INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 20–0403/2020/24–4–783–793 DOI: 10.17957/IJAB/15.1501 http://www.fspublishers.org Full Length Article Transcriptome Signatures Focusing on Immune-Related Genes in the Livers, Spleens and Kidneys of Male and Female Takifugu rubripes Jun Cui1,3, Lei Men2, Zhicheng Wang1, Xuemei Qiu1 and Xiuli Wang1* 1College of Fisheries and Life Science, Dalian Ocean University, Dalian, 116023, China 2College of Life Science, Dalian Minzu University, Dalian, 116600 China 3School of Bioengineering, Dalian University of Technology, Dalian 116024, China *For correspondence: [email protected] Received 14 March 2020; Accepted 07 May 2020; Published 16 August 2020 Abstract To obtain information regarding immune-related genes and provide a genetic basis for the immune response in fish, we performed transcriptomic comparison analysis of the livers, spleens and kidneys of male and female Takifugu rubripes. The GO terms associated with cell surface receptor signaling were the most significantly enriched, and among the KEGG pathways, cytokine-cytokine receptor interaction, an immune-related pathway, was present in all the male groups. In addition, expression level of complement C1q tumor necrosis factor-related protein 4-like (CTRP4-like) gene was upregulated in all the male samples, while the expression levels of voltage-dependent calcium channel subunit alpha-2/delta-2-like (CACNA2D2- like) and protein L-Myc-1b-like genes were upregulated in all the female samples. These results demonstrate the regulatory mechanisms of immune-related genes and provide candidates for the enhancement of pathogen resistance in T. rubripes. © 2020 Friends Science Publishers Keywords: Takifugu rubripes; Immune-related genes; Liver; Spleen; Kidney; RNA-Seq Introduction in immune response (Jiang et al. 2016). During channel catfish-Flavobacterium columnare interaction, the RNA- Pufferfish, Takifugu rubripes is one of marine commercial Seq results have shown that fish gill is involved in the fishes in Asia (Hamasaki et al. 2017; Kong et al. 2019). immune response to the pathogen (Peatman et al. 2013; This species has high nutritional and economic value. In Sudhagar et al. 2018). Meanwhile, some defense-related China, its production is approximately 70% of the total genes and pathways have been identified form Lateolabrax global production per year, and most are exported to Japan japonicus and Cynoglossus semilaevis challenged with and Korea (Guo et al. 2017). However, various diseases Vibrio anguillarum (Zhang et al. 2015; Zhao et al. 2016), caused by Edwardsiella tarda, Flexibacter maritimus, Larimichthys crocea, Megalobrama amblycephala and Myxococcus sp. and red sea bream iridovirus, have hindered Ctenopharyngodon idella challenged with A. hydrophila its production and caused economic losses (Cui et al. (Mu et al. 2010; Tran et al. 2015; Yang et al. 2016; Song et 2014a; Ma et al. 2014). Therefore, it is urgent and al. 2017), tilapia challenged with Streptococcus agalactiae significant to understand molecular immune-related and S. iniae (Zhu et al. 2017; Wang et al. 2016) and mechanisms of T. rubripes. zebrafish challenged with Salmonella typhimurium, E. tarda RNA-Seq sequencing has developed rapidly over the and Mycobacterium marinum (Stockhammer et al. 2010; past decade and has been widely used for transcriptome Ordas et al. 2011; Yang et al. 2012; Chen et al. 2017) by expression profiling (Zhou et al. 2016a, 2019), regulatory RNA-Seq. network construction (Zhao et al. 2016; Wang et al. 2018), In fishes, immune organs include liver, spleen, kidneys noncoding RNA identification (Cui et al. 2017, 2020; Jiang and gills. In our previous study, many immune-related genes et al. 2018), genetic diversity determination (Cui et al. and pathways have been found in the gills of T. rubripes 2014a; Zhou et al. 2016b) and other applications. RNA-Seq (Cui et al. 2014b), and it is also found that these immune- allows the identification of many immune-related genes and related genes contain a number of single-nucleotide elucidation of mechanisms of immune evasion in fishes. For polymorphisms (SNPs) (Cui et al. 2014c). In addition, fish example, after infection with Aeromonas hydrophila, 2,900 livers, spleens and kidneys, as important immune organs, differentially expressed genes (DEGs) are identified from play important roles in the response of fish against various common carp spleen, and these genes are mainly involved pathogens. Chemokines are key immune regulators, acting as To cite this paper: Cui J, L Men, Z Wang, X Qiu, X Wang (2020). Transcriptome signatures focusing on immune-related genes in the livers, spleens and kidneys of male and female Takifugu rubripes. Intl J Agric Biol 24:783‒793 Cui et al. / Intl J Agric Biol, Vol 24, No 4, 2020 bridges between innate and adaptive immunity. Some catfish study. The livers, spleens and kidneys of these fish were β and α chemokines are responsive to E. ictaluri infection in collected. The