- Open Access
RNA-seq analysis of Macrobrachium rosenbergii hepatopancreas in response to Vibrio parahaemolyticus infection
© Rao et al.; licensee BioMed Central. 2015
Received: 13 November 2014
Accepted: 13 February 2015
Published: 14 March 2015
The Malaysian giant freshwater prawn, Macrobrachium rosenbergii, is an economically important crustacean worldwide. However, production of this prawn is facing a serious threat from Vibriosis disease caused by Vibrio species such as Vibrio parahaemolyticus. Unfortunately, the mechanisms involved in the immune response of this species to bacterial infection are not fully understood. We therefore used a high-throughput deep sequencing technology to investigate the transcriptome and comparative expression profiles of the hepatopancreas from this freshwater prawn infected with V. parahaemolyticus to gain an increased understanding of the molecular mechanisms underlying the species’ immune response to this pathogenic bacteria.
A total of 59,122,940 raw reads were obtained from the control group, and 58,385,094 reads from the Vibrio-infected group. Via de novo assembly by Trinity assembler, 59,050 control unigenes and 73,946 Vibrio-infected group unigenes were obtained. By clustering unigenes from both libraries, a total of 64,411 standard unigenes were produced. The standard unigenes were annotated against the NCBI non-redundant, Swiss-Prot, Kyoto Encyclopaedia of Genes and Genome pathway (KEGG) and Orthologous Groups of Proteins (COG) databases, with 19,799 (30.73%), 16,832 (26.13%), 14,706 (22.83%) and 7,856 (12.19%) hits respectively, giving a final total of 22,455 significant hits (34.86% of all unigenes). A Gene Ontology (GO) analysis search using the Blast2GO program resulted in 6,007 unigenes (9.32%) being categorized into 55 functional groups. A differential gene expression analysis produced a total of 14,569 unigenes aberrantly expressed, with 11,446 unigenes significantly up-regulated and 3,103 unigenes significantly down-regulated. The differentially expressed immune genes fall under various processes of the animal immune system.
This study provided an insight into the antibacterial mechanism in M. rosenbergii and the role of differentially expressed immune genes in response to V. parahaemolyticus infection. Furthermore, this study has generated an abundant list of transcript from M.rosenbergii which will provide a fundamental basis for future genomics research in this field.
The Malaysian giant freshwater prawn, Macrobrachium rosenbergii (locally known as ‘udang galah’), belongs to the genus Macrobrachium, which is the largest genus of the family Palaemonidae . They are found in most inland freshwater areas, including lakes, rivers, swamps, estuarine areas, ponds, canals as well as in irrigation ducts . M. rosenbergii spends its adult life in fresh water, but requires brackish water during the initial stages of its life cycle . High demand from the aquaculture industry has led to large-scale farming of this prawn in many countries; the major producers being Bangladesh, Brazil, China, Ecuador, India, Thailand, Taiwan Province of China, and Malaysia .
The global production of this prawn had increased to over 200 000 tonnes/year by 2002, and income in Asia alone is now worth US$1 billion per annum [5,6]. In Malaysia, the production of cultured M. rosenbergii reached 281 metric tonnes by 1998 . Generally, M. rosenbergii is assumed to be less resistant towards diseases than penaeid shrimp . However, with the rise of large-scale high density prawn aquaculture techniques, production of this prawn worldwide is facing a serious threat from fatal diseases caused by nodaviruses and bacteria, particularly from the Vibrio species [8,9]. The emergence of these pathogens has had a detrimental impact on the M. rosenbergii farming industry, causing considerable economic losses.
Vibrio is a Gram-negative halophilic bacterium found abundantly in marine and estuarine environments [10,11]. Among the different species, Vibrio parahaemolyticus has emerged as an important pathogen for M. rosenbergii . Several other marine shrimps such as Penaeus monodon, Penaeus japonicas and Litopenaeus vannamei have also been found to be susceptible to Vibrio infection . Severe V. parahaemolyticus infection in prawns leads to a disease known as ‘Vibriosis’ [14,15]. M. rosenbergii suffering from vibriosis may appear black in colour on the carapace, with red discolouration of the exoskeleton and loss of appendages within six days, leading to an 80% mortality rate .
