Transcriptome analysis of juvenile genetically improved farmed tilapia (Oreochromis niloticus) livers by dietary resveratrol supplementation

Transcriptome analysis of juvenile genetically improved farmed tilapia (Oreochromis niloticus) livers by dietary resveratrol supplementation

Comparative Biochemistry and Physiology, Part C 223 (2019) 1–8 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Pa...

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Comparative Biochemistry and Physiology, Part C 223 (2019) 1–8

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc

Transcriptome analysis of juvenile genetically improved farmed tilapia (Oreochromis niloticus) livers by dietary resveratrol supplementation

T

Yao Zhenga, Gengdong Hua, Wei Wua, Zhixiang Zhaob, Shunlong Menga, Limin Fana, Chao Songa, ⁎ Liping Qiua, Jiazhang Chena,b,c, a Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences/Fishery Eco-Evironment Monitoring Center of Lower Reaches of Yangtze River, Ministry of Agriculture/Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Environmental Factors(Wuxi), Ministry of Agriculture, Wuxi, Jiangsu 214081, PR China b Wuxi Fishery College, Nanjing Agricultural University, Wuxi, Jiangsu 214081, PR China c Key Laboratory of Control of Quality and Safety for Aquatic Products, Ministry of Agriculture, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Resveratrol GIFT RNA sequencing Immune response Chinese medical herbs

Here we used RNA-Seq to explore the transcriptomic response and specific involvement of hepatic mRNA of juvenile Oreochromis niloticus (GIFT) as a result of dietary resveratrol supplementation (0.05 g/kg RES). More than 24,513,018 clean reads were reference genome guided assembly into 23,417 unigenes. 12,596 unigenes (29.64%) were annotated to GO database. There were 5, 179 and 1526 genes significantly differentially expressed genes at 15, 30 and 45 d respectively, and 8 KEGG pathways were enriched associated with this immune response. Hyperemia and compressed hepatic sinusoid, fibrosis of liver cell and abnormal hepatic epidermal cell revealed by H&E and SEM analysis respectively. Genes related with cytokine production (il12rb2, scfr), immune system (ig8l, hlfl, cd226, prf1l), autophagy regulation (atg4b), foxo signaling (ccnb2), steroid hormone biosynthesis (cyp3a40), fatty acid metabolism (scd1), metabolism (cacna1b) have been significantly decreased, while genes associated with such pathways above (leap-2, prdx4, mb, homer1, mif, sat1, cytbc1_8) and the pathway of protein processing in endoplasmic reticulum (cne1, tram1) have been significantly increased. These findings suggested RES activated some immune and biological process-related genes to enhance GIFT's innate immunity. It also suggested high concentration addition or long-time administration may bring negative effect in tilapia liver.

1. Introduction Traditional Chinese medical herbs have been used to cure various diseases in fish for thousands of years, with oral administration being the most common method of administration (Sham et al., 2014). Field investigations have revealed that floating bed cultivation of Polygonum cuspidatum (Huzhang in Chinese) in tilapia ponds not only improved the water quality, but also enhanced disease resistance to streptococcal infection (Zheng et al., 2019a, 2019b). P. cuspidatum contains anthraquinones, especially emodin polygonin, quercetin, and resveratrol (RES, distributed mostly in the root) and interestingly, tilapia often bites the roots of P. cuspidatum in aquatic ponds. Several studies shown that polyphenol (e.g. RES) has pharmacological activity when used as a

feed supplement (Kowalska et al., 2016). RES, a polyphenolic phytoalexin found in numerous plants, especially in the skin of grapes, has been reported to have antioxidative (Wilson et al., 2015) and vascular-targeting (Alex et al., 2010) in several fish models. RES dietary supplements are currently commercially available and marketed as anti-inflammatory “nutraceuticals.” RES is indicated to exert important modulatory effects on inflammatory responses in fish. Medaka (Oryzias latipes) fed a diet containing 40 and 80 mg/kg RES showed a significantly higher percentage of macrophages than untreated fish (Kowalska et al., 2016). Considering the importance of innate immunity in these vertebrates and their similarities with mammals, it has been suggested that it is possible to analyze the innate immune system of fish by examining the effects of substances