same tissue types from male or female fish the lever (Fu et al. 2017a, b). Meanwhile, fish toll-like were pooled. The tissues were placed in RNAlater receptors, which are important immune-related proteins, can (Ambion), stored at room temperature for 24 h, and then recognize pathogen-associated molecular patterns. During A. moved to -80℃ for storage until RNA isolation. hydrophila challenge, the expression levels of genes encoded toll-like receptors are significantly changed in the kidneys RNA extraction, library construction and sequencing and spleen of common carp, suggesting that the toll-like receptor genes affect in the host immune response to A. Total RNA of the livers, spleens and kidneys was extracted hydrophila infection (Gong et al. 2017). using RNAiso Plus (TaKaRa) according to the Previous studies have shown that immune responses manufacturer’s instructions. Total RNA was sent for next are different between males and females, which likely generation-sequencing to Biomarker Biotechnology contributes to differences in the response of the two sexes to Corporation (Beijing, China). The library was sequenced on pathogens (Pennell et al. 2012; Klein et al. 2015). Females an Illumina HiSeq 2000 with 101-bp paired-end reads. exhibit lower burdens of pathogen infection (Pennell et al. 2012). In previous studies, it has been found that a number Read mapping of immune-related genes are differentially expressed between males and females. For example, the expression Based on the method of Kong et al. (2018), the low-quality levels of some cytokines genes, including TNF-α, IL-1β, IL- reads were removed. The clean reads mapped to the T. 6 and CXCL10, are more up-regulated in male mice than in rubripes genome females (Kahlke et al. 2000; Asai et al. 2001; Moxley et al. (https://www.ncbi.nlm.nih.gov/genome/63?genome_assemb 2002; Marriott et al. 2006; Klein et al. 2015). However, the ly_id=22739) by TopHat. studies on the differences between the immune responses of Identification of new genes male and female fishes are limited. In this study, RNA-Seq is performed to investigate the To improve the annotation of the T. rubripes genome, we differences in genome-wide gene expression in important used the RNA-Seq data to identify new genes. First, organs, namely, livers, spleens and kidneys, between male Cufflinks was used to assemble all the mapped reads and and female T. rubripes. Sets of DEGs were identified. The map them to the genome. Then, sequences encoding short important pathways and key immune-related genes are peptide chains (less than 50 amino acid residues) and those screened by Gene Ontology (GO), KEGG pathway containing single exons were removed. Lastly, the new enrichment and protein-protein interaction (PPI) network genes identified above were used as query sequences to analysis. These results will not only provide insight into the search the Pfam, NR, eggnog, KOG, Swiss-Prot, GO, molecular immune-related mechanisms of T. rubripes but KEGG databases, and COGusing BLASTX. also help future molecular breeding. Analysis of DEGs Materials and Methods The expression levels of the genes were measured as read Ethics statement counts normalized to the respective lengths of the genes using Cufflinks. DEGs of nine groups, namely, ♂liver vs. All the fish experiments were conducted in accordance with ♂spleen, ♂liver vs. ♂kidney, ♂spleen vs. ♂kidney, ♀liver the Guidelines for Experimental Animals established by the vs. ♀spleen, ♀liver vs. ♀kidney, ♀spleen vs. ♀kidney, Ministry of Science and Technology. This study was ♂liver vs. ♀liver, ♂spleen vs. ♀spleen and ♂kidney vs. approved by the Animal Care and Use Committee of the ♀kidney, were identified using DEGseq with a false College of Fisheries and Life Science, Dalian Ocean discovery rate (FDR) < 0.01 and fold change > 2 or < -2. University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to Gene ontology (GO) and KEGG enrichment analyses minimize suffering. We used the GOseq R packages to conduct GO enrichment Sample collection analysis of the DEGs (Young et al. 2010), and KOBAS software was used to perform the KEGG pathway A total of 20 T. rubripes (10 males and 10 females) were enrichment analysis (Mao et al. 2005; Kanehisa et al. 2008). sampled from Dalian Tianzheng Industrial Co., Ltd. (Dalian, China). Each individual was 15–18 cm long. T. Protein-Protein interaction (PPI) analysis rubripes were transferred to experimental tanks. These individuals were kept in sea water at 20℃ for 7 days to The sequences of the DEGs were compared by BLASTX acclimate them to the experimental conditions before the against the genome of a related species (information about 784 Immune Genes in the Liver, Spleen and Kidney of Pufferfish/ Intl J Agric Biol, Vol 24, No 4, 2020 the PPIs of which can be found in the STRING database: http://string-db.org/) to obtain the predicted PPIs associated with these DEGs. Then, the PPIs associated with these DEGs were visualized using Cytoscape (Shannon et al.