Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/294578021

ATranscriptomeStudyonMacrobrachiumrosenbergiiHepatopancreasExperimentallyChallengedwithWhiteSpot...

ArticleinJournalofInvertebratePathology·February2016

DOI:10.1016/j.jip.2016.01.002

CITATIONS

0

READS

93

6authors,including:

Someoftheauthorsofthispublicationarealsoworkingontheserelatedprojects:

Host-pathogeninteractionsstudyandroleoffunctionalgeneincrustaceansViewproject

ZIKAvirustropisminAedesaegyptiViewproject

RamaRao

UniversityofMalaya

2PUBLICATIONS10CITATIONS

SEEPROFILE

SubhaBhassu

InstituteofBiologicalSciences,Universityof…

182PUBLICATIONS463CITATIONS

SEEPROFILE

TaherehAlinejad

UniversityofMalaya

4PUBLICATIONS44CITATIONS

SEEPROFILE

JunWang

UniversityofMichigan

413PUBLICATIONS1,507CITATIONS

SEEPROFILE

AllcontentfollowingthispagewasuploadedbyTaherehAlinejadon17March2016.

Theuserhasrequestedenhancementofthedownloadedfile.Allin-textreferencesunderlinedinblueareaddedtotheoriginaldocumentandarelinkedtopublicationsonResearchGate,lettingyouaccessandreadthemimmediately.

Journal of Invertebrate Pathology 136 (2016) 10–22

Contents lists available at ScienceDirect

Journal of Invertebrate Pathology

journal homepage: www.elsevier .com/ locate / j ip

A transcriptome study on Macrobrachium rosenbergii hepatopancreasexperimentally challenged with white spot syndrome virus (WSSV)

http://dx.doi.org/10.1016/j.jip.2016.01.0020022-2011/� 2016 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Genetics and Molecular Biology, Institute of BiologicalSciences, University Malaya, 53200 Kuala Lumpur, Malaysia.

E-mail addresses: ra2rulz_2004@yahoo.com (R. Rao), subhabhassu@gmail.com,subhabhassu@um.edu.my (S. Bhassu), robin.zhu@bgitechsolutions.com(R.Z.Y. Bing), talinejad698@gmail.com (T. Alinejad), sharifah.syedhassan@monash.edu (S.S. Hassan), wangjun30@gmail.com (J. Wang).

Rama Rao a, Subha Bhassu a,⇑, Robin Zhu Ya Bing b, Tahereh Alinejad a, Sharifah Syed Hassan c, Jun Wang a

aAnimal Genetics and Evolutionary Biology Laboratory and Terra-Aqua Lab, Centre for Research in Biotechnology for Agriculture (CEBAR), Institute of Biological Sciences, Facultyof Science, University of Malaya, 50603 Kuala Lumpur, MalaysiabBeijing Genomics Institute, Shenzhen, 11th Floor, Main Building, Beishan, Industrial Zone, Yantian District, Shenzhen 518083, Chinac Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Building 3, Jalan Lagoon Selatan, Bandar Sunway, 47500 Selangor Darul Ehsan, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 August 2015Revised 15 December 2015Accepted 4 January 2016Available online 12 February 2016

Keywords:TranscriptomicsMacrobrachium rosenbergiiWSSVDe novo assemblyImmune genesHost–pathogen interaction

The world production of shrimp such as the Malaysian giant freshwater prawn, Macrobrachium rosen-bergii is seriously affected by the white spot syndrome virus (WSSV). There is an urgent need to under-stand the host pathogen interaction between M. rosenbergii and WSSV which will be able to provide asolution in controlling the spread of this infectious disease and lastly save the aquaculture industry.Now, using Next Generation Sequencing (NGS), we will be able to capture the response of the M. rosen-bergii to the pathogen and have a better understanding of the host defence mechanism. Two cDNAlibraries, one of WSSV-challenged M. rosenbergii and a normal control one, were sequenced using theIllumina HiSeqTM 2000 platform. After de novo assembly and clustering of the unigenes from both libraries,63,584 standard unigenes were generated with a mean size of 698 bp and an N50 of 1137 bp. We success-fully annotated 35.31% of all unigenes by using BLASTX program (E-value <10–5) against NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genome pathway (KEGG) andOrthologous Groups of proteins (COG) databases. Gene Ontology (GO) assessment was conducted usingBLAST2GO software. Differentially expressed genes (DEGs) by using the FPKM method showed 8443 hostgenes were significantly up-regulated whereas 5973 genes were significantly down-regulated. The differ-entially expressed immune related genes were grouped into 15 animal immune functions. The presentstudy showed that WSSV infection has a significant impact on the transcriptome profile ofM. rosenbergii’shepatopancreas, and further enhanced the knowledge of this host-virus interaction. Furthermore, thehigh number of transcripts generated in this study will provide a platform for future genomic researchon freshwater prawns.

� 2016 Elsevier Inc. All rights reserved.

1. Introduction double-stranded DNA of about 300 kb (Chen et al., 2002; van

Viral diseases remain a thorn in the side of the shrimp aquacul-ture industry. Among the viruses reported for shrimps, the whitespot syndrome virus (WSSV) stands out as the most devastating,causing high mortality and severe economic losses in the shrimpfarming industry throughout the world (Flegel, 1997; Escobedo-Bonilla et al., 2008). WSSV, which was first reported in Fujian,China, in 1992, is classified under the genus Whispovirus of thevirus family Nimaviridae (Marks et al., 2004). This deadly virushas an enveloped, non-occluded, rod-shape that contains a circular,

Hulten et al., 2001). The virus has a diverse range of potential hostssuch as marine shrimps, crayfish, crab, spiny lobsters, insects andfreshwater prawns (Chang et al., 1998; Lo et al., 1996; Wanget al., 1998).

The giant freshwater prawn, Macrobrachium rosenbergii, is aneconomically important crustacean, being cultured on a large-scale in different parts of the world. M. rosenbergii is native toMalaysia and other Asian countries, including Vietnam, Cambodia,Thailand, Myanmar, Bangladesh, India, Sri Lanka, and the Philip-pines (Hameed, 2009). Being popular for its delicious flesh andhigh nutritive value, demand for this species has escalated, gener-ating revenue worth US$1 billion per annum in Asia alone(Schwantes et al., 2009). Generally M. rosenbergii is considered lessprone to various diseases in culture when compared to penaeidshrimps (Bonami and Widada, 2011). However, with exception ofadult stage, the other developmental stages of M. rosenbergii werefound to be more susceptible to WSSV (Kiran et al., 2002).

R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22 11

M. rosenbergii, like other crustaceans, possesses an innateimmune system which provides defence against pathogenic agents(Gross et al., 2001). A greater understanding of the immune systemin this species is necessary to develop methods to control and min-imise the loss of production due to infectious diseases. The under-standing of host pathogen interaction can be achieved by studyingthe transcriptome or the complete repertoire of expressed RNAtranscripts. Transcriptome profiling offers a strategy to assess therelative importance of gene products in any chosen cell, tissue,organism, or given condition (Mu et al., 2010). Considerable pro-gress has been made to understand the molecular mechanism ofthe shrimp-virus interaction using conventional transcriptometechniques such as suppression subtractive hybridization (SSH),differential hybridization (HD), microarrays and expressedsequence tag analysis (EST) (Zhao et al., 2007; James et al., 2010;Leu et al., 2007, 2011; Pongsomboon et al., 2011; Nanhai et al.,2005; Wang et al., 2006). However, microarrays and subtractivehybridization methods are hindered by background and cross-hybridization problems, and measure only the relative abundanceof transcripts (Moody, 2001; AC’t Hoen et al., 2008). Furthermore,only predefined sequences can be detected using this approach(Watson et al., 1998). The EST sequencing technique is moreoverlaborious; and it has limitations in the depth of the transcriptomethat can be sampled, which could possibly prevent the detection oftranscripts with low abundance (Hanriot et al., 2008).

A far superior technology that has revolutionised modern geno-mics research, known as ‘Next Generation Sequencing’ (NGS), wasintroduced in 2004. This platform provides a faster and cheaperway to sequence large amounts of data with greater depth andbreadth. These established NGS platforms such as the IlluminaGenome Analyzer, the Roche/454 Genome Sequencer FLX Instru-ment, and the ABI SOLiD System have proven to be powerful andcost-effective tools for advanced research in many areas such asgenome sequencing, metagenomics, epigenetics and the discoveryof non-coding RNAs, protein-binding sites and transcriptomicsanalysis (Morozova and Marra, 2008). Transcriptome analysisusing the NGS approach has been successfully utilised to identifyimmune-related genes in marine shrimps against the Taura Syn-drome Virus (TSV) and WSSV (Xue et al., 2013; Chen et al., 2013;Li et al., 2013; Zeng et al., 2013).

At present, there is limited evidence on the immune-relatedgenes ofM. rosenbergiiwhich are primarily involved inWSSV infec-tion. In this study, we performed a transcriptome analysis from thehepatopancreas of M. rosenbergii experimentally infected withWSSV, using a high-throughput sequencing method (IlluminaHiSeqTM 2000). The aim was to identify candidate immune-relatedgenes involved in WSSV infection which can provide a betterinsight into the virus-shrimp interaction between M. rosenbergiiand WSSV. In addition, the high-throughput sequencing methodrevealed a large number of new transcripts, which could pointthe way for future genomics research into the freshwater prawn.

2. Materials and methods

2.1. M. rosenbergii and WSSV challenge

Equal number of males and females juvenile M. rosenbergiiprawns aged 90 days (5–8 g body weight, were purchased from ahatchery at Kuala Kangsar, Perak, Malaysia. The prawns were accli-matized for 1 week prior to being challenged by WSSV in flat-bottomed glass tanks (300 L) with aerated and filtered freshwaterat 28 ± 1 �C in the laboratory. The prawns were randomly sampledand tested by PCR to ensure they were free fromWSSV. In the chal-lenge experiment, two prawn groups were used, each of 10prawns: one WSSV challenge group and one negative control

group. The challenged group were intramuscularly injected with100 ll filtered supernatant obtained from WSSV infected Penaeusmonodon (1 � 105 copies/ml, identified by qPCR). Similarly, thenegative control group were injected with 2% NaCl (1:10, w/v)solution. At 12 h after infection, the hepatopancreas tissues ofthe prawns were dissected and immediately frozen in liquid nitro-gen before storage at �80 �C until RNA extraction. Our previousstudies working on immune related genes from M. rosenbergii inresponse to infectious hypodermal and hematopoietic necrosisvirus (IHHNV) showed significant gene expression at 12 h timepoint, hence this time point was chosen for this study (Arockiarajet al., 2011a, 2011b, 2012).

2.2. Total RNA extraction

Total RNA (�20 mg) was extracted from the WSSV-challengedand negative control group hepatopancreases using a Macherey–Nagel NucleoSpin RNA II extraction kit in accordance with themanufacturers’ protocols. This was then dissolved in diethylpyro-carbonate (DEPC) treated water, to give a final concentration of>750 ng/ml. The integrity and size distribution of the dissolvedRNA samples was checked with Agilent 2100 Bioanalyzer (Agilenttechnologies, USA) before storage at �80 �C. The total RNA sampleswere equally pooled from 10 prawns in each negative control andWSSV-challenged groups prior to cDNA synthesis and sequencing.