Abbreviations: Nr, non-redundant protein database; Nt, non-redundant nucleotide database; GO, gene ontology; COG, Clusters of Orthologous Groups; KEGG, Kyoto Encyclopedia of Gene and Genomes; NGS, next-generation sequencing; RIN, RNA integrity number; NCBI, National Center for Biotechnology Information; DEG, differentially expressed gene; RES, resveratrol; ICP-OES, inductively coupled plasma optical emission spectrometry; qRT-PCR, real time quantitative PCR; Ct, threshold cycle; E, efficiency; HE, hematoxylin-eosin; SEM, scanning electron microscope; HMDS, hexamethyldisilizane; ANOVA, one-way analysis of variance ⁎ Corresponding author at: Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, No. 9 Shanshui east Rd., Wuxi, Jiangsu 214081, PR China. E-mail address: [email protected]ffrc.cn (J. Chen). https://doi.org/10.1016/j.cbpc.2019.04.011 Received 14 March 2019; Received in revised form 18 April 2019; Accepted 19 April 2019 Available online 24 April 2019 1532-0456/ © 2019 Published by Elsevier Inc.

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the present study. 0.05 g/kg dietary supplementation of RES was added to the feed made by our laboratory (Zheng et al., 2017c, 2018), and the components are presented in Table S1. Half of the fish were divided into 0.05 g/kg RES groups, and half without RES addition in triplicate. Fish were fed twice per day at 8:00 am and 15:00 pm, and the feeding rate was set 4% based on the total amount of fish (DeLong et al., 2009). There were no statistically significant differences in bodyweight or length in the exposure experiment. No abnormal behavior or mortality occurred during the experimental period. Fish cultivation water was sampled once every two days in triplicate dating from the initial exposure day. All experiments were approved by the Institutional Animal Care and Use Committee of the Ministry of Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences and were undertaken in accordance with the national legislation for fish welfare established by the Ministry of Science and Technology of the People's Republic of China (approval ID: 2011AA1004020012).

on their inflammatory responses (Zheng et al., 2017a, 2017b). Recent advances in the development of the next-generation sequencing (NGS) technology have been valuable tools for genomic research and have greatly improved the efficiency and speed of gene discovery. RNA-seq technology, the most commonly used for the transcriptome analysis over the last decade, facilitates functional genomic studies, including global gene expression, novel gene discovery, assembly of full-length genes, etc. (Huang et al., 2011), and has been applied to identify genes from aquaculture species, such as orangespotted grouper (Epinephelus coioides, Huang et al., 2011), tilapia (Oreochromis niloticus, Zhang et al., 2013). GIFT (O. niloticus) is sensitive to the stress of various treatments and which can be used it as a model for immunological experimentation (Zheng et al., 2019a, 2019b). The total global production of tilapia has reached five million tons; in China, the total production is 1.6 million tons (Fitzsimmons et al., 2011). The disease streptococcosis is caused by Streptococcus iniae and leads to high losses amounting to 23 billion dollars US per year to the tilapia market. A higher feed intake causes tilapia to grow faster between the months of July and September (Zheng et al., 2019b), and an effective strategy to reduce infection and disease is dietary supplementation with functional immunopotentiators. RES and its analogs have been used as potential candidates for the treatment and prevention of inflammatory responses (Kowalska et al., 2016). Liver has been used as the response tissue when tilapia facing different disease challenge (Yang et al., 2013; Zhan et al., 2015), and recently RNA-seq, miRNA-seq have been used in order to follow the molecular mechanism (Qiang et al., 2017; Zheng et al., 2017c). Our previous study showed that 0.05 g/kg dietary supplementation RES affected the metabolism pathway of peroxisome, proteasome and protein processing in endoplasmic reticulum (Zheng et al., 2017c), however, hepatic organic impairment has also revealed in our earlier reports (Zheng et al., 2017a, 2017b). Long-time duration with RES administration had different pathophysiological anti-neovascular effects (Richer et al., 2014). In the present study, the transcriptome of GIFT was characterized by means of Illumina/Hiseq-2500 RNA-seq technology and a comparative analysis of transcriptome data between the control and RES addition was performed to identify immune-related genes and biological processes involved in the innate response.

2.1.2. Fish sampling All fish liver samples of the control and 0.05 g/kg RES groups were collected at 15 d (T01_T07_T13_ and _T02_T08_T14 for control and 0.05 g/kg RES, respectively), 30 d (T19_T20_T21_, _T03_T09_T15, respectively) and 45 d (T22_T23_T24_, _T04_T10_T16, respectively). In each tank per sampling point, fish liver were sampled for gene expression (n = 3) and RNA-seq (n = 3). Samples for gene expression studies were homogenized using TRIzol reagent (Invitrogen, USA), frozen in liquid nitrogen and stored at −80 °C immediately until utilization.

