Fish and Shellfish Immunology 92 (2019) 348–355 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Differentially expressed genes of the shrimp Marsupenaeus japonicus in response to infection by white spot syndrome virus or Vibrio alginolyticus T ∗ Qingri Jin, Fei Zhu Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, College of Animal Science and Technology, Zhejiang Agriculture and Forestry University, Hangzhou, 311300, China ARTICLE INFO ABSTRACT Keywords: This is the first study to use a next-generation high-throughput DNA sequencing technique, the Illumina Differentially expressed genes Hiseq2000 method, to analyze the transcriptome from hemocytes of Marsupenaeus japonicus. A total of Marsupeneaus japonicus 80,929,652 Illumina reads, including 79,525,942 high quality reads were obtained in this study. From these, WSSV 40,231 unigenes with a mean length of 1557 bp were assembled using Trinity de novo software and 28,746 Vibrio alginolyticus cDNA were matched in the NCBI database. Then we compared the transcriptome changes after white spot syndrome virus (WSSV) or Vibrio alginolyticus infection. A total of 19,872 putative proteins were classified functionally into 25 molecular families in the cluster of orthologous groups. KEGG pathway analysis identified that the metabolic pathway possessed more unigenes (1358 unigenes), followed by biosynthesis of secondary metabolites, Huntington's disease and RNA transport. Important immune functions like apoptosis, phagocytosis, and lysosomes were in response to WSSV and V. alginolyticus early infection. Only 26 transcripts were sig- nificantly up-regulated or down-regulated after WSSV infection showed compared with V. alginolyticus. Crustin- like protein, endonuclease-reverse transcriptase, putative nuclease HARBI1-like, diphthamide biosynthesis protein 7 and hormone receptor 3 were involved in the immune response to WSSV or V. alginolyticus infection. These transcriptome datasets accelerate our understanding of the innate immune mechanisms in M. japonicus and other crustaceans. 1. Introduction discovered many immune-related genes responding to WSSV infection in crustaceans [9,10]. The kuruma shrimp (Marsupenaeus japonicus) is an economically Transcriptome analysis is widely used to identify novel and differ- important commercial farmed species of the Penaeidae family of dec- entially expressed genes in shrimp [11–14]. However, the hemocyte apod crustaceans in Southeast Asia. White spot syndrome virus (WSSV), transcriptome information for the shrimp response to WSSV and bac- an enveloped virus with a large double-stranded genome (∼300 kb) terial challenge is still limited, so we constructed and sequenced a [1,2], is a viral pathogen responsible for huge economic losses in the cDNA library from WSSV-challenged hemocytes in M. japonicus. The shrimp culture industry [3–5]. Vibriosis has also been implicated as the objective of this work was to identify and annotate more immune-re- cause of major mortality in juvenile penaeid shrimp [6]. Vibrio algino- lated genes in shrimp hemocytes and also provide information to elu- lyticus is an important bacterial pathogen in the shrimp farming in- cidate the mechanisms of innate immune response to WSSV and V. al- dustry. WSSV and V. alginolyticus often cause high production losses of ginolyticus challenge in crustaceans. In this study, we obtained 40,231 farmed shrimp in Southeast Asia. unigenes; most of these were novel for M. japonicus. In addition, many Crustaceans lack an adaptive immune system and rely totally on the genes were found to be related to the innate immune system in other innate immune system to resist pathogen invasion. The innate immune species, such as pattern recognition receptors, antimicrobial peptides, reaction comprises cellular reactions such as phagocytosis, nodule for- proteases and protease inhibitors, signal transduction proteins, apop- mation and encapsulation performed by hemocytes. Hemocytes are tosis-related proteins, and antioxidant proteins. crucial for the immune system in crustaceans as they are the main production site for immune recognition molecules and initiate the hu- moral reaction like phagocytosis [7,8]. Previous studies have ∗ Corresponding author. E-mail addresses: [email protected], [email protected] (F. Zhu). https://doi.org/10.1016/j.fsi.2019.06.034 Received 27 March 2019; Received in revised form 11 June 2019; Accepted 13 June 2019 Available online 16 June 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved. Q. Jin and F. Zhu Fish and Shellfish Immunology 92 (2019) 348–355 Table 1 mL). In addition, 30 shrimps were each injected with 20 μL PBS as the General information of the transcriptome from health shrimp. control