2.3. Illumina sequencing

The cDNA library preparation and sequencing were conductedusing Illumina sequencing technology (Beijing Genome Institute,Shenzhen, China). A Sera-magMagnetic Oligo (dT) Beads (Illumina)was used to isolate poly A+ mRNA from the total RNA. Small frag-ments of extracted RNA (�150–200 bp) were generated from theextracted RNA to avoid priming bias. These small fragments ofRNA were transcribed into double stranded cDNA using reversetranscriptase, RNase H (Invitrogen) and DNA polymerase I (NewEngland BioLabs) primed with random hexamers. Two sequencingcDNA libraries of pooled WSSV-infected and pooled negative con-trol hepatopancreas were prepared according to the manufac-turer’s protocols (Illumina), before being subjected to 90 bppaired-end sequencing using an Illumina HiSeqTM 2000 platform.

2.4. De novo assembly and assessment

After removing adaptor sequences, ambiguous ‘N’ nucleotides(with a ratio of ‘N’ more than 10%) and low quality sequences (witha quality score less than 20), the remaining clean reads wereassembled using Trinity software (Haas et al., 2013). The Trinitysoftware combines three independent software modules: Inch-worm, Chrysalis, and Butterfly, applied sequentially to processlarge volumes of RNA-seq reads. Trinity partitions the sequencedata into many individual de Bruijn graphs, each representingthe transcriptional complexity at a given gene or locus, and thenprocesses each graph independently to extract full-length splicingisoforms and to tease apart transcripts derived from paralogousgenes. The unigenes (UniGene computationally identifies tran-scripts from the same locus; analyzes expression by tissue, age,and health status; and reports related proteins (protEST) and cloneresources from both groups) were then clustered using the TIGRGene Indices clustering tools (TGICL) (Pertea et al., 2003), withdefault parameters. Bacterial sequence contamination was investi-gated using the web-based version of DeconSeq (Schmieder andEdwards, 2011), with a query coverage and sequence identitythreshold of 90%.

Table 1Summary of the control and WSSV-infected transcriptome sequencing.

Control WSSV Infected

Total number of reads 54,708,014 38,671,944Total base pairs (bp) 4,923,721,260 3,480,474,960Q20 value 97.73% 97.79%Total number of contigs 95,645 97,151Mean length of contigs (bp) 313 307Total number of unigenes 59,050 65,625Mean length of unigenes (bp) 479 427Total clustered Unigenes 63,584Mean length of clustered unigenes (bp) 698NCBI Nr annotated 19,799Swiss-Prot annotated 16,382KEGG annotated 14,706COG annotated 7856GO annotated 6935

12 R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22

2.5. Protein coding, gene identification and classification

The clustered unigene sequences were annotated using homol-ogy search (BLASTX) (Mount, 2007) with an E-value cut-off of 10�5

against an NCBI non-redundant (Nr) database, Swissprot(Boeckmann et al., 2003), Cluster of Orthologous Groups database(COG) (Tatusov et al., 2000) and Kyoto Encyclopedia of Genesand Genome (KEGG) database (Kanehisa and Goto, 2000). GeneOntology (GO) (Ashburner et al., 2000) assignment was conductedusing BLAST2GO software (Conesa et al., 2005) with default param-eters. The coding sequence and the direction of the annotated uni-genes were determined based on the BLAST results from the fourabove mentioned databases. For those sequences without annota-tion, the coding sequence and direction were predicted using anESTscan (Iseli et al., 1999), with BLAST predicted coding sequencedata as the training set.

2.6. Identification of differentially expressed unigenes

The transcript expression levels of the unigenes were measuredusing the FPKM method (Fragments Per kb per Million fragments).The calculation of Unigene expression uses FPKM method (Frag-ments Per kb per Million fragments), the formula is shown below:

FPKM ¼ 106C

NL=103

Set FPKM to be the expression of Unigene A, and C to be numberof fragments that uniquely aligned to Unigene A, N to be total num-ber of fragments that uniquely aligned to all Unigenes, and L to bethe base number in the CDS of Unigene A. The FPKMmethod is ableto eliminate the influence of different gene length and sequencinglevel on the calculation of gene expression. Therefore the calcu-lated gene expression can be directly used for comparing the dif-ference of gene expression between samples. A FDR (falsediscovery rate) of <0.001 was used as the threshold of the p-value in multiple tests to judge the degree of differences in geneexpression (Reiner et al., 2003). FDR (False Discovery Rate) controlis a statistical method used in multiple hypotheses testing to cor-rect for p-value. In practical terms, the FDR is the expected falsediscovery rate; for example, if 1000 observations were experimen-tally predicted to be different, and a maximum FDR for theseobservation was 0.1, then 100 out of these observations wouldbe expected to be false discovered. Genes were considered differ-entially expressed in a given library when the p-value was lessthan 0.001 and a greater than two-fold change in expression acrosslibraries was observed.

2.7. Phylogenetic analyses

Three immune genes, alpha-2-macroglobulin (Unigene12869_All), catalase (CL5831.Contig2_All) and heat shock protein 70(Unigene14757_All) were randomly selected for phylogeneticanalysis by using Maximum likelihood tree on MEGA 6 software(Tamura et al., 2013). The node support was assessed using a boot-strap procedure based on 10,000 replicates.

2.8. Quantitative RT–PCR analysis

Validation of our Illumina sequencing results involved theselection of ten differentially expressed M. rosenbergii unigenes(arginine kinase 1, anti-lipopolysaccharide factor, inhibitor ofapoptosis protein, caspase, chaperonin, heat shock protein 21, lec-tin 1, manganese superoxide dismutase, NF-kappa B inhibitoralpha and peroxiredoxin) for quantitative RT–PCR analysis(qRT–PCR). Primers for qRT–PCR were designed using Primer3

software http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/ (Table S1). The first strand of cDNA was synthe-sized from 1 lg of RNA (similar sample used for transcriptomesequencing) using the ImProm-IITM Reverse Transcription System(Promega). The qRT–PCR reaction (20 ll) consisted of 10 ll Taq-Man Universal RT–PCR Master Mix (Applied Biosystems, FosterCity, CA, USA), 1 ll of primers/probe set containing 900 nM of for-ward reverse primers, 300 nM probe and 2 ll of template cDNA.The RT–PCR program consisted of incubation at 50 �C for 2 min,and 40 cycles at 95 �C for 10 min, 95 �C for 15 s, and 60 �C for1 min with the Step One Plus Real-Time PCR System� (AppliedBiosystems). Concurrently, elongation factor 1-alpha primers wereapplied to normalize the amount of template cDNA added to eachreaction (Dhar et al., 2009). Each sample was analysed in triplicateand the comparative CT method (2�DDCT method) was used toobtain the expression level of the ten immune genes (Schmittgenand Livak, 2008).

3. Results

3.1. Illumina Sequencing and de novo assembly

The mission to identify immune-related genes which are crucialfor M. rosenbergii defence against WSSV infection involved prepar-ing two cDNA libraries using pooled mRNAs obtained from thehepatopancreases of control and WSSV-infected groups. The cDNAlibraries underwent sequencing using the Illumina HiSeqTM 2000platform, and the sequencing data were then subjected to de novoassembly using the Trinity program.

Once the adaptor sequence and the low-quality reads were fil-tered out, a total of 54,708,014 high quality clean reads of 90-bp inlength were generated, consisting of 4,923,721,260 nucleotides forthe control group and 38,671,944 reads with 3,480,474,960nucleotides for the WSSV-infected group. The sequencing readswere found to be high quality with the Average Q20 value of97.76% and their percentage of unknown nucleotide (N percentage)of 0%. All sequencing reads were deposited into the Short ReadArchive (SRA) of the National Center for Biotechnology Information(NCBI), and can be accessed under the accession numberSRR1424572 for control and SRR1424575 for WSSV-infected ones.An overview of the sequencing and assembly is shown in Table 1.The size and length distribution of the control and WSSV-infectedcontigs are shown in Fig. 1 in Rao et al. (submitted for publication).

3.2. Similarity searches

To achieve protein identification and gene annotation, a searchwas made on standard unigenes in the NCBI non-redundant (Nr)(19,799 unigenes, 31.14%), Swiss-Prot (16,832 unigenes, 26.47%),

Fig. 1. Species distribution of the BLASTX matches of the transcriptome unigenes.This figure shows the species distribution of unigene BLASTX matches against theNr protein database (cut-off value E < 10�5) and the proportions for each species.

R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22 13

Kyoto Encyclopedia of Genes and Genome pathway (KEGG)(14,706 unigenes, 23.12%) and Orthologous Groups of proteins(COG) databases (7856 unigenes, 12.36%) using the BLASTX pro-gram (E-value <10�5). This BLASTX search yielded a total of22,455 significant hits (35.31% of all unigenes). The size distribu-tion profile for the coding sequences (CDS) and identified proteinsare shown in Fig. 1 in Rao et al. (submitted for publication). A GOannotation search using the Blast2GO resulted in 6935 unigenes(10.9%) being categorized into 54 functional groups. The unigeneswith no hits in the BLASTX analysis were subjected to an ESTscan,producing 4977 unigenes (7.82%) predicted to contain coding

Fig. 2. Histogram presentation of Cluster of Orthologus Groups (COG) classification of 78classified into each of the 25 COG functional categories.

sequences. The size distribution of the ESTs and proteins are shownin Fig. 1 in Rao et al. (submitted for publication). The species distri-bution of the unigenes using the BLASTX results is shown in Fig. 1.

3.3. Functional annotation

The COG is a database containing classified orthologous geneproducts. Every protein in COG is assumed to originate from anancestor protein; thus the database is developed based on codingproteins with complete genomes as well as the system evolutionrelationships of bacteria, algae and eukaryotic creatures. The stan-dard unigenes were aligned to the COG database to predict andclassify their possible roles. A total of 7856 unigenes had COG clas-sifications, distributed among the 25 COG categories (Fig. 2).

Gene Ontology (GO) is a unified gene functional classificationsystem which offers a systematic and updated vocabulary in astrictly defined concept to describe the properties of genes andtheir products in any organism. GO has three ontologies: molecularfunction, cellular component and biological process. The basic unitof GO is ‘GO-term’, which is a type of ontology. The unigenes withNr annotations were subjected to GO annotations using the BLAS-T2GO program for functional unigene classification and to obtainthe macro-level gene functional distribution for this species. TheGO annotations produced 6935 unigenes (Biological process:2477 unigenes; Cellular component: 1896 unigenes; and Molecu-lar function: 2562 unigenes), which were assigned to 54 GO ontol-ogy (Fig. 3).

The understanding of biological functions and gene interactioncan be achieved by performing a pathway-based analysis. TheKyoto Encyclopedia of Genes and Genomes (KEGG) offers detailedinformation on networks related to intracellular molecular interac-tions and their organism-specific variations. The biological path-ways involved in WSSV infection were identified when wemapped the annotated sequences to the reference canonical path-

56 known protein annotated unigenes. Each bar represents the number of unigenes

Fig. 3. Gene ontology (GO) classification of the 6935 protein annotated unigenes. Unigenes sequences were systematically classified into GO sub-categories under thebiological process, cellular component and molecular function gene ontology catalogue system. Each bar represents the relative abundance of unigenes classified under eachsub-category.

