyte Transcriptome analysis transendothelial migration and the phagosome pathways might play important roles in mediating immunity in grouper at the three developmental stages. This information will increase our understanding of the metamor- phosis process in grouper larvae, and shed light on the development of antimicrobial strategy during larval development. 1. Introduction colonized by bacteria, archaea and eukaryotes, all together known as the gut microbiota [5]. The quantity of microorganisms living in the gut The orange-spotted grouper, Epinephelus coioides, is a commercially may reach over 95% of the total population in the whole body [6,7]. important fish in many Asian countries [1]. In grouper larval devel- The gastrointestinal microbiota is known to produce beneficial com- opment, metamorphosis is an essential and critical stage involved in the ponents related to, for example, enhancing the immune system, pro- morphological and physiological changes [2]. During metamorphosis, tecting against pathogen, nutrition, and modulating host metabolism the digestive system goes through significant changes, including the [8,9]. The gut microbiota produce short-chain fatty acids (SCFAs), such development of a functional stomach and increase in digestive capacity as propionate and butyrate, by fermentation of the non-digestible [3]. The metamorphosis process begins with the appearance of pro- substrates like dietary fibers and endogenous intestinal mucus [10]. longed second dorsal section at 12 days post hatching (dph), and is SCFAs can induce apoptosis of cancer cells, and regulate the intestinal complete by 50 dph. From 13 to 18 dph, the second dorsal section and gluconeogenesis; they also play an important role to maintain colon pelvic fin spines become clearly visible and reach to their maximal healthy [11]. Numerous evidences have demonstrated a network called lengths (Fig. 1). During the early developmental stage, the adaptive “gut-brain axis”, in which bidirectional interactions between the gut immunity is not yet fully established and functional. Hence, the innate microbiota and the central nervous system exist. Not only may the brain immunity plays an important role in protecting the host against pa- influence the function of the gut microbiota [12,13], but also altera- thogens at the early developmental stage [4]. tions of microbiota composition may be associated with several dis- The intestinal mucosal immune system encounters more antigens orders of the nervous system, including neuropsychiatric, neurode- than any other parts of the body do. The gastrointestinal tract is richly generative, and neuroinflammatory disorders [14]. As a result, gut ∗ Corresponding author. Department of Aquaculture, National Taiwan Ocean University, No.2, Beining Rd., Keelung, Taiwan. E-mail address: [email protected] (M.-W. Lu). https://doi.org/10.1016/j.fsi.2019.03.063 Received 4 January 2019; Received in revised form 26 March 2019; Accepted 26 March 2019 Available online 02 May 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved. J.T. Xiao Joe, et al. Fish and Shellfish Immunology 90 (2019) 141–149 Fig. 1. The morphology of E. coioides during metamorphosis. (A) 12 days after hatching, the second dorsal started to lengthen. (B) 18dph, the second dorsal and pelvic fin spines were clearly visible and reach to the maximal length. (C) 50dph, transformation of larvae into juveniles occurred. microbiota ultimately have a significant impact on the immunity, me- manufacturer's recommendations, and the index codes were added. The tabolism, diseases, brain and behaviors [15,16]. library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo In recent years, metagenomics has become the major technique to Scientific) and Agilent Bioanalyzer 2100 system (Agilent Technologies, study the intestinal microbiota in host [17]. To investigate the changes Inc). At last, the library was sequenced on an Illumina platform and 250 in intestinal microbiota and the development of immune system asso- bp paired-end reads were generated. ciated with metamorphosis in E. coioides, we have performed metage- Data split paired-end reads was assigned to samples based on their nomic and transcriptomic analyses on the intestines of fish at the pre-, unique barcode and truncated by cutting off the barcode and primer mid and post-metamorphosis stages. The metagenomics data showed sequence. Sequence assembly paired-end reads were processed using drastic changes in the microbial communities at different develop- FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/) (Caporaso et al., mental stages. The transcriptomic data revealed pathways and mole- 2010) to merge paired-end reads when at least some of the reads cules that might play important roles in mediating immunity in grouper overlap the read generated from the opposite end of the same DNA at the three developmental stages. The new information will increase fragment. The splicing sequences were afterwards called raw tags. Data our understanding of the development of grouper larvae into juveniles, Filtration Quality filtering on the raw tags were performed under spe- and further assist in the development of antimicrobial strategy in larvae cific filtering conditions to obtain the high-quality clean tags [18] ac- going through the metamorphosis process. cording to the QIIME (V1.7.0, http://qiime.org/index.html)[19] quality-controlled process. Chimera removal the tags were compared with the reference database (Gold database, http://drive5.com/ 2. Materials and methods uchime/uchime_download.html) using UCHIME algorithm (UCHIME Algorithm, http://www.drive5.com/usearch/manual/uchime_algo. 2.1. Experimental animals html)[20] to detect chimera sequences, and then the chimera se- quences were removed [21] to obtain Effective Tags. In this study, fertilized eggs of orange-spotted grouper were col- OTU Production Sequences analysis were performed by Uparse lected from hatchery at Pingtung, Taiwan. The animal experiment was software (Uparse v7.0.1001, http://drive5.com/uparse/)[22]. Se- conducted at National Taiwan Ocean University (Keelung, Taiwan) by quences with ≥97% similarity were assigned to the same OTUs. Re- following the institutional IACUC guideline (Approve number: presentative sequence for each OTU was screened for further annota- 106009). The tanks were stocked with Chlorella spp. prior to the in- tion. Species annotation for each representative sequence, the stallment of fertilized eggs. One gram of eggs was placed into a 100L GreenGene Database (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) tank with slightly aeration. The temperature was around room tem- [23] was used based on RDP 3 classifier (Version 2.2, http:// perature with little photoperiod. Before 3dph, the first live feed pro- sourceforge.net/projects/rdp-classifier/)[24] algorithm to annotate vided to the larvae was ss-type live rotifers (Brachionus plicatilis), which taxonomic information. Data Normalization OTUs abundance in- were fed with Chlorella spp. From 3dph to 25dph, the larvae were fed formation was normalized using a standard of sequence number cor- with s-type rotifer; the L-type rotifers were fed to 25dph throughout responding to the sample with the least sequences. Subsequent analysis 35dph juveniles. Subsequently, the juveniles were fed with copepod of alpha diversity and beta diversity were all performed based on this nauplii from 35dph to 50dph. output normalized data. The KEGG pathway used an OTU table that had already been generated for use with PICRUSt. PICRUSt currently can 2.2. DNA extraction and 16S rRNA gene sequencing only use an OTU table with GreenGene OTU identifiers which is the output from closed-reference picking or by filtering out de-novo OTUs The samples (12dph: n = 5group (150 fish pool together), 18dph: