Journal of Invertebrate Pathology 135 (2016) 22–33

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Journal of Invertebrate Pathology

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De novo transcriptome assembly of Perkinsus olseni trophozoite stimulated in vitro with Manila clam (Ruditapes philippinarum) plasma

Abul Farah Md. Hasanuzzaman a,b, Diego Robledo c, Antonio Gómez-Tato d, Jose A. Alvarez-Dios e, ⇑ Peter W. Harrison f, Asunción Cao g, Sergio Fernández-Boo g, Antonio Villalba g, Belén G. Pardo a, , Paulino Martínez a a Departamento de Xenética, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo 27002, Spain b Fisheries and Marine Resource Technology Discipline, Khulna University, Khulna 9208, Bangladesh c Departamento de Xenética, Facultade de Bioloxía, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain d Departamento de Xeometría e Topoloxía, Facultade de Matemáticas, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain e Departamento de Matemática Aplicada, Facultade de Matemáticas, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain f Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, United Kingdom g Centro de Investigacións Mariñas (CIMA), Consellería do Medio Rural e do Mar, Xunta de Galicia, 36620 Vilanova de Arousa, Spain article info abstract

Article history: The protistan parasite Perkinsus olseni is a deadly causative agent of perkinsosis, a molluscan disease Received 16 September 2015 affecting Manila clam (Ruditapes philippinarum), having a significant impact on world mollusc production. Revised 18 January 2016 Deciphering the underlying molecular mechanisms in R. philippinarum-P. olseni interaction is crucial for Accepted 24 January 2016 controlling this parasitosis. The present study investigated the transcriptional expression in the parasite Available online 25 January 2016 trophozoite using RNA-seq. Control and treatment (in vitro challenged with Manila clam-plasma) P. olseni trophozoite RNA were extracted and sequenced on the Illumina HiSeq 2000 instrument using a 100-bp Keywords: paired-end sequencing strategy. Paired reads (64.7 million) were de novo assembled using Trinity, and Perkinsus olseni the resultant transcripts were further clustered using CAP3. The re-constructed P. olseni transcriptome Transcriptome RNA-seq contains 47,590 unique transcripts of which 23,505 were annotated to 9764 unique proteins. A large Gene expression number of genes were associated with Gene Ontology terms such as stress and immune-response, cell Pathogenicity homeostasis, antioxidation, cell communication, signal transduction, signalling and proteolysis. Among Response to host-immunity annotated transcripts, a preliminary gene expression analysis detected 679 up-regulated and 478 down-regulated genes, linked to virulence factors, anti-oxidants, adhesion and immune-response molecules. Genes of several metabolic pathways such as DOXP/MEP, FAS II or folate biosynthesis, which are potential therapeutic targets, were identified. This study is the first description of the P. olseni transcriptome, and provides a substantial genomic resource for studying the molecular mechanisms of the host-parasite interaction in perkinsosis. In this sense, it is also the first evaluation of the parasite gene expression after challenge with clam extracellular products. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Abbreviations: AA, arachidonic acid; AARS, aminoacyl-tRNA synthetase; bp, base pair; BP, Biological Process; CC, Cellular Component; DEG, differentially expressed Perkinsosis is a disease caused by protozoan parasites of the gene; DOXP/MEP, 1-deoxy-D-xylulose-5-phosphate/methylerythritol phosphate; genus Perkinsus which has long been known to devastate marine FAS II, fatty acid synthase type II; FC, fold change; GPI, glycosylphosphatidylinos- itol; HSP, heat shock protein; HSSP, High-Scoring Segment Pair; Fe-S, iron-sulfur; molluscs of socio-economic and ecological importance. Perkinsus MF, Molecular Function; MSP, merozoite surface protein; MIF, migration inhibitory spp. are included in the Rhizaria and, within this group, into the factor; RNA-seq, RNA sequencing; PE, paired-end; GO, Gene Ontology; KEGG, Kyoto Alveolata, Protalveolata and Perkinsidae (Adl et al., 2012). Seven Encyclopedia of Genes and Genomes; SOD, superoxide dismutases; SRA, sequence species are currently accepted within this genus: P. marinus, read archive; TRIM, tripartite motif-containing protein. P. olseni, P. qugwadi, P. chesapeaki, P. mediterraneus, P. honshuensis ⇑ Corresponding author at: Departamento de Xenética, Universidade de Santiago de Compostela, Lugo 27002, Spain. and P. beihaiensis (Villalba et al., 2011). Among them, P. olseni E-mail address: [email protected] (B.G. Pardo). and P. marinus appear on the list of reportable diseases of the http://dx.doi.org/10.1016/j.jip.2016.01.009 0022-2011/Ó 2016 Elsevier Inc. All rights reserved. Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33 23