2.2. RNA-seq 2.2.1. RNA quantification and qualification RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit® RNAAssay Kit in Qubit®2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

2. Materials and methods 2.1. Experimental design

2.2.2. Library preparation for transcriptome sequencing A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonucleases/polymerases. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 200–250 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

2.1.1. Animals Fertilized eggs of O. niloticus were obtained from Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences, Yixing. The fish fry has been cultivated in the pond (20 m × 30 m), and the acclimatization period lasted for two weeks before the experiment. Two-month old O. niloticus juveniles were used in the experiment. Fish were acclimatized in the aquarium facility with dechlorinated tap water at 25 ± 1 °C, with 14 h:10 h light/dark cycle. The experimental fish were offered feed once a day (Jiangsu Zhe Ya Food. Co. Ltd., China). Fish (from 41.2 g to 50.9 g, from 10.5 cm to 11.4 cm) were randomly selected for exposure experiments. Throughout the experimental period, water samples were taken before and after each water change, and the experimental conditions were as follows: pH, 7.1 ± 0.5 units; dissolved oxygen (tested by YSI 556MPS, USA), 7.16 ± 0.16 mg/L; total phosphate, 2.16 ± 0.17 mg/L; total nitrogen and ammonia nitrogen (by Nessler's reagent spectrophotometry), 0.52 ± 0.15 mg/L and 0.44 ± 0.06 mg/L respectively; total water hardness (ICP-OES, Optima 7000, PerkinElmer, USA), 194.3 ± 13.0 mg/L CaCO3. The GIFT juveniles (n = 120) were assigned to six tanks (n = 20 per aquarium, 1500 L, 1.5 m length x 1.0 m width x 1.0 m height). RES was purchased from Sigma-Aldrich (St Louis, MO, USA). It was revealed that 0.05 g/kg dietary supplementation of RES induced hepatic immune response, and hence, the concentrations were also used in the feed in 2

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2.5. Expression verification

2.2.3. Clustering and sequencing The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PECluster Kit v4-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated. The reference genomes were chosen tilapia (PRJNA354796 and PRJNA59571) and the assembly numbers were GCA_001858045.2 and GCA_000188235.2 respectively. In the present study, genetic data have been deposited in the NCBI Short Read Archive (SRA) under the project accession PRJNA383255.

2.5.1. RNA isolation and reverse transcription (RT) Livers of fish at different sampling points were homogenized in TRIzol reagent (Invitrogen, USA) and total RNAs were extracted and treated with RNase-free DNase I (Fermentas, Canada) to remove genomic DNA contamination as previously described (Zheng et al., 2017b). RNA isolation, RNA quality checking, and reverse transcription were performed as previously described (Zheng et al., 2017b).

2.5.2. qRT-PCR, DEGs selection and validation The qRT-PCR was performed using CFX96 thermocycler (Bio-Rad, USA) and SYBR Premix ExTaq II kit (TaKaRa, Japan). The qRT-PCR reactions were carried out in a final volume of 25 μL, using 1× SYBR Premix Ex Taq™, 0.4 μM of each primer, and 2.5 μL RT reaction solution. Cycling parameters were: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s. Each individual sample was run in triplicate. A melt curve analysis was performed at the end of each PCR thermal profile to verify the specificity of each amplicon. Analysis of SYBR green I density and determination of threshold cycle (Ct) values were carried out by CFX Manager software (Bio-Rad, USA). The efficiency (E) of each PCR reaction was determined by the slope generated by 10-fold diluted cDNA series with five dilution points measured in triplicate. The equation was E = 10 (−1/slope) (Pfaffl, 2001). Gene expression changes were detected in livers in the current study, and the qRT-PCR primers are presented in Table S4. With the respect to find the molecular mechanism on immune system or metabolism in GIFT by dietary resveratrol supplementation, gene expression profiles of the selected pathways have been detected in the sampled liver tissues. These pathways included cytokine il12rb2 (interleukin-12 receptor subunit beta-2-like), scfr (mast/stem cell growth factor receptor kita-like isoform X1); immune system pathway: ig8l (immunoglobulin superfamily member 8-like), hlfl (hepatic leukemia factor-like), cd226 (CD226 antigen-like isoform X2), prf1l (perforin-1like), leap-2 (liver-expressed antimicrobial peptide 2-like), prdx4 (peroxiredoxin-4-like isoform X1), mb (myoglobin); regulation of autophagy pathway: atg4b (cysteine protease ATG4B-like isoform X1); foxo signaling pathway: ccnb2 (G2/mitotic-specific cyclin-B2-like isoform X1), homer1 (homer protein homolog 1-like isoform X1); steroid hormone biosynthesis pathway: cyp3a40 (cytochrome P450 3A40-like); fatty acid metabolism pathway: scd1 (acyl-CoA desaturase-like); protein processing in endoplasmic reticulum pathway: cne1 (calnexin-like), tram1 (translocating chain-associated membrane protein 1-like 1-like isoform X1); metabolism pathway: cacna1b (voltage-dependent N-type calcium channel subunit alpha-1B-like), mif (macrophage migration inhibitory factor-like), sat1 (diamine acetyltransferase 1-like) and cytbc1_8 (cytochrome b-c1 complex subunit 8-like). β-actin was chosen as reference gene in the current study following the method reported in our previous study (Zheng et al., 2017a, 2017b, 2017c). The cDNA for gene expression analysis were normalized with β-actin (n = 9) together with no-template controls and no-reverse transcription controls. Changes of mRNA levels for these genes were calculated using the method described by Livak and Schmittgen (2001).