Fig. 4. Top 20 KEGG biological pathway classification histograms for annotated unigenes. The x-axis shows pathways from KEGG classification and the y-axis shows thenumber of the matched unigenes.

14 R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22

ways contained in the KEGG database. A total of 14,706 unigeneswere assigned into 253 pathways. The highly represented cate-gories were ‘‘metabolic pathways’’ (2192 members, 14.91%), ‘‘reg-ulation of actin cytoskeleton” (557 members, 3.79%),‘‘spliceosome” (509 members, 3.46%), ‘‘ubiquitin mediated proteol-ysis (508 members, 3.45%) and ‘‘RNA transport” (498 members,3.39%). The least represented pathways with less than 10 unigenesfound in each pathway were ‘‘biotin metabolism”, ‘‘vitamin B6metabolism”, ‘‘cyanoamino acid metabolism”, ‘‘phenylalanine, tyr-

osine and tryptophan biosynthesis”, ‘‘D-glutamine and D-glutamatemetabolism” and ‘‘type I diabetes mellitus”. The top twenty ofthese KEGG biological pathway classifications are shown in Fig. 4.

3.4. Identification of aberrantly expressed genes

In order to identify the genes expressed differentially betweenthese two groups, a comparison of the relative transcript abun-dance in each unigene was carried out using the FPKM method

Fig. 5. Digital gene expression between control group and WSSV infected group.Each point represents a unigene. The x- and y-axis are the log10 of the normalizedexpression level (FPKM) of unigene between the two groups. Red and green pointsindicate significant change at the absolute value of log2 (FPKM ratio in two groups)P1 and FDR = 0.001. Red points indicate up-regulated unigenes and green pointsindicate down-regulated unigenes in the two groups which its expression level isrepresented by the y-axis. Blue points indicate insignificant differentially expressedunigenes. (For interpretation of the references to colour in this figure legend, thereader is referred to the web version of this article.)

Fig. 6. Comparison of the expression profiles of selected genes as determined byIllumina HiSeqTM 2000 sequencing (orange) and qRT–PCR (yellow). Target geneabbreviations are as follows: AK1 – arginine kinase, ALF – anti-lipopolysaccharidefactor, IAPs – apoptosis inhibitor, Casp – caspase, Chap – chaperonin, HSP21 – heatshock protein 21, LT1 – lectin 1, MnSOD –manganese superoxide dismutase, NFjBI-a – NF-kappa B inhibitor alpha, PRDX – peroxiredoxin. Error bars indicated standarddeviations of averages from three replicates. For interpretation of the references tocolour in this figure legend, the reader is referred to the web version of this article.)

R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22 15

(Fragments Per kb per Million fragments). A total of 14,416 unige-nes were found to be aberrantly expressed; with 8443 unigenessignificantly up-regulated and 5973 unigenes significantly down-regulated (Fig. 5). The differentially expressed genes were anno-tated against the NR, Swiss-Prot, GO, COG and KEGG databasesby BLASTX, with a cutoff E-value of 10�5. The resulting annotationanalysis is presented in additional Table S3. The 9469 (65%) genescontaining low sequence homology to known sequences in publicdatabases could represent non-coding RNA, misassembled unige-nes, or unknown genes of M. rosenbergii which responded toWSSV-infection.

3.5. Phylogenetic analyses

The reason for performing the phylogenetic analyses by usingcontigs or unigenes was to test the accuracy of assembly(Sadamoto et al., 2012). Maximum likelihood tree for three unige-nes, alpha-2-macroglobulin (Unigene12869_All), catalase (CL5831.Contig2_All) and heat shock protein 70 (Unigene14757_All) werefound to be closely related with other crustacean and was evolu-tionarily distant from that of other phylum represented in the tree(Fig. S2).

3.6. qRT–PCR analysis

Ten unigenes were chosen randomly for quantitative real time–PCR (qRT–PCR) analysis, to validate the sequencing results. Theresulting qRT–PCR results showed similar trends for all the genesto those of the sequencing data (Fig. 6). For example, based onthe Illumina sequencing analysis, arginine kinase 1, anti-lipopolysaccharide factor, inhibitor of apoptosis protein, caspase,heat shock protein 21, lectin 1, manganese superoxide dismutase,NF-kappa B inhibitor alpha and peroxiredoxin were up-regulated5.33, 1.66, 2.26, 2.41, 4.61, 2.83, 1.61, 1.12, and 1.35 log2-foldchanges respectively; and 6.51, 2.5, 1.53, 1.8, 3.35, 2.05, 1.38,

2.68 and 1.8 log2-fold changes respectively in the qRT–PCR analy-sis. The chaperonin unigene meanwhile was down-regulated by�2.01 log2-fold changes in our transcriptome data, and was simi-larly found to be down-regulated by �3.33 log2-fold changes inthe qRT–PCR analysis. Although the results from both analysesdid not match perfectly, perhaps due to sequencing biases (AC’tHoen et al., 2008; Grabherr et al., 2011), the qRT–PCR analysis sub-stantially confirmed the direction of changes obtained from theIllumina sequencing analysis.

3.7. Potential immune-related genes involved in M. rosenbergiiimmune responses

Numerous genes found to be aberrantly expressed in theWSSV-infected groups compared to the control groups were known to bepart of various processes clustered under the animal immune sys-tem (Table 2). These immune genes are grouped into 15 functions,which include antiviral proteins (2 unigenes), antimicrobial pro-teins (4 unigenes), proteases (10 unigenes), protease inhibitors (3unigenes), blood clotting system (2 unigenes), transcriptional con-trol (4 unigenes), cell death (11 unigenes), cell adhesion (4 unige-nes), heat shock proteins (6 unigenes), RNA silencing (3 unigenes),oxidative stress (7 unigenes), pathogen recognition immune recep-tors (9 unigenes), prophenoloxidase system (2 unigenes), sig-nalling pathway; (Toll pathway-5 unigenes, Wnt signallingpathway-3 unigenes, MAPK signalling pathway-7 unigenes, JAK/STAT pathway-2 unigenes, Ubiquitin Proteasome Pathway-10 uni-genes) and other immune genes (17 unigenes). Overall a total of 74immune related unigenes were found to be significantly up-regulated whereas 37 were significantly down-regulated. The roleof these groups is elaborated in detail in Section 4.

4. Discussion

The white spot syndrome disease resulting fromWSSV infectionis the most serious viral disease in cultured commercial shrimpsworldwide (Flegel, 1997; Escobedo-Bonilla et al., 2008). The dis-ease has caused high mortality and consequent economic lossesto the shrimp culture industry in Thailand (Wongteerasupaya

Table 2Candidate genes involved in M. rosenbergii immune response against WSSV.

Category or gene id Homologues function Species FCa

AntiviralUnigene19164_All Zinc finger CCCH-type antiviral protein 1 Anolis carolinensis 1.20Unigene10846_All Antiviral protein Litopenaeus vannamei �1.19

AntimicrobialUnigene26338_All Lysozyme Portunus trituberculatus 2.24Unigene13073_All NF-kappa B inhibitor alpha Macrobrachium rosenbergii 1.12Unigene4120_All Anti-lipopolysaccharide factor Macrobrachium rosenbergii 1.66Unigene25224_All Crustin type I Macrobrachium rosenbergii �14.90

Blood clotting systemUnigene13048_All Clottable protein Marsupenaeus japonicus 3.67Unigene6536_All Transglutaminase Fenneropenaeus chinensis 3.05

ProteasesCL2946.Contig3_All Clip domain serine proteinase 1 Penaeus monodon 11.30CL5686.Contig1_All Cathepsin L Penaeus monodon �3.59CL1587.Contig2_All Cathepsin D Penaeus monodon �2.83CL1474.Contig1_All Cathepsin C Fenneropenaeus chinensis 1.08CL6038.Contig1_All 26S protease regulatory Nasonia vitripennis �1.44Unigene7896_All ATP-dependent Clp protease proteolytic Caenorhabditis briggsae �2.46Unigene12869_All Alpha-2-macroglobulin Macrobrachium rosenbergii 2.90Unigene13293_All Astacin Strongylocentrotus purpuratus �1.03Unigene29347_All Serine carboxypeptidase 1 Saccoglossus kowalevskii �4.21Unigene13633_All Serine protease Fenneropenaeus chinensis 2.37

Protease inhibitorCL1127.Contig2_All Kazal-type serine proteinase inhibitor 4 Procambarus clarkii �4.57Unigene22656_All Pacifastin-related serine protease inhibitor Portunus trituberculatus 6.79CL816.Contig12_All Serpin B Amblyomma americanum �1.68

Signal transductionToll pathwayUnigene25507_All Toll-like receptor 3 Daphnia pulex 3.86CL2421.Contig1_All Toll-like receptor 8 Ixodes scapularis 2.11Unigene11753_All Toll protein Litopenaeus vannamei 11.00CL2438.Contig1_All Caspase Eriocheir sinensis 1.54Unigene25799_All Myd88 protein Artemia sinica 1.35

Wnt signalling pathwayUnigene23651_All Ring box protein Bombyx mori �2.21Unigene16346_All Supernumerary limbs Tribolium castaneum 1.27Unigene25990_All Casein kinase II subunit beta Acromyrmex echinatior �1.58

MAPK signalling pathwayCL1473.Contig2_All Heat shock protein 70 cognate Fenneropenaeus chinensis 4.11CL4432.Contig2_All Max protein Daphnia pulex �5.67Unigene29777_All Serine/threonine-protein kinase PAK 1 Harpegnathos saltator 3.43Unigene9636_All Serine/threonine-protein kinase TAO1 Acromyrmex echinatior 1.44CL3877.Contig1_All MAPK kinase 1 interacting protein 1 Penaeus monodon �1.62CL5614.Contig1_All JNK-interacting protein 3 Apis mellifera 4.99Unigene102_All Map kinase-interacting serine/threonine Scylla paramamosain 1.46

JAK/STAT pathwayUnigene25562_All STAT long form Penaeus monodon 2.78CL1133.Contig5_All Domeless Tribolium castaneum 1.38

Ubiquitin proteasome pathwayUnigene548_All Ubiquitin Procambarus clarkii �1.61Unigene22971_All Cullin-2 Harpegnathos saltator 1.29Unigene6699_All Ubiquitin-protein ligase E3C Camponotus floridanus 1.03CL5401.Contig1_All E3 ubiquitin-protein ligase HUWE1 Acromyrmex echinatior 10.58Unigene6165_All Rho-related BTB domain-containing protein 2 Acromyrmex echinatior 4.44Unigene29739_All Baculoviral IAP repeat-containing protein 6 Crassostrea gigas 2.80Unigene10240_All Cullin-associated NEDD8-dissociated protein 1 Crassostrea gigas 1.66CL1584.Contig1_All Beta 1,4-endoglucanase Cherax quadricarinatus 1.98Unigene3861_All U88 Brugia malayi 1.39Unigene20413_All RING finger protein Eriocheir sinensis �1.75

Transcriptional controlCL965.Contig3_All RNA-binding region-containing protein Danaus plexippus 3.03Unigene7122_All DNA-directed RNA polymerase III subunit RPC2-like Cavia porcellus �2.02CL5643.Contig1_All Microphthalmia-associated transcription factor-like Megachile rotundata 3.43CL4672.Contig1_All Replication protein A Callorhinchus milii 1.65

Cell deathUnigene28705_All Ribosomal protein S3 Plecoglossus altivelis �1.03CL1843.Contig2_All ALG-2 interacting protein x Penaeus monodon Penaeus monodon 2.88CL6385.Contig2_All Caspase Harpegnathos saltator 2.41Unigene2555_All Beclin 1 Megachile rotundata 11.10

16 R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22

Table 2 (continued)

Category or gene id Homologues function Species FCa

Unigene14045_All Program cell death 5-like Penaeus monodon 1.36Unigene325_All Prohibitin Penaeus monodon �2.17Unigene10321_All Oncoprotein nm23 Litopenaeus vannamei 1.18Unigene19853_All Deoxyribonuclease I Marsupenaeus japonicus �2.63CL6019.Contig2_All Inhibitor of apoptosis protein Litopenaeus vannamei 2.26Unigene16154_All Zinc finger protein ush-like Nasonia vitripennis 5.08Unigene9836_All Autophagy protein 5 Callinectes sapidus �1.16

Cell adhesionUnigene13411_All ADP-ribosylation factor 1 Marsupenaeus japonicus �1.52Unigene16833_All Adhesion regulating molecule 1 Scylla paramamosain �1.63CL2046.Contig1_All Integrin Litopenaeus vannamei 1.85Unigene23158_All Tetraspanins-like protein-8 Fenneropenaeus chinensis �1.72

Oxidative stressUnigene7354_All Thioredoxin 1 Bombus impatiens �2.06Unigene20577_All Peroxiredoxin Eriocheir sinensis 1.35CL353.Contig2_All Glutathione S transferase D1 Procambarus clarkii �2.94CL1891.Contig1_All O-methyltransferase Litopenaeus vannamei �1.71Unigene26204_All Glutathione peroxidase Metapenaeus ensis 1.85CL5831.Contig2_All Catalase Macrobrachium rosenbergii 3.56Unigene19405_All Manganese superoxide dismutase Macrobrachium rosenbergii 1.61

Heat shock proteinsUnigene20010_All Chaperonin 10 Scylla paramamosain �2.87CL4288.Contig1_All Heat shock protein 90 Macrobrachium nipponense 1.43Unigene13644_All Chaperonin Macrobrachium rosenbergii �2.01Unigene14757_All Heat shock protein 70 Portunus trituberculatus 2.43Unigene16617_All Heat shock protein 60 Litopenaeus vannamei 1.20Unigene3736_All Heat shock protein 21 Macrobrachium rosenbergii 4.61

RNA silencingCL1315.Contig3_All Dicer 2 Litopenaeus vannamei 1.48Unigene26246_All Argonaute 1 isoform B Marsupenaeus japonicus 4.03CL3019.Contig2_All Argonaute 2 Marsupenaeus japonicus 1.56

PRPsUnigene1391_All Hemolectin Papilio xuthus 5.85CL1124.Contig1_All Glucan pattern-recognition lipoprotein Fenneropenaeus chinensis 1.53Unigene17200_All Lectin B isoform 1 Marsupenaeus japonicus �1.65CL4516.Contig1_All Lectin 2 Macrobrachium rosenbergii �1.16CL51.Contig1_All Lectin D Marsupenaeus japonicus �1.82CL5230.Contig3_All C-type lectin 5 Fenneropenaeus chinensis 2.00Unigene10978_All Lectin 1 Macrobrachium rosenbergii 2.83Unigene23671_All Lipopolysaccharide and beta-1,3-glucan binding protein Macrobrachium rosenbergii 1.20CL3039.Contig4_All Lectin 4 Macrobrachium rosenbergii 2.41

ProPO systemUnigene12734_All Prophenoloxidase Macrobrachium rosenbergii 4.54Unigene7353_All Prophenoloxidase activating factor Fenneropenaeus chinensis 1.83

Other immune genesUnigene26739_All Metallothionein I Macrobrachium rosenbergii 1.12CL4071.Contig2_All Peritrophin Macrobrachium nipponense 2.56Unigene7335_All Profilin Penaeus monodon 1.15Unigene1349_All Copper-specific metallothionein CuMT-II Callinectes sapidus �3.19Unigene10835_All Selenoprotein W Cricetulus griseus �2.76CL4159.Contig2_All Selenoprotein M Penaeus monodon �1.16CL6324.Contig2_All Arginine kinase 1 Macrobrachium rosenbergii 5.33CL5195.Contig1_All Hemocyanin Macrobrachium nipponense 3.04Unigene12758_All Tetraspanins-like protein-8 Fenneropenaeus chinensis �1.53CL1939.Contig4_All Ferritin Litopenaeus vannamei 1.84CL6297.Contig1_All E cadherin Tenebrio molitor 1.45CL2214.Contig1_All Chitinase Pandalopsis japonica 1.97CL6026.Contig5_All Actin Marsupenaeus japonicus 3.69Unigene13021_All Calponin Chironomus riparius 1.41CL2339.Contig2_All Adenosine deaminase Caligus rogercresseyi �1.23Unigene19713_All Pacifastin heavy chain Macrobrachium rosenbergii 1.27CL4920.Contig1_All Calmodulin Procambarus clarkii 1.26

a Fold changes (log2 ratio) in gene expression. PRPs – pattern recognition proteins, ProPO – prophenoloxidase.

R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22 17

et al., 1995), Taiwan (Wang et al., 1995), India (Rajan et al., 2000;Sunarto, 2001) and Malaysia (Wang et al., 1999). Knowledge aboutthe interaction between M. rosenbergii and the virus is limited,despite the numerous published studies on the characterisationand detection of WSSV available (Huang et al., 2001; van Hultenet al., 2001; Pradeep et al., 2008; Sithigorngul et al., 2006; Otta

et al., 1999). A better understanding of viral-host interaction isurgently needed to help develop a pro-active approach to fightingand suppressing this disease.

The application of NGS platforms such as Illumina HiSeqTM 2000has allowed the fast and cost-effective generation of massiveamounts of sequence data, which has revolutionised the field of

18 R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22

transcriptomics. This platform, together with the de novo assem-bler, has been successfully utilised to discover novel genes as wellas to reveal the expression levels of genes in non-model organismswhich lack a complete genome database (Mohd-Shamsudin et al.,2013; Xu et al., 2013; Li et al., 2012; Shen et al., 2011;Kawahara-Miki et al., 2011). The introduction of NGS technologyhas led to studies on host–viral interaction in shrimps in order toidentify candidate immune-related genes (Xue et al., 2013; Chenet al., 2013; Li et al., 2013; Zeng et al., 2013). In the present study,we utilised a similar approach to elucidate the transcriptionalresponses of M. rosenbergii towards WSSV infection.

The total RNA for sequencing was sampled from pooled hep-atopancreases of both groups, taken at 12th hour time point. Theoverall analysis yielded 14,416 unigenes differentially expressed,with 8443 unigenes significantly up-regulated and 5973 unigenessignificantly down-regulated. The number of differentiallyexpressed unigenes obtained was higher than from Litopenaeusvannamei (315 unigenes in hemocytes and 1496 unigenes in hep-atopancreas) (Xue et al., 2013; Chen et al., 2013). This could bedue to a number of reasons: the type of tissue samples used, differ-ent time-points of tissue sampling, the use of different NGS plat-forms, different de novo assembly approaches, and lastly differentspecies of shrimp. However, the number of up-regulated genesobtained was much larger than the down-regulated genes, aswas the case with studies done on L. vannamei challenged byWSSV.

Crustaceans likeM. rosenbergii rely on their innate immune sys-tem to defend themselves against various pathogens and environ-mental stress. This defence system is initiated by detecting theinvading pathogen through pattern recognition proteins (PRPs)(Akira et al., 2006). The hepatopancreas of M. rosenbergii, similarto L. vannamei and Litopenaeus setiferus, seems to be the primaryproduction site for two PRPs molecules, namely the beta-1,3-glucan binding protein (LGBP) and C-type lectins (Gross et al.,2001). The LGBP protein functions to recognize and eliminatepathogens, whereas C-type lectins facilitate recognition and patho-gen phagocytosis through opsonisation (Cerenius and Söderhäll,2004; Vargas-Albores and Yepiz-Plascencia, 2000). In this study,PRPs expression was affected by the WSSV challenge, similar towhat has been reported in previous studies (Roux et al., 2002;Ma et al., 2007; Song et al., 2010). The initial immune responsetowards pathogen invasion also activates the melanization process.Melanization is achieved by the prophenoloxidase-activating(proPO-AS) system, which is an enzymatic cascade involving sev-eral enzymes including the key enzyme phenoloxidase (PO)(Söderhäll and Cerenius, 1998; Ratcliffe et al., 1984). The up-regulation of proPO-AS components in our results showed that thissystem was activated by WSSV infection.

Besides the melanization process, anti-microbial peptides(AMPs) also function as the first line of defence to fight againstinvading pathogens (Hancock and Diamond, 2000). Several fami-lies of shrimp AMPs, such as penaeidins, lysozymes, crustins,anti-lipopolysaccharide factors (ALFs) and stylicins, have beenidentified and characterised previously (Rolland et al., 2010;Tassanakajon et al., 2011; Destoumieux et al., 2000; Bartlettet al., 2002). In our data, we found only three members of theAMPs, lysozymes, ALFs and crustins. The lysozymes and ALFs weresignificantly up-regulated, whereas the crustins were significantlydown-regulated. An increased expression of lysozyme and ALF wasalso reported in Fenneropenaeus chinensis, Litopenaeus stylirostrisand L. vannamei, suggesting that these two genes are involved inthe innate response of shrimps to WSSV (Wang et al., 2006; Maiand Wang, 2010; De la Vega et al., 2008). A reduced expressionof crustin type 1 was also noted in P. monodon challenged by WSSV(Antony et al., 2011a, 2011b). Since crustin type 1 was reported tobe down-regulated in both P. monodon and M. rosenbergii during

WSSV infection, therefore this isoform of crustin may not berequired for antiviral defence of shrimp against WSSV (Antonyet al., 2011b).

Blood coagulation is a conserved defence mechanism amonginvertebrates, which possess open circulatory systems and hencerely more extensively on this system than vertebrates (Theopoldet al., 2004). Transglutaminase (TGase) and clotting protein (CP)are molecules responsible for blood clotting in shrimps(Maningas et al., 2008). The clotting process involves the polymer-ization of the plasma clotting protein catalyzed by a Ca2+ depen-dent transglutaminase (TGase) released from the haemocytes inresponse to tissue damage or the invasion of microbial pathogens.The released TGase produces a covalent cross-linkage between glu-tamine and lysine of the clottable protein (CP) (Kopácek et al.,1993). Higher expression levels for both components of bloodcoagulation were observed in this study. Both the TGase geneand clotting protein were observed to be differentially expressedin response to viral and bacterial pathogens, which suggests thatthese genes have an important role in shrimp immunity (Songet al., 2003; Liu et al., 2007; Wang et al., 2010). Several familiesof heat shock proteins (Hsps) – Hsp90s (83–99 kDa), Hsp70s (68–80 kDa), Hsp60s, and the small Hsps (25–28 kDa), function aschaperones, correcting the folding of proteins and repairing andforming multiprotein assemblies (Stewart and Young, 2004;Pockley, 2003). The differential expression of heat shock proteinsin our data is similar to that reported by other researchers inWSSV-infected shrimps (Wang et al., 2006; Yan et al., 2010). Allthese results point to a potential role for heat shock proteins asanti-virus responses to WSSV in shrimps.