Acquiring and establishing knowledge regarding host pathogen interactions is necessary to unlock the pathogenesis of a particular disease. Host pathogen interactions can result in acute and adaptive immune responses against an invader; however, this has been lacking in M. rosenbergii . The species defends itself against pathogen invasion using an innate immune system involving the cellular and humoral mechanisms [17,18]. Recently, some progress has been made in analysing the molecular mechanisms of shrimp-pathogen interactions, and several immune genes from shrimp have been discovered such as lectins, antimicrobial peptides, prophenoloxidase and manganese superoxide dismutase, using methods such as suppression subtractive hybridization (SSH) and expressed sequence tags (EST) [19-21]. However, these two methods have been found to be laborious and costly, which limits their use for the production of large-scale transcripts .
A cutting edge technology has emerged recently, known as Next Generation Sequencing technology (NGS). Currently, there are four established platforms which uses NGS technology: the Illumina Genome Analyzer, the Roche/454 Genome Sequencer FLX Instrument, and the ABI SOLiD System [23,24]. These platforms have proven versatile and cost-effective tools for advanced research in various genomic areas, such as genome sequencing and re-sequencing, DNA methylation analysis, miRNA expression profiling, and also in non-model organisms as the de novo transcriptome sequencing . By using the NGS platform, transcriptome analysis can be performed faster and more easily, because it does not require any bacterial cloning of cDNAs . NGS sequencing has the further advantage of generating greater depth of short reads with minimum error rates . Moreover, it is more reliable and efficient than previous methods in measuring transcriptome composition, revealing RNA expression patterns, and discovering new genes on a larger scale . The superiority of this technology also lies in its sensitivity, which allows the detection of low-abundance transcripts .
Previous studies have been performed on whole transcriptome sequencing of the hepatopancreas, gill and muscle tissues of M. rosenbergii using the Illumina Genome Analyzer IIx platform (Illumina). They successfully produced a comprehensive transcript data for this freshwater prawn, leading to the discovery of new genes . This present study utilised a similar approach to analyse transcriptome data obtained from the hepatopancreas of M. rosenbergii experimentally infected with V. parahaemolyticus. The aim was to discover, and determine the role of, immune genes in M. rosenbergii involved in V. parahaemolyticus infection, which in turn could provide insights into the host-pathogen interactions between these two organisms.
Material and methods
M. rosenbergii and V. parahaemolyticus PCV08-7 challenge
M. rosenbergii prawns (5-8 g body weight) purchased from a local hatchery (Kuala Kangsar, Perak, Malaysia) were acclimatized at 28 ± 1°C in aerated and filtered freshwater for one week prior to challenge with V. parahaemolyticus. During the challenge experiment, the prawns (n = 10) were intramuscularly injected with 100 μl 1X105 cfu cultured V. parahaemolyticus  whereas another batch of prawns (n = 10) were injected with 100 μl 2% NaCl (1:10, w/v) solution which serves as negative control group. The hepatopancreas tissues of the prawns were dissected at 12 hours post-infection. The tissues were rapidly frozen in liquid nitrogen and stored at −80°C until total RNA extraction. The 12 hour time point was chosen based on our previous work regarding immune related genes from M. rosenbergii in response to pathogen such as viruses showing significant gene expression at this time point [32-35].
Total RNA extraction and next-generation sequencing
Total RNA (~20 mg) was isolated from both the V. parahaemolyticus-challenged and negative control group hepatopancreases. The RNA extraction process was performed by using the Macherey-Nagel NucleoSpin RNA II extraction kit in accordance with the manufacturer’s protocols and stored at −80°C prior to RNA sequencing. The purity and integrity of the RNA was assessed by using the Bioanalyzer 2100 (Agilent technologies, USA). In each group, the total RNA samples were pooled from 10 prawns after which cDNA was synthesized followed by sequencing. The sequencing run was conducted on an Illumina HiSeq™ 2000 platform at the Beijing Genome Institute, Shenzhen, China. The sequencing data constituted 90 bp paired end read data, with ~117 million raw reads.
Assembly and functional annotation
The raw reads were primarily quality filtered to remove adaptor sequences followed by removal of ambiguous ‘N’ nucleotides (with a ratio of ‘N’ more than 10%) and sequences with a phred quality score of less than 20 before proceeding to de novo assembly by using the Trinity software . The Trinity programme assembles the reads into contigs and these contigs were assembled to unigenes. Finally, the TIGR Gene Indices clustering tools (TGICL)  with default parameters was applied to cluster the unigenes from both groups which produces non-redundant unigenes.
The non-redundant unigene sequences were aligned to databases which included NCBI non-redundant (Nr), Swissprot , Cluster of Orthologous Groups (COG)  and Kyoto Encyclopaedia of Genes and Genome (KEGG)  using BLASTX  with an E-value cut-off of 10−5. Gene Ontology (GO) was conducted utilizing default parameters using the BLAST2GO software [42,43]. It was from the above mentioned databases that the gene direction of the unigenes which were annotated and the coding sequence were determined from the BLAST results. The prediction for the coding sequence and the gene direction was performed by ESTscan  for those sequences with no defined annotation by using BLAST predicted coding sequence data as the training set.
Identification of differentially expressed unigenes
The FPKM method (Fragments Per kb per Million fragments) was used to calculate the transcript expression levels . An FDR (false discovery rate) of <0.001 was used as the threshold p-value in multiple tests to judge the degree of differences in gene expression . In a given library when the p-value was less than 0.001 and when the expression level showed greater than two-fold change between two groups genes were considered as differentially expressed.
Quantitative RT-PCR analysis
We selected seven differentially expressed M. rosenbergii unigenes (arginine kinase 1, anti-lipopolysaccharide factor, inhibitor of apoptosis protein, caspase, heat shock protein 21, lectin 1, and NF-kappa B inhibitor alpha) for quantitative RT-PCR analysis (qRT-PCR) to evaluate our Illumina sequencing result. The primer design for the seven unigenes was performed by using Primer3 software http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/ and listed in Additional file 1. Using 1 μg of RNA, first strand cDNA synthesis was carried out (similar to the sample used for transcriptome sequencing) by using the ImProm-II™ Reverse Transcription System (Promega). The qRT-PCR reaction (20 μl) consisted of a 10 μl TaqMan Universal RT-PCR Master Mix (Applied Biosystems, Foster City, CA, USA), a 1 μl of primers/probe set containing 900 nM of forward reverse primers, a 300 nM probe and 2 μl of template cDNA. The qRT-PCR program was set with an incubation step at 50°C for 2 min, 40 cycles at 95°C for 10 min, 95°C for 15 sec, and 60°C for 1 min, carried out by using Step One Plus Real-Time PCR System® (Applied Biosystems). Similar qRT-PCR cycle profile was applied for the internal control gene, Elongation factor 1-alpha (primer sequences are listed in Additional file 1). The expression level of the seven immune genes were analysed by using the comparative CT method (2 -ΔΔCT method) .
Illumina sequencing and assembly
The task of profiling all the immune-related genes involved in V .parahaemolyticus infection began with sequencing the two cDNA libraries prepared from pooled mRNAs obtained from the hepatopancreases of the control and infected groups using the Illumina HiSeq™ 2000 platform. A total of 59,122,940 raw reads were obtained from the control group, and 58,385,094 reads from the Vibrio-infected group. The raw reads were further filtered to remove adaptor sequences, ambiguous reads and low quality reads, thereby generating 90-bp of 54,708,014 and 54,295,342 clean reads for the control and infected groups respectively ( Q20 ~ 98% and percentage of unknown nucleotide is 0%). All sequencing reads were deposited into the Short Read Archive (SRA) of the National Centre for Biotechnology Information (NCBI), and can be accessed under the accession number SRR1424572 for control and SRR1424574 for Vibrio-infected ones.
Summary of the control and V. parahaemolyticus infected transcriptome sequencing
V. parahaemolyticus infected
Total number of clean reads
Total base pairs (bp)
Total number of contigs
Mean length of contigs (bp)
Total number of unigenes
Mean length of unigenes (bp)
NCBI Nr annotated
The standard unigenes were annotated by searching the sequences using BLASTX against the NCBI non-redundant, Swiss-Prot, Kyoto Encyclopaedia of Genes and Genome pathway (KEGG) and Orthologous Groups of Proteins (COG) databases, which produced 19,799 (30.73%), 16,832 (26.13%), 14,706 (22.83%) and 7,856 (12.19%) hits respectively, giving a final total of 22,455 significant hits (34.86% of all unigenes). The size distribution profile for the coding sequences (CDS) and identified proteins are shown in Additional file 2. A Gene Ontology (GO) analysis search using the Blast2GO program resulted in 6,007 unigenes (9.32%) being categorized into 55 functional groups. The unigenes without hits using the BLASTX analysis were subjected to an ESTScan, producing 4,977 unigenes (7.82%) predicted to contain coding sequences. The size distribution of the ESTs and proteins are shown in Additional file 2.
Identification of aberrantly expressed genes
Potential immune-related genes involved in M. rosenbergii immune response
Candidate genes involved in M. rosenbergii immune response against V. parahaemolyticus
Category or gene id
Anti-lipopolysaccharide factor 2
Anti-lipopolysaccharide factor 3
NF-kappa B inhibitor alpha
Blood clotting system
Coagulation factor XII
C-type lectin 5
Lectin B isoform 2
Glucan pattern-recognition lipoprotein
lipopolysaccharide and beta-1,3-glucan binding protein
Proteinases and proteinases inhibitors
Serpin serine protease inhibitor
26S protease regulatory
Serine proteinase inhibitor 6
Hemocyte kazal-type proteinase inhibitor
Kazal-type serine proteinase inhibitor 4
Masquerade-like serine proteinase-like protein 2
Serine proteinase inhibitor
Heat shock proteins
Heat shock protein 21
Heat shock protein 40
Heat shock protein 70
Heat shock protein 90
Small heat shock protein
Glutathione S transferase
Cu/Zn superoxide dismutase
Farnesoic acid O-methyltransferase
Toll interacting protein
Toll receptor 2
Toll-like receptor 3
Toll-like receptor 8
Wnt signaling pathway
Low-density lipoprotein receptor-related protein 6
Secreted frizzled-related protein 5
MAPK signaling pathway
Mitogen-activated protein kinase kinase kinase 1
Mitogen-activated protein kinase 8 interacting protein 3
Mitogen-activated protein kinase kinase kinase kinase 4
Mitogen-activated protein kinase kinase kinase 7
Mitogen-activated protein kinase kinase kinase 13
Heat shock protein 70
Protein phosphatase 5
Map kinase-interacting serine/threonine
Raf homolog serine/threonine-protein kinase phl-like
STAT long form
Signal transducing adaptor molecule
Tumor susceptibility gene 101 protein
Pediculus humanus corporis
Other signal transduction genes
cAMP-dependent protein kinase type II regulatory subunit
Casein kinase II subunit alpha
Pediculus humanus corporis
TBC1 domain family member 10B
REM2- and Rab-like small GTPase 1-like
Prophenoloxidase activating factor
Prophenoloxide activating enzyme III
ALG-2 interacting protein x Penaeus monodon
Program cell death 5-like
inhibitor of apoptosis protein
DNA fragmentation factor subunit beta
Other immune genes
Arginine kinase 1
Tetraspanin-like protein CD9
Apart from viral diseases, Vibrio infections causing Vibriosis is another factor hindering the shrimp aquaculture industry worldwide . This fatal disease has contributed to mass mortality and severe economic losses in India, Thailand, Philippines, Japan, Ecuador, Peru, Colombia and Central America . Knowledge about the interaction between M. rosenbergii and Vibrio species is in its infancy, and in-depth study is urgently needed to address this issue. Discovery of the molecular mechanisms surrounding the innate immune system against Vibrio infection in freshwater prawns should be beneficial to both scientific research and the aquaculture industry. Identifying and quantifying immune-related gene expression on a large scale is a promising method to investigate the host response against pathogens and provide a platform for further studies in this area.
Microarray and EST analyses have long been used to study the molecular mechanisms underlying the innate immune system and to identify genes aberrantly expressed during infection [48-50]. However, the most recent NGS platforms such as the Illumina HiSeq™ 2000 appear much better at quantifying transcripts expressed at low levels than microarrays or EST analysis . This is because this revolutionary technique verifies direct transcript profiling without compromise or bias, unlike previous methods . Furthermore, this technology has been successfully used in transcriptome profiling studies on non-model organisms where there is no complete genome database [51-53]. The introduction of NGS technology has led to various studies on host-viral interactions in shrimps to identify potential immune-related genes [54-56] – but not so far on the interaction between the freshwater prawn and Vibrio species. To our knowledge, this study could be the very first to use the Illumina HiSeq™ 2000 platform to explore the immune-related gene response in M. rosenbergii against V. parahaemolyticus.