2.3. Sequencing and reference genome guided assembly Using Illumina paired-end sequencing technology, a total of 52,678,371 raw reads were generated from the control group (con) and 63,400,875 from the RES addition group (RES) at 15 d (Table S2). Of the raw reads (filtered before mapping) from 15 d-con, > 57.07%–60.43% has been mapped, while 55.49%–58.93% has been mapped in the 15 d-0.05 g/kg RES groups. Of the mapped reads in the 15 d-con group, 29,951,277 (55.31%) have been revealed as unique mapped reads, 961,396 as multiple map reads, and 26,339,186 clean reads (trimmed reads that meet the minimum Phred score and length, Liao et al., 2017) were reached. The clean reads were used for reference genome (PRJNA374766) guided assembly (Table S2). The Trinity software generated all-transcripts with a length from 300 to 1000 bp and > 1000 bp (Table S3). All-transcripts contributed to 12,596 reads (putative protein coding genes), with Clusters of Orthologous Groups (COG)_Annotation 6940, Gene Ontology database (GO)_Annotation 12,596, etc. Transcripts longer than 1000 bp were assigned the amount of the value (Table S3). For annotation, all unigenes were searched using the BLAST algorithm against non-redundant protein sequence (Nr), non-redundant nucleotide (Nt), Swiss-Prot, and COG databases. The number of unigenes with significant similarity to sequences in COG, GO, Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot and Nr databases were 6940; 12,596; 13,641; 16,313 and 23,417, respectively (Table S3). 2.4. Differential expression analysis Differential expression analysis of two RES groups (0, 0.05 g/kg) was performed using the DESeq R package (1.10.1), and the comparison at 15, 30 and 45 d were named as G0, G1 and G2 respectively. DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value of < 0.05 found by DESeq were assigned as differentially expressed. 2.4.1. GO enrichment analysis GO enrichment analysis of the differentially expressed genes (DEGs) was implemented by the GO seq R packages based Wallenius noncentral hyper-geometric distribution (Young et al., 2010), which can adjust for gene length bias in DEGs. 2.4.2. KEGG pathway enrichment analysis KEGG (Kanehisa et al., 2008) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). We used KOBAS (Mao et al., 2005) software to test the statistical enrichment of differential expression genes in KEGG pathways.

2.6. Histology test After all the samples of liver (treatment and control groups) were fixed in formaldehyde solution, the samples were further dehydrated through ascending grades of alcohol series, cleared in xylene and embedded in paraffin blocks. Seven-micrometer-thick paraffin sections were cut with a rotary microtome (Leica RM2235, Leica Microsystems, Germany) and stained using the hematoxylin-eosin (H&E) method employing standard procedures (n = 9). 3

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the various categories of biological process, the most dominant groups were cellular process, single-organism process, and metabolic process, followed by biological regulation, response to stimulus, localization, etc. Within the cellular component category, cell part, and cell were the most dominant groups, followed by membrane, organelle, membrane part, and so on. Within molecular function, the unigenes were assigned to binding, followed by catalytic activity, molecular transducer activity and transporter activity (Fig. 1). From the results of the COG annotation, a total of 10,018 unigenes were successfully annotated to 25 categories (Fig. S1). Among them, the cluster of General Functional Prediction only (R) represented the largest group (2787, 27.82%), followed by Signal Transduction (T) (994, 9.92%), Transcription (K) (975, 9.73%), Replication, recombination and repair (L) (918, 9.16%) and Post-translational modification, protein turnover, chaperone (O) (550, 5.49%). In addition, 65 unigenes (0.65%) were assigned to Defense mechanisms (V) (Fig. S1). A BLAST search against the KEGG database revealed that the data were assigned to 50 KEGG pathways. While among them, one pathway was related to metabolism, involving one unigene at 15 d; 50 pathways were related to immune functions, involving 76 unigenes at 30 d; 50 pathways were related to immune functions, involving 711 unigenes at 45 d (Fig. S2).

2.7. Scanning electron microscope (SEM) To determine how RES affected the response in the liver, we used modified SEM methods for these tissues (Peng et al., 2013) as described (Ramesh et al., 2013). Treated samples were prepared for SEM as follows: a) fixed in gluteraldehyde (3%) in Sorensen's buffer overnight; b) dehydrated (15 min each) with a graded ethanol series (50%, 60%, 70%, 80%, 90%, 100%), mixture of ethanol and hexamethyldisilizane (HMDS) (1:1 ratio), and then finally with pure HMDS (100%); c) dried overnight in a fume hood. Samples were placed on an aluminum stub, sputtered with gold, and then visualized using a JEOL JSM scanning electron microscope (15 and 20 kV). To assess the morphological changes (n = 9), we scanned each sample from anterior to posterior and obtained digital images of the affected region using Semaphore software. 2.8. Statistical analyses All data were expressed as mean ± standard deviation (SD), a oneway analysis of variance (ANOVA) was used to test for differences among treatments. In data sets where there were significant difference, Kruskal-Wallis analysis and Dunn's post hoc test were carried out with SPSS Statistics 18.0 software (P < 0.05 for significant using asterisk).

3.2. Identification and enrichment analysis of differentially expressed genes (DEGs)

3. Results

According to the normalized gene expression level, the genes significantly differentially expressed after RES addition were identified, and a total of 5 (15 d), 179 (30 d) and 1526 (45 d) genes were detected as significant DEGs (Table 1). Compared with the control group at 15 d (T01_T07_T13_), three genes were significantly up-regulated (accounting for 60.00% of all significant DEGs), while two genes were significantly down-regulated (accounting for 40.00%). Compared with the control group at 30 d (T19_T20_T21_), 72 genes were significantly up-regulated (accounting for 40.22% of all significant DEGs), while 107

3.1. Functional annotation and classification The count of unique mapped reads and multiple map reads in 30 d0.05 g/kg RES groups were significantly lower than those in controls, while the count of clean reads in 45 d-0.05 g/kg RES groups were significantly lower than those in controls (Table S2). GO functional analysis based on sequence homology showed that 12,596 unigenes were categorized into 59 sub-categories of three major categories: biological process, cellular component, and molecular function (Fig. 1). Among

Fig. 1. Gene Ontology classifications of assembled unigenes. A total of 12,596 unigenes were categorized into 3 functional categories: biological process, cellular component and; molecular function. 4

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Table 1 DEGs comparative analysis at different sample days. Sampling time

DEG Set

DEG Number

Up-regulated

Down-regulated

15 d 30 d 45 d

T02_T08_T14_vs T01_T07_T13_, G0 T03_T09_T15_vs T19_T20_T21_, G1 T04_T10_T16_vs T22_T23_T24_, G2

5 179 1526

3 72 691

2 107 835

Fig. 2. The verification of DEGs in different KEGG pathways following 0.05 g/kg RES feed addition at 30 and 45 d by qRT-PCR method (n = 9). Cytokine pathway: il12rb2 (ENSONIG00000002601, interleukin-12 receptor subunit beta-2-like), scfr (ENSONIG00000008788, mast/stem cell growth factor receptor kita-like isoform X1); immune system pathway: ig8l (ENSONIG00000005111, immunoglobulin superfamily member 8-like), hlfl (ENSONIG00000008480, hepatic leukemia factorlike), cd226 (ENSONIG00000014324, CD226 antigen-like isoform X2), prf1l (ENSONIG00000015062, perforin-1-like), leap-2 (O. niloticus_newGene_2488, liverexpressed antimicrobial peptide 2-like), prdx4 (ENSONIG00000005145, peroxiredoxin-4-like isoform X1), mb (ENSONIG00000018018, myoglobin); regulation of autophagy pathway: atg4b (ENSONIG00000019280, cysteine protease ATG4B-like isoform X1); foxo signaling pathway: ccnb2 (ENSONIG00000015677, G2/mitoticspecific cyclin-B2-like isoform X1), homer1 (ENSONIG00000013747, homer protein homolog 1-like isoform X1); steroid hormone biosynthesis pathway: cyp3a40 (ENSONIG00000003249, cytochrome P450 3A40-like); fatty acid metabolism pathway: scd1 (ENSONIG00000018500, acyl-CoA desaturase-like); protein processing in endoplasmic reticulum pathway: cne1 (ENSONIG00000004319, calnexin-like), tram1 (ENSONIG00000014261, translocating chain-associated membrane protein 1-like 1-like isoform X1); metabolism pathway: cacna1b (ENSONIG00000004915, voltage-dependent N-type calcium channel subunit alpha-1B-like), mif (O. niloticus_newGene_2581, macrophage migration inhibitory factor-like), sat1 (O. niloticus_newGene_3368, diamine acetyltransferase 1-like), cytbc1_8 (O. niloticus_newGene_6522, cytochrome b-c1 complex subunit 8-like).

tram1, mif, sat1, cytbc1_8. The DGEs were verified using the qRT-PCR method, successfully mapping to mRNA-seq results (Fig. 2).

genes were significantly down-regulated (accounting for 59.78%). Compared with the control group at 45 d (T22_T23_T24_), 691 genes were significantly up-regulated (accounting for 45.28% of all significant DEGs), while 835 genes were significantly down-regulated (accounting for 55.72%).

3.4. H&E and SEM analysis Previous studies have observed necrosis, compressed sinusoids and apoptotic cells in the 0.3 g/kg RES feeding groups by H&E staining, while the SEM results showed the deformation of epidermal cells in the liver of the treated groups (Zheng et al., 2017b). Then hepatic transcriptome revealed that genes related with immune response has been affected to support this phenomena (Zheng et al., 2017c). Results of HE revealed hyperemia (n = 6, Fig. 3-b), distribution of lower ratio of blood cells (n = 6, Fig. 3-b), compressed hepatic sinusoid (n = 5, Fig. 3d), dark nucleolus, and visible cell boundary (n = 5, Fig. 3-d) in the 0.05 g/kg RES addition groups (Fig. 3). SEM analysis demonstrated fibrosis of liver cell (n = 7, Fig. 3-f) and abnormal hepatic epidermal cell (round fully in controls and long flat in treatments; n = 9, Fig. 3-h).

3.3. DEGs selection associated with immune system and validation At 15 d, COG functional enrichment analysis results showed that five DEGs associated with immune system could be categorized into coenzyme transport and metabolism (Fig. 2), which has been associated with folate biosynthesis. At 30 and 45 d, it showed that General Functional Prediction only (R) represented the largest group, followed by Signal Transduction (T), Transcription (K), Replication, recombination and repair. At 15 d and 45 d, cell surface A33 antigen-like was significantly down-regulated (Table S5), while the genes from newGene_2625 to gene:ENSONIG00000019941 were significantly downregulated both at 30 d and 45 d, and the genes from newGene_2488 to gene:ENSONIG00000018018 were significantly up-regulated both at 30 d and 45 d. At 45 d, immune-related genes were enriched in the ‘biological process’ category, and in which eight KEGG terms including 20 genes were involved, such as ‘il12rb2’, ‘scfr’, ‘ig8l’, ‘hlfl’, cd226, prf1l, atg4b, ccnb2, cyp3a40, scd1, cacna1b; leap-2, prdx4, mb, homer1, cne1,

4. Discussion 4.1. Transcriptome analysis The 5

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Fig. 3. Morphological change in GIFT liver by dietary 0.05 g/kg resveratrol supplementation at 45 d. 1 and 3 stand for control liver (n = 9), while 2 and 4 stand for liver after feeding 0.05 g/kg RES. a, effects of RES addition on histological changes in liver of juvenile GIFT (O. niloticus). In the first figure, gridlines shows normal distribution of blood cells, while in 2, gridlines shows distribution of lower ratio of blood cells, hyperaemia means the increased blood in an organ, the dotted arrow shows hyperaemia (n = 6); In the third figure, the dotted arrow shows normal nucleolus, the solid arrow stands for hepatic sinusoid, while in 4, the dotted arrow shows dark nucleolus (stain being darken), the solid arrow stands for compressed hepatic sinusoid (n = 5). When compressed hepatic sinusoid occurs, it means the emergence of liver cell necrosis and apoptosis reveals in the higher concentration of RES groups. b, Scanning electron micrographs showing the surface morphology of tilapia liver. 1 shows normal liver cell for 10 μm, Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. 2 reveals fibrosis of liver cell (n = 7, the red dotted arrow); 3 shows round fully grown hepatic epidermal cell, 4 stands for long flat hepatic epidermal cell (n = 9, the red dotted arrow showed cell deformation).

process, environmental information processing, genetic information processing, human diseases, and organismal systems pathways. These results are the same as those in previous studies performed in pearl oyster (Pinctada fucata, Wang et al., 2015) and banana shrimp (Fenneropenaeus merguiensis, Powell et al., 2015). These functional annotations provide a valuable resource for investigating specific processes, functions, and pathways in O. niloticus.

transcriptome is still scarce, especially for the information of immunerelated genes involved in innate response. In this context, the goal of the present study was to characterize the transcriptome of O. niloticus liver by means of Illumina RNA-Seq technology, and identify immunerelated transcripts and biological processes involved in RES addition group. In the present study, the Illumina sequencing yielded > 1 billion reads with 480 G clean bases from liver of O. niloticus. After assembly, 12,596 unigenes (Annotated in GO, 53.79%) were generated, which were smaller than those achieved in previous studies of Pinctada fucata (23,513, Wang et al., 2015), Paphia textile (14,571, Chen et al., 2016), Ctenopharyngodon idella (34,027, Xu et al., 2014) and Fenneropenaeus merguiensis (16,866, Powell et al., 2015). The poor annotation efficiency could be due to the difference among the genus (GIFT, Qiang et al., 2017, Zheng et al., 2017c; Nile tilapia, Zhang et al., 2013, Yang et al., 2013; Oreochromis mossambicus, Lam et al., 2014), or even the inadaptable bioinformatics analysis. ‘General Functional Prediction only’ occupied the largest component of unigenes under COG classification, which was the same as the previous studies (Xu et al., 2014; Powell et al., 2015; Wang et al., 2015; Chen et al., 2016). This result further supports that Illumina/Hiseq-2500 RNA-Seq is an ideal method for the transcriptome analysis with high efficiency and massive data output. Our results indicate that although a comprehensive transcriptome of one species can be obtained by NGS, transcriptome and functional resources associated with the immune system for O. niloticus need to be addressed with some effective efforts (Qiang et al., 2017; Zheng et al., 2017c). Therefore, the unigenes that had no hits to any sequences in the public databases may represent novel genes, which will be important for further research. By KEGG analysis, functions of over 46.6% unigenes were annotated. A large percentage of unigenes were mapped into KEGG pathways associated with metabolism, and most were involved in cellular

4.2. DEGs selection and validation The significant DEGs between 15 d (G0), 30 d (G1) and 45 d (G2) revealed a tendency to increase over time. At 30 d and 45 d, unique mapped reads, multiple map reads, and clean reads were significantly lower than those in controls. The significant DEGs have been gathered on ‘cytokine’, ‘immune system’, ‘regulation of autophagy’, ‘foxo signaling pathway’, ‘steroid hormone biosynthesis’, ‘fatty acid metabolism’, ‘protein processing in endoplasmic reticulum’ and ‘metabolism’, and 53 down-regulated and 24 up-regulated genes have been revealed (Table S5). Regardless of the data revealed by transcriptome analysis or qRTPCR verification, we identified many other DEGs in the transcriptome dataset, more PCR primers could be developed to validate them (Table S4). The current study showed that il12rb2 has been reduced in 0.05 g/ kg RES groups at 30 d and 45 d, and cytokine receptor related genes were not reduced in the miiuy croaker (Miichthys miiuy, Yang et al., 2017), which are in the category of apoptosis (Wei et al., 2017); nevertheless, decreased genes involved in FOXO signaling pathway (ccnb2, homer1) may lead to the activation of antioxidant and apoptosis (Jin et al., 2017). Immunoglobulin (ig8l), hepatic leukemia factor (hlfl), CD226 antigen-like isoform X2 (cd226), and perforin-1-like (prf1l) expression reduced, while that of liver-expressed antimicrobial peptide 2like (leap-2), peroxiredoxin-4-like isoform X1 (prdx4), myoglobin (mb) 6

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Interestingly, the number of DEGs after RES treatment increased with treatment time (Table 1) in the present study, and it raised another question: whether high concentration RES alert harmful effect to tilapia organic development? Previous study showed the limit feed addition rate was 0.025 g/kg, meanwhile, the result of histological observations indicated that hyperemia and compressed hepatic sinusoid, fibrosis of liver cell and abnormal hepatic epidermal cell occurred. Combining with the result of inflammation, oxidative stress, metabolism-related transcriptional changes, thus, these results suggested high concentration addition or long-time administration may bring negative effect in tilapia liver. While looking at transcriptional/micro RNA responses to Streptococcosis infection in tilapia or other fish (Zhang et al., 2013), il1r was upregulated (Zhang et al., 2013) and il12rb2 was down-regulated in the present study. It is demonstrated that RES may disturb different pathways associated with immune system or metabolism. Qiang et al. (2017) showed that the decreased micro-RNA expression may enhance inflammation response, with the phenomenon of inflammation response observed in the present study, and it hinted that micro-RNA and transcriptional regulation in such this immune response related pathway. To conclude, we infer that the high occurrence of immune and metabolism-related genes was involved in the response of GIFT to RES addition, which may result in innate responses and lead to morphological change.