In our transcriptome data, three unigenes identified as impor-tant components of the RNA interference (RNAi) pathway, Arg-onaute 1, Argonaute 2 and Dicer 2 were highly expressed.Previous research reported a significant inhibition of virus replica-tion via the dsRNA (Robalino et al., 2005; Yodmuang et al., 2006;Sarathi et al., 2008; Ongvarrasopone et al., 2008) and siRNA(Westenberg et al., 2005;Wu et al., 2007; Xu et al., 2007) of specificgenes, suggesting that an RNAi mechanism against virus infectionpossibly exists in shrimps. Subsequently, the genes encoding Arg-onaute (Unajak et al., 2006), Dicer (Su et al., 2008) and other mem-bers of the RNAi pathway were revealed in shrimps. The increasedexpression of these genes in our data might imply that they havean antiviral role against WSSV. The zinc finger CCCH-type antiviralprotein 1-like was found to be significantly up-regulated in ourdata. This protein has been reported to inhibit the replication ofseveral viruses which could possibly act similarly to WSSV infec-tion (Gao et al., 2002; Bick et al., 2003; Müller et al., 2007).

Apoptosis also plays a major role in regulating the innate cellu-lar response, by minimising virus replication and suppressing thespread of progeny virus in the host (Roulston et al., 1999). Caspase,an effector molecule that mediates the apoptotic process (Wanget al., 2008), was found to be up-regulated in our data. We notethat the up-regulation of this gene has been similarly observedwhen M. rosenbergii are challenged by the infectious hypodermaland haematopoietic necrosis virus (IHHNV) (Arockiaraj et al.,2012b) and in other shrimps in response to WSSV as well(Wongprasert et al., 2007; Phongdara et al., 2006). The inhibitorsof apoptosis proteins (IAPs) regulate the apoptosis mechanism byblocking the activity of caspase (Deveraux and Reed, 1999). Fur-thermore, IAPs are also reported to participate in signal transduc-tion regulation during immune responses in insects andmammals (Deveraux and Reed, 1999; Earnshaw et al., 1999; Silkeand Meier, 2013). IAPs were highly expressed in our transcriptomedata, in M. rosenbergii challenged with IHHNV (Arockiaraj et al.,2011b) and in L. vannamei challenged by WSSV (Wang et al.,2013), which means they could perhaps play a protective roleagainst viral infection. We observed the expression of oncoprotein

R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22 19

nm23, beclin, prohibitin and deoxyribonuclease I in our data sim-ilarly to previous reports, which may reaffirm the role of thesegenes in controlling the extent of apoptosis in response to WSSV(Chen et al., 2013). The role of other genes in this group remainsvague and needs further study. The differentially expressed genesin cell adhesion, transcriptional control, proteases and proteasesinhibitors groups are generally assumed to modulate phagocyticevents, the recruitment of immune cells to sites of infection, cellu-lar remodelling, and extracellular immune cascades such as themelanization response (Robalino et al., 2007).

Antioxidant enzymes play a crucial role in shrimp defencesagainst pathogens by removing potentially harmful excess reactiveoxygen species generated during the immune response (De laFuente and Victor, 2000). In this study we reported seven unigenesin this group to be differentially expressed after WSSV infection.Aberrant expression levels of these genes have been noted in othershrimps as well, but in a differential manner according to the typesof pathogen (Mohankumar and Ramasamy, 2006; Castex et al.,2010; Mathew et al., 2007; Zhang et al., 2007). We also observedthat the four signal transduction pathways, Toll, Janus kinase-signal transducer and activator of transcription (JAK/STAT),mitogen-activated protein kinase (MAPK) and Wnt signalling path-way were greatly affected by WSSV infection. These pathways areknown to be activated to play important roles in viral recognitionand replication (Chen et al., 2008; Andrade et al., 2004; Gan et al.,2001; Wang et al., 2012; Li and Xiang, 2013). These Toll, MAPK andWnt pathways were also found to be activated in L. vannamei hep-atopancreas when infected with WSSV (Chen et al., 2013). How-ever, the underlying molecular mechanisms of these pathways inresponse to WSSV infection still remains unclear. Therefore furtherstudies are necessary to uncover the roles of those pathways in M.rosenbergii innate immune system.

In addition to the four above mentioned pathways, we alsoobserved the activation of ubiquitin proteasome pathway (UPP)in M. rosenbergii with E3 ubiquitin-protein ligase HUWE1 (+10.58fold) showing the highest fold changes. The UPP pathway regulatesdegradation of short-lived proteins in various cellular processessuch as cell cycle control, transcriptional regulation, signal trans-duction, antigen presentation and induction of the inflammatoryresponse, degradation from the ER, membrane trafficking, receptorendocytosis and down-regulation, apoptosis, and development invertebrate (Loureiro and Ploegh, 2006). Furthermore, this pathwayis also involved in viral infection and facilitates activities requiredfor various aspects of virus life cycle, from entry through replica-tion and enhanced cell survival to viral release (Chen and Gerlier,2006). Lately, various reports have emerged showing the abilityof WSSV to hijack the host UPP pathway to modulate cellularintrinsic antiviral activities and innate immunity (Jeena et al.,2012; Keezhedath et al., 2013; Vidya et al., 2013). The higherexpression of E3 ubiquitin-protein ligase HUWE1 could possiblybe due to ability of virus to redirect host ubiquitin E3 ligases to tar-get new substrate proteins for its own gain (Banks et al., 2003).However further study is required to fully understand the interac-tion between WSSV and this protein.

Several of the differentially expressed genes in other immunegene groups have been reported to be involved in immuneresponses in shrimps against WSSV. Hemocyanins, the oxygen-transporting proteins in arthropods and molluscs, play a defence-related role mediated through phenoloxidase activity (Markl andDecker, 1992; Zhang et al., 2004). Several previous studies havefound hemocyanins in shrimp and crayfish to be highly expressedduring WSSV infection (Pan et al., 2005; Lei et al., 2008; Wanget al., 2013). Ferritin, a major iron storage protein involved in ironmetabolism, was up-regulated in the hepatopancreases of theshrimps challenged by the white spot syndrome virus (WSSV)(Pan et al., 2005; Zhang et al., 2006). Profilins are a group of ubiq-

uitous proteins commonly found in animals, plants and viruses.They activate the sequestering of actin monomers and preventactin polymerization. Besides their actin binding activity, profilinsare also involved in cellular processes such as membrane traffick-ing, small-GTPase signalling and nuclear activities, in addition toneurological diseases and tumour formation (Rawe et al., 2006;Witke, 2004). The up-regulation of this gene has also beenobserved in L. vannamei and F. chinensis challenged with WSSV(Kong et al., 2009; Clavero-Salas et al., 2007). Calmodulin, animportant intracellular Ca2+ sensor and a Ca-binding proteininvolved in calcium-dependent signal transduction pathways, hasbeen induced in L. vannamei challenged with WSSV (Ji et al.,2011). Arginine kinase (AK) is a phosphotransferase that plays acrucial role in the coupling of energy production during energymetabolism in invertebrates (Arockiaraj et al., 2011a; Yao et al.,2009). Recently, shrimp AK has been reported to play an importantrole in WSSV infections by interacting with the VP14 protein ofWSSV in infected L. vannamei (Ma et al., 2014). All of these geneswere up-regulated in our transcriptome data, indicating that theymay play important roles in M. rosenbergii defence against WSSV.

5. Conclusion

In this study, we investigated the transcriptome profile of M.rosenbergii hepatopancreas when infected with WSSV using theIllumina HiSeqTM 2000 technology. We observed that WSSV infec-tion has effected the transcriptome profile of M. rosenbergii’s hep-atopancreas, with 8443 host genes were significantly up-regulatedwhereas 5973 genes were significantly down-regulated. The differ-entially expressed immune related genes were grouped into 15animal immune functions. However to further enhance our under-standing regarding molecular interactions between M. rosenbergiiand WSSV, further studies on the functionality of these genes willprovide valuable information to find effective strategies to preventviral disease. Besides, with whole genome sequence of this speciesis still unavailable, large number of transcripts obtained from thisstudy could provide a strong basis for future genomic research onM. rosenbergii.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by a High Impact Research (HIR)Grant, H-23001-G000006 and Postgraduate Research Grant (PPP)of the University Malaya, Malaysia (PG088-2012B).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jip.2016.01.002.

References

AC’t Hoen, P., Ariyurek, Y., Thygesen, H.H., Vreugdenhil, E., Vossen, R.H., de Menezes,R.X., Boer, J.M., van Ommen, G.-J.B., den Dunnen, J.T., 2008. Deep sequencing-based expression analysis shows major advances in robustness, resolution andinter-lab portability over five microarray platforms. Nucleic Acids Res. 36,e141–e141.

Akira, S., Uematsu, S., Takeuchi, O., 2006. Pathogen recognition and innateimmunity. Cell 124, 783–801.

Andrade, A., Silva, P., Pereira, A., De Sousa, L., Ferreira, P., Gazzinelli, R., Kroon, E.,Ropert, C., Bonjardim, C., 2004. The vaccinia virus-stimulated mitogen-activatedprotein kinase (MAPK) pathway is required for virus multiplication. Biochem. J.381, 437–446.

20 R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22

Antony, S.P., Singh, I., Sudheer, N., Vrinda, S., Priyaja, P., Philip, R., 2011a. Molecularcharacterization of a crustin-like antimicrobial peptide in the giant tigershrimp, Penaeus monodon, and its expression profile in response to variousimmunostimulants and challenge with WSSV. Immunobiology 216, 184–194.

Antony, S.P., Philip, R., Joseph, V., Singh, I.B., 2011b. Anti-lipopolysaccharide factorand crustin-III, the anti-white spot virus peptides in Penaeus monodon: controlof viral infection by up-regulation. Aquaculture 319, 11–17.

Arockiaraj, J., Vanaraja, P., Easwvaran, S., Singh, A., Alinejaid, T., Othman, R.Y.,Bhassu, S., 2011a. Gene profiling and characterization of arginine kinase-1(mrak-1) from freshwater giant prawn (Macrobrachium rosenbergii). FishShellfish Immunol. 31, 81–89.

Arockiaraj, J., Vanaraja, P., Easwvaran, S., Singh, A., Othman, R.Y., Bhassu, S., 2011b.Bioinformatic characterization and gene expression pattern of apoptosisinhibitor from Macrobrachium rosenbergii challenged with infectioushypodermal and hematopoietic necrosis virus. Fish Shellfish Immunol. 31,1259–1267.

Arockiaraj, J., Easwvaran, S., Vanaraja, P., Singh, A., Othman, R.Y., Bhassu, S., 2012a.Molecular cloning, characterization and gene expression of an antioxidantenzyme catalase (MrCat) from Macrobrachium rosenbergii. Fish ShellfishImmunol. 32, 670–682.

Arockiaraj, J., Easwvaran, S., Vanaraja, P., Singh, A., Othman, R.Y., Bhassu, S., 2012b.Effect of infectious hypodermal and haematopoietic necrosis virus (IHHNV)infection on caspase 3c expression and activity in freshwater prawnMacrobrachium rosenbergii. Fish Shellfish Immunol. 32, 161–169.

Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P.,Dolinski, K., Dwight, S.S., Eppig, J.T., 2000. Gene ontology: tool for theunification of biology. Nat. Genet. 25, 25–29.

Banks, L., Pim, D., Thomas, M., 2003. Viruses and the 26s proteasome: hacking intodestruction. Trends Biochem. Sci. 28, 452–459.

Bartlett, T.C., Cuthbertson, B.J., Shepard, E.F., Chapman, R.W., Gross, P.S., Warr, G.W.,2002. Crustins, homologues of an 11.5-kda antibacterial peptide, from twospecies of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus.Mar. Biotechnol. 4, 278–293.

Bick, M.J., Carroll, J.-W.N., Gao, G., Goff, S.P., Rice, C.M., MacDonald, M.R., 2003.Expression of the zinc-finger antiviral protein inhibits alphavirus replication. J.Virol. 77, 11555–11562.

Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M.-C., Estreicher, A., Gasteiger, E.,Martin, M.J., Michoud, K., O’Donovan, C., Phan, I., 2003. The Swiss-PROT proteinknowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res. 31, 365–370.

Bonami, J.-R., Widada, J. Sri, 2011. Viral diseases of the giant fresh water prawnMacrobrachium rosenbergii: a review. J. Invertebr. Pathol. 106, 131–142.

Castex, M., Lemaire, P., Wabete, N., Chim, L., 2010. Effect of probiotic Pediococcusacidilactici on antioxidant defences and oxidative stress of Litopenaeusstylirostris under vibrio Nigripulchritudo challenge. Fish Shellfish Immunol.28, 622–631.

Cerenius, L., Söderhäll, K., 2004. The prophenoloxidase-activating system ininvertebrates. Immunol. Rev. 198, 116–126.

Chang, P.-S., Chen, H.-C., Wang, Y.-C., 1998. Detection of white spot syndromeassociated baculovirus in experimentally infected wild shrimp, crab andlobsters by in situ hybridization. Aquaculture 164, 233–242.

Chen, M., Gerlier, D., 2006. Viral hijacking of cellular ubiquitination pathways as ananti-innate immunity strategy. Viral Immunol. 19, 349–362.

Chen, L.L., Wang, H.C., Huang, C.J., Peng, S.E., Chen, Y.G., Lin, S.J., Chen, W.Y., Dai, C.F.,Yu, H.T., Wang, C.H., et al., 2002. Transcriptional analysis of the DNA polymerasegene of shrimp white spot syndrome virus. Virology 301, 136–147.

Chen, W.Y., Ho, K.C., Leu, J.H., Liu, K.F., Wang, H.C., Kou, G.H., Lo, C.F., 2008. WSSVinfection activates stat in shrimp. Dev. Comp. Immunol. 32, 1142–1150.

Chen, X.H., Zeng, D.G., Chen, X.L., Xie, D.X., Zhao, Y.Z., Yang, C.L., Li, Y.M., Ma, N., Li,M., Yang, Q., et al., 2013. Transcriptome analysis of Litopenaeus vannamei inresponse to white spot syndrome virus infection. PLoS ONE 8.

Clavero-Salas, A., Sotelo-Mundo, R.R., Gollas-Galván, T., Hernández-López, J.,Peregrino-Uriarte, A.B., Muhlia-Almazán, A., Yepiz-Plascencia, G., 2007.Transcriptome analysis of gills from the white shrimp Litopenaeus vannameiinfected with white spot syndrome virus. Fish Shellfish Immunol. 23, 459–472.

Conesa, A., Götz, S., García-Gómez, J.M., Terol, J., Talón, M., Robles, M., 2005.Blast2GO: a universal tool for annotation, visualization and analysis infunctional genomics research. Bioinformatics 21, 3674–3676.

De la Fuente, M., Victor, V., 2000. Anti-oxidants as modulators of immune function.Immunol. Cell Biol. 78, 49–54.

De la Vega, E., O’Leary, N.A., Shockey, J.E., Robalino, J., Payne, C., Browdy, C.L., Warr,G.W., Gross, P.S., 2008. Anti-lipopolysaccharide factor in Litopenaeus vannamei(LvALF): a broad spectrum antimicrobial peptide essential for shrimp immunityagainst bacterial and fungal infection. Mol. Immunol. 45, 1916–1925.

Destoumieux, D., Munoz, M., Bulet, P., Bachere, E., 2000. Penaeidins, a family ofantimicrobial peptides from penaeid shrimp (Crustacea, Decapoda). Cell. Mol.Life Sci. (CMLS) 57, 1260–1271.

Deveraux, Q.L., Reed, J.C., 1999. IAP family proteins—suppressors of apoptosis.Genes Dev. 13, 239–252.

Dhar, A.K., Bowers, R.M., Licon, K.S., Veazey, G., Read, B., 2009. Validation ofreference genes for quantitative measurement of immune gene expression inshrimp. Mol. Immunol. 46, 1688–1695.

Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspases:structure, activation, substrates, and functions during apoptosis. Annu. Rev.Biochem. 68, 383–424.

Escobedo-Bonilla, C., Alday-Sanz, V., Wille, M., Sorgeloos, P., Pensaert, M.,Nauwynck, H., 2008. A review on the morphology, molecular characterization,morphogenesis and pathogenesis of white spot syndrome virus. J. Fish Dis. 31,1–18.

Flegel, T.W., 1997. Major viral diseases of the black tiger prawn (Penaeus monodon)in Thailand. World J. Microbiol. Biotechnol. 13, 433–442.

Gan, D.-D., Reiss, K., Carrill, T., Del Valle, L., Croul, S., Giordano, A., Fishman, P.,Khalili, K., 2001. Involvement of Wnt signaling pathway in murinemedulloblastoma induced by human neurotropic JC virus. Oncogene 20,4864–4870.

Gao, G., Guo, X., Goff, S.P., 2002. Inhibition of retroviral RNA production by ZAP, aCCCH-type zinc finger protein. Science 297, 1703–1706.

Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis,X., Fan, L., Raychowdhury, R., Zeng, Q., 2011. Full-length transcriptomeassembly from RNA-seq data without a reference genome. Nat. Biotechnol.29, 644–652.

Gross, P.S., Bartlett, T.C., Browdy, C.L., Chapman, R.W., Warr, G.W., 2001. Immunegene discovery by expressed sequence tag analysis of hemocytes andhepatopancreas in the Pacific white shrimp, Litopenaeus vannamei, and theAtlantic white shrimp, l. Setiferus. Dev. Comp. Immunol. 25, 565–577.

Gross, P., Bartlett, T., Browdy, C., Chapman, R., Warr, G., 2001. Immune genediscovery by expressed sequence tag analysis of hemocytes and hepatopancreasin the Pacific white shrimp, Litopenaeus vannamei, and the Atlantic whiteshrimp, l. Setiferus. Dev. Comp. Immunol. 25, 565–577.

Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P.D., Bowden, J.,Couger, M.B., Eccles, D., Li, B., Lieber, M., 2013. De novo transcript sequencereconstruction from RNA-seq using the trinity platform for reference generationand analysis. Nat. Protoc. 8, 1494–1512.

Hameed, A.S.S., 2009. Viral infections of Macrobrachium spp.: global status ofoutbreaks, diagnosis, surveillance, and research. Isr. J. Aquac. – Bamidgeh 61,240–247.

Hancock, R.E., Diamond, G., 2000. The role of cationic antimicrobial peptides ininnate host defences. Trends Microbiol. 8, 402–410.

Hanriot, L., Keime, C., Gay, N., Faure, C., Dossat, C., Wincker, P., Scoté-Blachon, C.,Peyron, C., Gandrillon, O., 2008. A combination of LongSAGE with Solexasequencing is well suited to explore the depth and the complexity oftranscriptome. BMC Genom. 9, 418.

Huang, C.-H., Zhang, L.-R., Zhang, J.-H., Xiao, L.-C., Wu, Q.-J., Chen, D.-H., Li, J.K.-K.,2001. Purification and characterization of white spot syndrome virus (WSSV)produced in an alternate host: crayfish, Cambarus clarkii. Virus Res. 76, 115–125.

Iseli, C., Jongeneel, C.V., Bucher, P., 1999. ESTscan: a program for detecting,evaluating, and reconstructing potential coding regions in EST sequences. ISMB,138–148.

James, R., Thampuran, N., Lalitha, K.V., Rajan, L.A., Joseph, T.C., 2010. Differentialgene expression profile of the hepatopancreas of white spot syndrome virusinfected Fenneropenaeus indicus by suppression subtractive hybridization. FishShellfish Immunol. 29, 884–889.

Jeena, K., Prasad, K.P., Pathan, M.K., Babu, P.G., 2012. Expression profiling of WSSVorf 199 and shrimp ubiquitin conjugating enzyme in WSSV infected Penaeusmonodon. Asian–Aust. J. Anim. Sci. 25, 1184.

Ji, P.-F., Yao, C.-L., Wang, Z.-Y., 2011. Two types of calmodulin play different roles inPacific white shrimp (Litopenaeus vannamei) defenses against vibrioparahaemolyticus and WSSV infection. Fish Shellfish Immunol. 31, 260–268.

Kanehisa, M., Goto, S., 2000. Kegg: Kyoto encyclopedia of genes and genomes.Nucleic Acids Res. 28, 27–30.

Kawahara-Miki, R., Wada, K., Azuma, N., Chiba, S., 2011. Expression profilingwithout genome sequence information in a non-model species, pandalidshrimp (Pandalus latirostris), by next-generation sequencing. PLoS ONE 6,e26043.

Keezhedath, J., Kurcheti, P.P., Pathan, M.K., Babu, G.P., Tripathi, G., Sudhagar, A., Rao,S.P., 2013. Expression profile of Penaeus monodon ubiquitin conjugatingenzyme (PmUbc) at protein level in white spot syndrome virus challengedshrimp. Ind. J. Virol. 24, 48–53.

Kiran, R., Rajendran, K., Jung, S., Oh, M., 2002. Experimental susceptibility ofdifferent life-stages of the giant freshwater prawn, Macrobrachium rosenbergii(de Man), to white spot syndrome virus (WSSV). J. Fish Dis. 25, 201–207.

Kong, H., Hong, G., Cho, H., Nam, B., Kim, Y., Kim, W., Lee, S., Kim, K., 2009. Cloning ofprofilin (FcPFN) from the shrimpFenneropenaeus chinensis, a highly expressedprotein in white spot syndrome virus (WSSV)-infected shrimp. J. Appl. Genet.50, 245–250.

Kopácek, P., Hall, M., Söderhäll, K., 1993. Characterization of a clotting protein,isolated from plasma of the freshwater crayfish Pacifastacus leniusculus. Eur. J.Biochem. 213, 591–597.

Lei, K., Li, F., Zhang, M., Yang, H., Luo, T., Xu, X., 2008. Difference betweenhemocyanin subunits from shrimp Penaeus japonicus in anti-WSSV defense.Dev. Comp. Immunol. 32, 808–813.

Leu, J.H., Chang, C.C., Wu, J.L., Hsu, C.W., Hirono, I., Aoki, T., Juan, H.F., Lo, C.F., Kou, G.H., Huang, H.C., 2007. Comparative analysis of differentially expressed genes innormal and white spot syndrome virus infected Penaeus monodon. BMCGenom. 8, 120.