Taking advantage of the Illumina HiSeq™ 2000 platform’s capability to sequence with a high throughput data providing more candidate genes, the total RNA extracted at the 12th hour time point from a pool of control and infected hepatopancreases was sequenced and assembled using the Trinity assembler. The overall analysis yielded 14,549 differentially expressed unigenes, with 11,446 unigenes significantly up-regulated and 3,103 unigenes significantly down-regulated. The sequencing data analyses obtained clearly showed a significant impact of V. parahaemolyticus infection on the M. rosenbergii transcriptome.
M. rosenbergii possesses an innate immune system, consisting of cellular and humoral components which work individually or cooperatively to protect the species from invading pathogens such as V. parahaemolyticus [17,18]. This immune response is activated when the animal detects the invading pathogen through pattern recognition proteins (PRPs) . Two important PRPs molecules identified in shrimp are lectins and the beta-1,3- glucan binding protein (LGBP) [58-61]. Besides pathogen recognition, lectins are also involved in phagocytosis through opsonisation in crustaceans . Our data showed that challenge by V. parahaemolyticus greatly affects the expression of PRPs, as observed in previous studies using different Vibrio strains [63-67]. The activation of LGBP molecules triggers the melanisation process, a prophenoloxidase-activating system (proPO-AS) which is an enzymatic cascade involving several enzymes, including the key enzyme phenoloxidase (PO) [68-70]. The active PO converts phenols into quinones. These build a non-specific crosslink between neighbouring molecules to form melanin, which provides defence against invading microorganisms . Increased activity of prophenoloxidase against Vibrio species has also been noted in Fenneropenaeus indicus , L. vannamei  and P. monodon .
Antimicrobial peptides (AMPs) play a pivotal role in killing or clearing infected pathogens, especially Vibrio species . Notable shrimp AMPs, such as penaeidins, lysozymes, crustins, anti-lipopolysaccharide factors (ALFs) and stylicins have been identified and characterized previously in shrimps [76-80]. However, only four types of AMPs – lysozyme, crustin, NF-kappa B inhibitor alpha and ALF (3 isoforms) – were detected in our transcriptome data and found to be highly expressed. The up-regulation of these AMPs correlated with previous studies showing their antimicrobial properties against Vibrio species and other bacteria [81-84]. Blood clotting is vital in crustaceans to prevent excess blood loss from a wound and prevent micro-organisms from invading the wound . We found four molecules of the blood clotting system – transglutaminase, clottable protein, proclotting enzyme and coagulation factor XII – to be highly induced in our transcriptome data after challenge by V. parahaemolyticus. A similar expression of these molecules after bacterial challenge has been reported in previous studies [72,86,87].
Stress conditions such as bacterial infections lead to an accumulation of reactive oxygen species (ROS) in a cell . Increased levels of ROS causes oxidative damage to important cellular macromolecules (lipids, proteins, carbohydrates and nucleotides) which are components of the membranes, cellular enzymes and DNA . In order to restrict the production of ROS, antioxidant genes are activated to produce antioxidant enzymes which eliminate ROS. Several antioxidant enzymes have been isolated and characterised in the penaeid shrimp in previous studies [90-92]. In this study, we found six antioxidant unigenes to be over-expressed after V. parahaemolyticus challenge – the exception being glutamine synthetase. High expression levels of these genes have similarly been observed in other shrimps and scallops after Vibrio challenge [93-96]. The up-regulation of actin and tubulin genes play a crucial role in a wide range of cellular functions such as nodule formation, phagocytosis, encapsulation, as well as cell shape change, cell motility and adhesion, all of which may aid in clearing the pathogen .
Heat shock proteins (HSPs) are highly generated when induced by stress. They are known to play a major role in protein folding, the protection of proteins from denaturation or aggregation, and aiding protein transport through membrane channels [98,99]. In addition to molecular chaperones, HSPs have been reported to play important roles in innate immune responses, and have been well studied in crustaceans [100-102]. In this study, we noted higher levels of expression of all heat shock proteins in M. rosenbergii when challenged by V. parahaemolyticus. The increased expression of these HSPs is in line with previous reports, which tends to confirm the important role of these proteins in protecting this species from the stress induced by Vibrio challenge [97,103,104]. The general higher expression of proteinases and their inhibitors was to be expected in our data, as these are known to modulate elements of the innate immune system such as haemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanisation .
Bacteria like Vibrio are known to induce cell apoptosis through a variety of mechanisms such as pore-forming proteins, secretion of protein synthesis inhibitors, molecules activating the endogenous death machinery in an infected cell, lipopolysaccharides, and other superantigens . Increased levels of apoptosis contribute to the degradation of DNA and RNA, which may contribute to shrimp mortality . In our transcriptome data, an up-regulation of genes involved in apoptosis was observed, similar to the trends reported in previous studies [74,108,109]. Apoptosis may also serve as host defence against bacterium by allowing other healthy cells to phagocytise apoptotic bodies containing bacteria from target cells, effectively clearing the pathogen .
The signalling pathways involved in the M. rosenbergii innate immune response against V. parahaemolyticus were observed to be highly induced in our transcriptome data. The Toll protein initially identified in Drosophila had been reported to play a key role in the anti-fungal and anti-Gram-positive bacterial responses of flies in the Toll pathway . Other Toll components have also been found to be activated in penaeid shrimps when challenged with Vibrio species [109,112,113]. In Caenorhabditis elegans, the mitogen-activated protein kinase (MAPK) pathways are transcriptionally up-regulated by the pore-forming toxin released by Bacillus thuringiensis, which provide a cellular defence against this toxin . This could explain the higher expression of this signalling pathway in our data, as V. parahaemolyticus is known to release a thermostable direct haemolysin, which is a pore-forming toxin . The Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathways have been reported to be activated when Fenneropenaeus chinensis is challenged with Vibrio anguillarum, which suggest that these pathways are important for immune responses against bacteria . In addition, Rab-related proteins have been reported to regulate the hemocytic phagocytosis of bacteria in Marsupenaeus japonicas .
Several genes in the other immune gene group were found to be aberrantly expressed in our transcriptome data. Calmodulin, which plays an important role in calcium-dependent signal transduction pathways, was over-expressed in our data – as was the case in L. vannamei when challenged with V. parahaemolyticus . Ferritin, an iron storage protein crucial for the metabolism of iron and maintaining iron homeostasis in a cell, was found to be down-regulated. The reduced expression of this gene could possibly lead to prawn mortality, as increased expression of this gene has been found to protect P. monodon from Vibrio harveyi . Arginine kinase (AK), a phosphagen kinase in the invertebrate energy metabolism, has previously been reported to play an immune role against viral infection . However, we observed a higher expression of this gene in M. rosenbergii after challenge by V. parahaemolyticus, which could suggest that AK plays a similar role in bacterial infection. Haemocyanin, an important immune gene in crustaceans, is involved in prophenoloxidase activity . It has antiviral properties against WSSV , and its increased expression in our transcriptome data tends to bear out its importance as a defence molecule against challenge by V. parahaemolyticus. Finally, metallothioneins, a metal-binding protein, was found to be highly expressed in our transcriptome data. The increased expression of this gene was to be expected, as it is known as a scavenger of reactive oxygen intermediates and generally shows higher expression levels during immune responses in invertebrates against pathogens .
We utilised the Illumina HiSeq™ 2000 platform and Trinity assembler package to perform a de novo transcriptome profiling of the hepatopancreases isolated from M. rosenbergii challenged with V. parahaemolyticus. The differential expression analysis between V. parahaemolyticus-infected and control groups revealed significant differences in the gene expression with 11,446 unigenes found to be significantly up-regulated and 3,103 unigenes observed to be significantly down-regulated. This study provided a valuable insight into antibacterial mechanisms of freshwater prawn against V. parahaemolyticus with majority of the differentially expressed unigenes were grouped into 11 animal immune system categories. Furthermore, this study has generated an abundant list of transcript from M.rosenbergii which will provide a fundamental basis for future genomics research in this field.
This work was supported by a Flagship Grant No: FL002-201 granted by Cluster-Bio, University of Malaya and Postgraduate Research Grant (PPP) of the University of Malaya, Malaysia (PG088-2012B) and publication assisted by Grant No 57-02-03-1015.
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