significantly increased in the RES addition groups. RES enhances antioxidative (Zheng et al., 2010) and aerobic respiration (Zhao et al., 2014) in tilapia, and atg4b has been reported the vital role in compensatory growth and protein degradation in triploid juvenile rainbow trout (Oncorhynchus mykiss, Cleveland and Weber, 2013); the reduction of this autophagy-related gene indicates reduced proteolysis in the RES addition groups. Meanwhile, scd1 and cyp3a40 significantly reduced in RES addition groups, and RES seems as estrogen analogues (Robb and Stuart, 2014) and may disturb the cross-talk in steroidogenesis (20 mg/ kg·d, Aluru and Vijayan, 2006). Some annotated new genes (mif, sat1, cytbc1_8) have demonstrated increases in the RES addition groups, which suggests RES may cause exogenous substance-induced immune responses (Qiu et al., 2013) through intestinal absorption from feed (Zheng et al., 2017a, 2017b). This explains why the majority of interesting transcripts found in this study were detected. These data indicate the liver plays an extensive role in the immune system of this species. These findings confirmed that RES activates some immune-related genes and biological processes that may be involved in the innate response in tilapia. 4.3. Morphological change Unlike zebrafish, the information about GIFT has not been well provided despite some useful efforts (Qiang et al., 2017). Recent studies have shown that RES may cause inflammatory response in mice (Wang et al., 2014) and in different fish species (Medina et al., 2010); however, the molecular mechanism may be associated with different pathways, such as tumor necrosis factor-alpha (TNF-α, Leiro et al., 2010) and AMP-activated protein kinase (Chang et al., 2011). According to the reference performed on immunity system of eastern oyster (Crassostrea virginica, Zhang et al., 2014), the present study revealed that all the pathways reveal RES disturbs innate immunity of fish. Previous studies found that necrosis, compressed sinusoids and apoptotic cells revealed in 0.3 g/kg RES feeding groups by H&E method, while the results from SEM showed deformation of epidermal cells in the liver of the treated groups (Zheng et al., 2017b). The current study showed hyperemia, compressed hepatic sinusoid and fibrosis of liver cells have been revealed in 0.05 g/kg RES feeding groups, which was consistent with its role in vascular function through oxidative phosphorylation pathway (Pollack et al., 2017). When we compared with short-time administration with RES, long-time duration had antineovascular effects (Richer et al., 2014). Cytochrome b-c1 complex subunit 8-like (cytbc1_8) is associated with the oxidative phosphorylation pathway, the transcripts of which were enhanced by 0.05 g/kg RES addition at 30 and 45 d in the current study (Fig. 3). Macrophage migration inhibitory factor (Mif) is a pro-inflammatory cytokine, which prevented Akt1 phosphorylation reduction and then resulted in the decrease in oxidative stress (Zhu et al., 2018). The gene profile of oxidative stress and inflammatory reaction revealed that such inflammation related genes (e.g. TNFα, IL-1β) had the same tendency with mif (Hu et al., 2017), and the former one has been demonstrated in our previous study with RES addition (Zheng et al., 2017b). mif significantly increased by 0.05 g/kg RES addition at both 45 and 30 d in the current study. Abnormal hepatic epidermal cells were observed in the current study, as in our previous study (Fig. 3, Zheng et al., 2017b). In teleosts, dietary resveratrol may regulate fat metabolism through Sirt1-PGC1α-PPARα pathway (Milton-Laskibar et al., 2018) or posttranscriptional regulation. Recent study showed resveratrol pretreatment ameliorated liver injury and accelerated regeneration of the hepatic remnant (Jin et al., 2019). However, the relationship between cytbc1_8, mif and such hepatic morphological change has not been determined. Nevertheless, dark nucleoli were observed in the RES groups, which may be related to the greater number of significant DEGs at 45 d when compared with those at 15 or 30 d. Previous studies showed that rutin and RES addition may disturb the process of cytokine pathway and result in hepatic impairment (Zheng et al., 2017a, 2017b).

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