Leu, J.H., Chen, S.H., Wang, Y.B., Chen, Y.C., Su, S.Y., Lin, C.Y., Ho, J.M., Lo, C.F., 2011. Areview of the major penaeid shrimp EST studies and the construction of ashrimp transcriptome database based on the ESTs from four penaeid shrimp.Mar. Biotechnol. (NY) 13, 608–621.

R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22 21

Li, F., Xiang, J., 2013. Recent advances in researches on the innate immunity ofshrimp in China. Dev. Comp. Immunol. 39, 11–26.

Li, C., Weng, S., Chen, Y., Yu, X., Lü, L., Zhang, H., He, J., Xu, X., 2012. Analysis ofLitopenaeus vannamei transcriptome using the next-generation DNAsequencing technique. PLoS ONE 7, e47442.

Li, S.H., Zhang, X.J., Sun, Z., Li, F.H., Xiang, J.H., 2013. Transcriptome analysis onChinese shrimp Fenneropenaeus chinensis during WSSV acute infection. PLoSONE 8.

Liu, Y.-C., Li, F.-H., Wang, B., Dong, B., Zhang, Q.-L., Luan, W., Zhang, X.-J., Xiang, J.-H.,2007. A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis),full-length cdna cloning, tissue localization and expression profile afterchallenge. Fish Shellfish Immunol. 22, 576–588.

Lo, C.-F., Ho, C., Peng, S., Chen, C.H., Hsu, H., Chiu, Y., Chang, C., Liu, K., Su, M., Wang,C., 1996. White spot syndrome baculovirus (WSBV) detected in cultured andcaptured shrimp, crabs and other arthropods. Dis. Aquat. Org. 27, 215–225.

Loureiro, J., Ploegh, H.L., 2006. Antigen presentation and the ubiquitin-proteasomesystem in host–pathogen interactions. Adv. Immunol. 92, 225–305.

Ma, T.H.T., Tiu, S.H.K., He, J.-G., Chan, S.-M., 2007. Molecular cloning of a C-typelectin (LvLT) from the shrimp Litopenaeus vannamei: early gene down-regulation after WSSV infection. Fish Shellfish Immunol. 23, 430–437.

Ma, F.-f., Liu, Q.-h., Guan, K.-k., Li, C., Huang, J., 2014. Arginine kinase of Litopenaeusvannamei involved in white spot syndrome virus infection. Gene.

Mai, W.-J., Wang, W.-N., 2010. Protection of blue shrimp (Litopenaeus stylirostris)against the white spot syndrome virus (WSSV) when injected with shrimplysozyme. Fish Shellfish Immunol. 28, 727–733.

Maningas, M.B.B., Kondo, H., Hirono, I., Saito-Taki, T., Aoki, T., 2008. Essentialfunction of transglutaminase and clotting protein in shrimp immunity. Mol.Immunol. 45, 1269–1275.

Markl, J., Decker, H., 1992. Molecular structure of the arthropod hemocyanins. Bloodand Tissue Oxygen Carriers. Springer, pp. 325–376.

Marks, H., Goldbach, R.W., Vlak, J.M., van Hulten, M.C.W., 2004. Genetic variationamong isolates of white spot syndrome virus. Arch. Virol. 149, 673–697.

Mathew, S., Kumar, K. Ashok, Anandan, R., Nair, P. Viswanathan, Devadasan, K.,2007. Changes in tissue defence system in white spot syndrome virus (WSSV)infected Penaeus monodon. Comp. Biochem. Physiol. C: Toxicol. Pharmacol.145, 315–320.

Mohankumar, K., Ramasamy, P., 2006. White spot syndrome virus infectiondecreases the activity of antioxidant enzymes in Fenneropenaeus indicus.Virus Res. 115, 69–75.

Mohd-Shamsudin, M.I., Kang, Y., Lili, Z., Tan, T.T., Kwong, Q.B., Liu, H., Zhang, G.,Othman, R.Y., Bhassu, S., 2013. In-depth tanscriptomic analysis on giantfreshwater prawns. PLoS ONE 8, e60839.

Moody, D., 2001. Genomics techniques: an overview of methods for the study ofgene expression. J. Anim. Sci. 79, E128–E135.

Morozova, O., Marra, M.A., 2008. Applications of next-generation sequencingtechnologies in functional genomics. Genomics 92, 255–264.

Mount, D.W., 2007. Using the basic local alignment search tool (blast). Cold SpringHarbor Protoc. (pdb top17).

Mu, Y., Ding, F., Cui, P., Ao, J., Hu, S., Chen, X., 2010. Transcriptome and expressionprofiling analysis revealed changes of multiple signaling pathways involved inimmunity in the large yellow croaker during Aeromonas hydrophila infection.BMC Genom. 11, 506.

Müller, S., Möller, P., Bick, M.J., Wurr, S., Becker, S., Günther, S., Kümmerer, B.M.,2007. Inhibition of filovirus replication by the zinc finger antiviral protein. J.Virol. 81, 2391–2400.

Nanhai, H., Qiwei, Q., Xun, X., 2005. Differential profile of genes expressed inhemocytes of white spot syndrome virus resistant shrimp (Penaeus japonicus)by combining suppression subtractive hybridization and differentialhybridization. Antiviral Res. 66, 39–45.

Ongvarrasopone, C., Chanasakulniyom, M., Sritunyalucksana, K., Panyim, S., 2008.Suppression of PmRab7 by dsRNA inhibits WSSV or YHV infection in shrimp.Mar. Biotechnol. 10, 374–381.

Otta, S., Shubha, G., Joseph, B., Chakraborty, A., Karunasagar, I., Karunasagar, I., 1999.Polymerase chain reaction (PCR) detection of white spot syndrome virus(WSSV) in cultured and wild crustaceans in India. Dis. Aquat. Org. 38, 67–70.

Pan, D., He, N., Yang, Z., Liu, H., Xu, X., 2005. Differential gene expression profile inhepatopancreas of WSSV-resistant shrimp (Penaeus japonicus) by suppressionsubtractive hybridization. Dev. Comp. Immunol. 29, 103–112.

Pertea, G., Huang, X., Liang, F., Antonescu, V., Sultana, R., Karamycheva, S., Lee, Y.,White, J., Cheung, F., Parvizi, B., 2003. TIGR gene indices clustering tools (TGICL):a software system for fast clustering of large EST datasets. Bioinformatics 19,651–652.

Phongdara, A., Wanna, W., Chotigeat, W., 2006. Molecular cloning and expression ofcaspase from white shrimp Penaeus merguiensis. Aquaculture 252, 114–120.

Pockley, A.G., 2003. Heat shock proteins as regulators of the immune response. TheLancet 362, 469–476.

Pongsomboon, S., Tang, S., Boonda, S., Aoki, T., Hirono, I., Tassanakajon, A., 2011. Acdna microarray approach for analyzing transcriptional changes in Penaeusmonodon after infection by pathogens. Fish Shellfish Immunol. 30, 439–446.

Pradeep, B., Shekar, M., Karunasagar, I., Karunasagar, I., 2008. Characterization ofvariable genomic regions of Indian white spot syndrome virus. Virology 376,24–30.

Rajan, P., Ramasamy, P., Purushothaman, V., Brennan, G., 2000. White spotbaculovirus syndrome in the Indian shrimp Penaeus monodon and P. indicus.Aquaculture 184, 31–44.

Rao, R., Zhu, Y.B., Alinejad, T., Hassan, S.S., Wang, J., Bhassu, S., 2016. Supplementarydata for transcriptomics studies on Macrobrachium rosenbergii hepatopancreasexperimentally challenged with white spot syndrome virus (WSSV). Data Brief(submitted for publication).

Ratcliffe, N.A., Leonard, C., Rowley, A.F., 1984. Prophenoloxidase activation: nonselfrecognition and cell cooperation in insect immunity. Science 226, 557–559.

Rawe, V.Y., Payne, C., Schatten, G., 2006. Profilin and actin-related proteins regulatemicrofilament dynamics during early mammalian embryogenesis. Hum.Reprod. 21, 1143–1153.

Reiner, A., Yekutieli, D., Benjamini, Y., 2003. Identifying differentially expressedgenes using false discovery rate controlling procedures. Bioinformatics 19, 368–375.

Robalino, J., Bartlett, T., Shepard, E., Prior, S., Jaramillo, G., Scura, E., Chapman, R.W.,Gross, P.S., Browdy, C.L., Warr, G.W., 2005. Double-stranded RNA inducessequence-specific antiviral silencing in addition to nonspecific immunity in amarine shrimp: convergence of RNA interference and innate immunity in theinvertebrate antiviral response? J. Virol. 79, 13561–13571.

Robalino, J., Almeida, J.S., McKillen, D., Colglazier, J., Trent, H.F., Chen, Y.A., Peck, M.E., Browdy, C.L., Chapman, R.W., Warr, G.W., 2007. Insights into the immunetranscriptome of the shrimp Litopenaeus vannamei: tissue-specific expressionprofiles and transcriptomic responses to immune challenge. Physiol. Genomics29, 44–56.

Rolland, J.-L., Abdelouahab, M., Dupont, J., Lefevre, F., Bachère, E., Romestand, B.,2010. Stylicins, a new family of antimicrobial peptides from the Pacific blueshrimp Litopenaeus stylirostris. Mol. Immunol. 47, 1269–1277.

Roulston, A., Marcellus, R.C., Branton, P.E., 1999. Viruses and apoptosis. Ann. Rev.Microbiol. 53, 577–628.

Roux, M.M., Pain, A., Klimpel, K.R., Dhar, A.K., 2002. The lipopolysaccharide and b-1,3-glucan binding protein gene is upregulated in white spot virus-infectedshrimp (Penaeus stylirostris). J. Virol. 76, 7140–7149.

Sadamoto, H., Takahashi, H., Okada, T., Kenmoku, H., Toyota, M., Asakawa, Y., 2012.De novo sequencing and transcriptome analysis of the central nervous systemof mollusc Lymnaea stagnalis by deep RNA sequencing. PLoS ONE 7, e42546.

Sarathi, M., Simon, M.C., Ahmed, V.I., Kumar, S.R., Hameed, A.S., 2008. Silencingvp28 gene of white spot syndrome virus of shrimp by bacterially expresseddsRNA. Mar. Biotechnol. 10, 198–206.

Schmieder, R., Edwards, R., 2011. Fast identification and removal of sequencecontamination from genomic and metagenomic datasets. PLoS ONE 6, e17288–e17288.

Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparativeCT method. Nat. Protoc. 3, 1101–1108.

Schwantes, V.S., Diana, J.S., Yi, Y., 2009. Social, economic, and productioncharacteristics of giant river prawn Macrobrachium rosenbergii culture inThailand. Aquaculture 287, 120–127.

Shen, G.-M., Dou, W., Niu, J.-Z., Jiang, H.-B., Yang, W.-J., Jia, F.-X., Hu, F., Cong, L.,Wang, J.-J., 2011. Transcriptome analysis of the oriental fruit fly (Bactroceradorsalis). PLoS ONE 6, e29127.

Silke, J., Meier, P., 2013. Inhibitor of apoptosis (IAP) proteins–modulators of celldeath and inflammation. Cold Spring Harbor Perspect. Biol. 5, a008730.