for the challenge experiments. The hemolymph was withdrawn fi Dataset name health shrimp from the ventral sinus located at the rst abdominal segment using an equal volume of modified Alsever's solution anticoagulant [15] and Total raw reads (paired-end) 80929652 subsequently centrifuged for 10 min at 1000×g, 4 °C. Hemocytes was Total clean reads 79525942 collected at various times after infection (12 and 24 h) and immediately Q20% 98.94% N percentage 0% stored in liquid nitrogen for later RNA extraction. GC percentage 44.33% Total unigenes 40231 2.2. RNA isolation and illumina sequencing Total genes 28746 Mean length (bp) 1557.55 N50 (bp) 3208 Total RNAs were isolated from the hemocytes of the infected and Min-Max length (bp) 351–18945 non-infected shrimps at different times after infection using Unizol re- agent (UnionGene, China) and treated with DNase I. The mRNAs were purified from the total RNAs using the PolyATtract mRNA isolation 2. Methods system (Promega, Madison, USA) following the manufacturer's in- structions. The quantity and purity of total RNAs were monitored using 2.1. Shrimp culture for challenge experiments a NanoDrop ND-1000 spectrophotometer (NanoDrop, DE, USA). cDNA libraries were generated using Illumina TruSeq™ RNA Sample Cultures of M. japonicus shrimps, each approximately 10 g and Preparation Kit (Illumia, USA) following manufacturer's instructions 10 cm in length, were maintained in groups of 20 individuals in 80 L and the index codes were added to attribute sequences to each sample. aquariums at 22 °C. Hemolymph from cultured shrimps was subjected Briefly, mRNA was purified from total RNA using poly-T oligo-attached to random PCR detection with WSSV-specific primers to ensure that the magnetic beads. Fragmentation was carried out using divalent cations shrimps were WSSV-free before experimental infection. The WSSV in- under elevated temperature in Illumina proprietary fragmentation oculum was prepared from WSSV-infected shrimps according to Zhu buffer. First strand cDNA was synthesized using random oligonucleo- μ et al. [9]. The WSSV-challenged group received 20 L injections of tides and SuperScript II. Second strand cDNA synthesis was subse- 5 WSSV (GenBank accession no. AF 332093.1, 10 copies/mL). In the quently performed using DNA Polymerase I and RNase H. Remaining μ Vibrio challenge experiment, 30 shrimps were each injected with 20 L overhangs were converted into blunt ends via exonuclease/polymerase 6 of V. alginolyticus (ATCC17749) suspended in PBS solution (10 cells/ activities and enzymes were removed. After adenylation of 3′ ends of Fig. 1. Contig length distribution of M. japonicus transcriptomic ESTs. 349 Q. Jin and F. Zhu Fish and Shellfish Immunology 92 (2019) 348–355 Fig. 2. Cluster of orthologous groups (COG) function classification in the transcriptome of hemocytes in healthy M. japonicus. Fig. 3. Gene ontology classification of the hemocyte transcriptome in healthy M. japonicus. The left y-axis indicates the percentage of a specific category of genes existing in the main category, whereas the right y-axis indicates the number of a specific category of genes existing in the main category. DNA fragments, Illumina PE adapter oligonucleotides were ligated to 2.3. De novo assembly and functional annotation prepare for hybridization. In order to select cDNA fragments of pre- ferentially 300 bp, the library fragments were purified with AMPure XP Raw sequencing reads were quality trimmed, and adaptor sequences system (Beckman Coulter, USA). DNA fragments with ligated adaptor were removed before the assembly. The processed reads were as- molecules on both ends were selectively enriched using Illumina PCR sembled using Trinity software with default parameters [16]. The de- Primer Cocktail in a 10 cycle PCR reaction. Products were purified rived unigene sequences were searched against the NCBI nonredundant (AMPure XP system) and quantified using the Agilent high sensitivity protein sequence database for annotation, with an e-value cutoff of − DNA assay on the Agilent Bioanalyzer 2100 system. 1×10 10. For Gene Ontology (GO) annotation, InterproScan was used for obtaining GO terms (http://nar.oxfordjournals.org/content/43/D1/ 350 Q. Jin and F. Zhu Fish and Shellfish Immunology 92 (2019) 348–355 Fig. 4. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification of hemocytes transcriptome in healthy M. japonicus. Table 2 Candidate genes involved in the immune response of M. japonicus at 12 h after challenged with WSSV or VA. Gene ID Fold change Homologous function Species WSSV VA comp16935_c0 2.97 23.41 crustin-like protein Litopenaeus vannamei comp6853_c0 2.5 0 conserved hypothetical protein Tribolium castaneum comp150797_c0