Sithigorngul, W., Rukpratanporn, S., Pecharaburanin, N., Longyant, S.,Chaivisuthangkura, P., Sithigorngul, P., 2006. A simple and rapidimmunochromatographic test strip for detection of white spot syndromevirus (WSSV) of shrimp. Dis. Aquat. Org. 72, 101.

Söderhäll, K., Cerenius, L., 1998. Role of the prophenoloxidase-activating system ininvertebrate immunity. Curr. Opin. Immunol. 10, 23–28.

Song, Y.-L., Yu, C.-I., Lien, T.-W., Huang, C.-C., Lin, M.-N., 2003. Haemolymphparameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taurasyndrome virus. Fish Shellfish Immunol. 14, 317–331.

Song, K.-K., Li, D.-F., Zhang, M.-C., Yang, H.-J., Ruan, L.-W., Xu, X., 2010. Cloning andcharacterization of three novel WSSV recognizing lectins from shrimpMarsupenaeus japonicus. Fish Shellfish Immunol. 28, 596–603.

Stewart, G.R., Young, D.B., 2004. Heat-shock proteins and the host–pathogeninteraction during bacterial infection. Curr. Opin. Immunol. 16, 506–510.

Su, J., Oanh, D.T., Lyons, R.E., Leeton, L., van Hulten, M.C., Tan, S.-H., Song, L.,Rajendran, K., Walker, P.J., 2008. A key gene of the RNA interference pathway inthe black tiger shrimp, Penaeus monodon: Identification and functionalcharacterisation of dicer-1. Fish Shellfish Immunol. 24, 223–233.

Sunarto, A., 2001. Characterization of White Spot Syndrome Virus (WSSV) fromIndonesian Shrimp Farms and Development of Polymerase Chain Reaction(PCR) Assay for its Detection. Universiti Putra Malaysia.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecularevolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.

Tassanakajon, A., Amparyup, P., Somboonwiwat, K., Supungul, P., 2011. Cationicantimicrobial peptides in penaeid shrimp. Mar. Biotechnol. 13, 639–657.

Tatusov, R.L., Galperin, M.Y., Natale, D.A., Koonin, E.V., 2000. The COG database: atool for genome-scale analysis of protein functions and evolution. Nucleic AcidsRes. 28, 33–36.

Theopold, U., Schmidt, O., Söderhäll, K., Dushay, M.S., 2004. Coagulation inarthropods: defence, wound closure and healing. Trends Immunol. 25, 289–294.

Unajak, S., Boonsaeng, V., Jitrapakdee, S., 2006. Isolation and characterization ofcdna encoding Argonaute, a component of RNA silencing in shrimp (Penaeusmonodon). Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 145, 179–187.

van Hulten, M.C.W., Witteveldt, J., Peters, S., Kloosterboer, N., Tarchini, R., Fiers, M.,Sandbrink, H., Lankhorst, R.K., Vlak, J.M., 2001. The white spot syndrome virusDNA genome sequence. Virology 286, 7–22.

22 R. Rao et al. / Journal of Invertebrate Pathology 136 (2016) 10–22

van Hulten, M.C., Witteveldt, J., Peters, S., Kloosterboer, N., Tarchini, R., Fiers, M.,Sandbrink, H., Lankhorst, R.K., Vlak, J.M., 2001. The white spot syndrome virusDNA genome sequence. Virology 286, 7–22.

Vargas-Albores, F., Yepiz-Plascencia, G., 2000. Beta glucan binding protein and itsrole in shrimp immune response. Aquaculture 191, 13–21.

Vidya, R., Gireesh-Babu, P., Prasad, K.P., 2013. White spot syndrome virusmanipulates ubiquitin gene expression in Penaeus monodon. Ind. J. Virol. 24,82–84.

Wang, C., Lo, C., Leu, J., Chou, C., et al., 1995. Purification and genomic analysis ofbaculovirus associated with white spot syndrome (WSBV) of Penaeus monodon.Dis. Aquat. Org. 23, 239–242.

Wang, Y.-C., Lo, C.-F., Chang, P.-S., Kou, G.-H., 1998. Experimental infection of whitespot baculovirus in some cultured and wild decapods in Taiwan. Aquaculture164, 221–231.

Wang, Y., Hassan, M., Shariff, M., Zamri, S., Chen, X., 1999. Histopathology andcytopathology of white spot syndrome virus (WSSV) in cultured Penaeusmonodon from peninsular Malaysia with emphasis on pathogenesis and themechanism of white spot formation. Dis. Aquat. Org. 39, 1–11.

Wang, B., Li, F., Dong, B., Zhang, X., Zhang, C., Xiang, J., 2006. Discovery of the genesin response to white spot syndrome virus (WSSV) infection in Fenneropenaeuschinensis through cDNA microarray. Mar. Biotechnol. 8, 491–500.

Wang, L., Zhi, B., Wu, W., Zhang, X., 2008. Requirement for shrimp caspase inapoptosis against virus infection. Dev. Comp. Immunol. 32, 706–715.

Wang, K. Han-Ching, Tseng, C.-W., Lin, H.-Y., Chen, I., Chen, Y.-H., Chen, Y.-M., Chen,T.-Y., Yang, H.-L., 2010. RNAi knock-down of the Litopenaeus vannamei Tollgene (LvToll) significantly increases mortality and reduces bacterial clearanceafter challenge with Vibrio harveyi. Dev. Comp. Immunol. 34, 49–58.

Wang, P.-H., Liang, J.-P., Gu, Z.-H., Wan, D.-H., Weng, S.-P., Yu, X.-Q., He, J.-G., 2012.Molecular cloning, characterization and expression analysis of two novel tolls(LvToll2 and LvToll3) and three putative Spätzle-like toll ligands (LvSpz1–3)from Litopenaeus vannamei. Dev. Comp. Immunol. 36, 359–371.

Wang, P.-H., Wan, D.-H., Gu, Z.-H., Qiu, W., Chen, Y.-G., Weng, S.-P., Yu, X.-Q., He, J.-G., 2013. Analysis of expression, cellular localization, and function of threeinhibitors of apoptosis (IAPs) from Litopenaeus vannamei during WSSVinfection and in regulation of antimicrobial peptide genes (AMPs). PLoS ONE8, e72592.

Wang, D.-L., Sun, T., Zuo, D., Wang, L.-M., Wang, Q., Zhao, Y.-L., 2013. Cloning andtissue expression of hemocyanin gene in Cherax quadricarinatus during whitespot syndrome virus infection. Aquaculture 410, 216–224.

Watson, A., Mazumder, A., Stewart, M., Balasubramanian, S., 1998. Technology formicroarray analysis of gene expression. Curr. Opin. Biotechnol. 9, 609–614.

Westenberg, M., Heinhuis, B., Zuidema, D., Vlak, J.M., 2005. siRNA injection inducessequence-independent protection in Penaeus monodon against white spotsyndrome virus. Virus Res. 114, 133–139.

Witke, W., 2004. The role of profilin complexes in cell motility and other cellularprocesses. Trends Cell Biol. 14, 461–469.

View publication statsView publication stats

Wongprasert, K., Sangsuriya, P., Phongdara, A., Senapin, S., 2007. Cloning andcharacterization of a caspase gene from black tiger shrimp (Penaeus monodon)-infected with white spot syndrome virus (WSSV). J. Biotechnol. 131, 9–19.

Wongteerasupaya, C., Vickers, J., Sriurairatana, S., Nash, G., Akarajamorn, A.,Boonsaeng, V., Panyim, S., Tassanakajon, A., Withyachumnarnkul, B., Flegel, T.,1995. A non-occluded, systemic baculovirus that occurs in cells of ectodermaland mesodermal origin and causes high mortality in the black tiger prawnPenaeus monodon. Dis. Aquat. Org. 21, 69–77.

Wu, Y., Lü, L., Yang, L.-S., Weng, S.-P., Chan, S.-M., He, J.-G., 2007. Inhibition of whitespot syndrome virus in Litopenaeus vannamei shrimp by sequence-specificsiRNA. Aquaculture 271, 21–30.

Xu, J., Han, F., Zhang, X., 2007. Silencing shrimp white spot syndrome virus (WSSV)genes by siRNA. Antiviral Res. 73, 126–131.

Xu, J., Ji, P., Wang, B., Zhao, L., Wang, J., Zhao, Z., Zhang, Y., Li, J., Xu, P., Sun, X., 2013.Transcriptome sequencing and analysis of wild Amur ide (Leuciscus waleckii)inhabiting an extreme alkaline–saline lake reveals insights into stressadaptation. PLoS ONE 8, e59703.

Xue, S.X., Liu, Y.C., Zhang, Y.C., Sun, Y., Geng, X.Y., Sun, J.S., 2013. Sequencing and denovo analysis of the hemocytes transcriptome in Litopenaeus vannameiresponse to white spot syndrome virus infection. PLoS ONE 8.

Yan, F., Xia, D., Hu, J., Yuan, H., Zou, T., Zhou, Q., Liang, L., Qi, Y., Xu, H., 2010. Heatshock cognate protein 70 gene is required for prevention of apoptosis inducedby WSSV infection. Arch. Virol. 155, 1077–1083.

Yao, C.-L., Ji, P.-F., Kong, P., Wang, Z.-Y., Xiang, J.-H., 2009. Arginine kinase fromLitopenaeus vannamei: cloning, expression and catalytic properties. FishShellfish Immunol. 26, 553–558.

Yodmuang, S., Tirasophon, W., Roshorm, Y., Chinnirunvong, W., Panyim, S., 2006.YHV-protease dsRNA inhibits YHV replication in Penaeus monodon andprevents mortality. Biochem. Biophys. Res. Commun. 341, 351–356.

Zeng, D.G., Chen, X.L., Xie, D.X., Zhao, Y.Z., Yang, C.L., Li, Y.M., Ma, N., Peng, M., Yang,Q., Liao, Z.P., et al., 2013. Transcriptome analysis of Pacific white shrimp(Litopenaeus vannamei) hepatopancreas in response to Taura syndrome virus(TSV) experimental infection. PLoS ONE 8.

Zhang, X., Huang, C., Qin, Q., 2004. Antiviral properties of hemocyanin isolated fromshrimp Penaeus monodon. Antiviral Res. 61, 93–99.

Zhang, J., Li, F., Wang, Z., Zhang, X., Zhou, Q., Xiang, J., 2006. Cloning, expression andidentification of ferritin from Chinese shrimp, Fenneropenaeus chinensis. J.Biotechnol. 125, 173–184.

Zhang, Q., Li, F., Wang, B., Zhang, J., Liu, Y., Zhou, Q., Xiang, J., 2007. Themitochondrial manganese superoxide dismutase gene in Chinese shrimpFenneropenaeus chinensis: cloning, distribution and expression. Dev. Comp.Immunol. 31, 429–440.

Zhao, Z.Y., Yin, Z.X., Weng, S.P., Guan, H.J., Li, S.D., Xing, K., Chan, S.M., He, J.G., 2007.Profiling of differentially expressed genes in hepatopancreas of white spotsyndrome virus-resistant shrimp (Litopenaeus vannamei) by suppressionsubtractive hybridisation. Fish Shellfish Immunol. 22, 520–534.