World Organization for Animal Health (OIE), which makes stop- 2. Materials and methods ping this disease from happening of the outmost interest. P. olseni (Lester and Davis, 1981), synonymous to P. atlanticus (Murrell 2.1. Sampling and in vitro challenge et al., 2002), infects clams, abalones, oysters, cockles and arks (Villalba et al., 2004; Dang et al., 2015). It also causes lesions that The in vitro challenges between P. olseni trophozoites and Man- may interfere with respiration and other physiological processes ila clam R. philippinarum plasma were performed at the facilities of such as reproduction and/or growth, possibly causing death. Thus CIMA, Spain. Clams were collected from a P. olseni-free bed this parasite can have a deep impact on fishery productivity (Camariñas, A Coruña, Spain; regional government laboratories (Park et al., 2006; Casas and Villalba, 2012; Dang et al., 2013). High have never found Perkinsus spp. in this area following the criteria mortality rates have been reported in R. philippinarum when of framework 2006/88/CE) confirmed by PCR diagnosis of 30 col- affected by this parasitosis (Choi et al., 2002; Shimokawa et al., lected clams (Abollo et al., 2006) and incubation of gill tissue in 2010; Pretto et al., 2014). RFTM (Ray’s fluid thioglycollate medium). Clam haemolymph was Knowledge of host-parasite interaction is the basis for formulat- collected from the adductor muscle using a syringe with a 23 gauge ing effective control strategies against parasitosis. P. marinus is the needle, and samples contaminated with gametes and/or bacteria, most studied causative species for perkinsosis, and its virulence detected using light microscopic examination, were discarded. factors and interaction with the host have been extensively Haemolymph-samples from various clams were transferred into addressed (Bushek and Allen, 1996; Joseph et al., 2010, and refer- 1.5 mL tubes kept on crushed ice and subsequently centrifuged ences therein; Allam et al., 2013; Pales Espinosa et al., 2013, (800 g, 4 °C, 15 min) to obtain plasma as supernatant. Plasma 2014). Additionally, treatments of P. marinus cells with oyster was pooled and filtered with 0.45 lm filters in order to make it plasma, mucus, and tissue extracts induce functional changes in haemocyte-free. the parasite that mimic its behaviour during in vivo infections P. olseni trophozoites were obtained from the gills of parasitised (Earnhart et al., 2004; Pales Espinosa et al., 2013, 2014). In P. olseni R. decussatus clams, and in vitro cultured for 1–2 months following several aspects of biology have been studied: proliferation capacity the procedure described by Casas et al. (2002). A volume of culture (Elandalloussi et al., 2003, 2005a; Araujo et al., 2013), metabolic containing 5  106 trophozoites was taken from each culture and pathways (Elandalloussi et al., 2005b), pathogenicity in clam centrifuged (1000g,25°C, 10 min) to concentrate cells. The concen- (Shimokawa et al., 2010), karyotype (Teles-Grilo et al., 2007; trated trophozoites were resuspended in 2.5 mL filtered sea water Marques et al., 2012), genetic markers and population genetic and added to IWAKI 6-well plates. A permeable insert (0.2 lm Ano- structure (Pardo et al., 2011; Vilas et al., 2011), and more recently, poreÒ membrane NUNC 25 mm) was set in the plate-wells and gene and protein expression (Ascenso, 2011; Ascenso et al., 2007; 2.5 mL of plasma were added into it. Three challenge periods (1, 8 Leite et al., 2008; Fernández-Boo et al., 2014, 2015). Nevertheless, and 24 h) were assayed, each of them in triplicate for both treat- there is still a dearth of data regarding gene expression and the ment and control (filtered sea water instead of plasma) groups. molecular mechanisms involved in the infectivity of P. olseni and Once the incubation period finished, the inserts were removed from its interaction with the host. the wells. The trophozoites were collected from the wells and con- The development of in vitro culture of P. olseni (Casas et al., centrated by centrifuging (1000g, room temperature, 10 min). The 2002; Robledo et al., 2002) has greatly facilitated conducting resultant pellets were resuspended in RNAlater (Qiagen) solution in vitro and in vivo experiments on the underlying mechanisms and then stored at À80 °C until RNA extraction. of Perkinsus-host interaction, particularly using the trophozoite, the stage that proliferates through host tissues, reported as an 2.2. RNA extraction and sequencing effective agent for disease transmission in the American oyster C. virginica (Volety and Chu, 1994; Ford et al., 2002). However, Total RNA was extracted from treatment and control samples the lack of genomic resources has limited the comprehension of using the RNeasy mini kit (Qiagen) with DNase treatment following host-P. olseni interaction at a molecular level. Recent advances in the manufacturer’s instructions. The quality and quantity of RNA sequencing technologies have boosted the genomic and transcrip- were evaluated using a Bioanalyzer (Bonsai Technologies) and a tomic resources of many non-model species, offering an excellent NanoDropÒ ND-1000 spectrophotometer (NanoDropÒ Technologies opportunity to characterize them from a genomic perspective, Inc), respectively. Afterwards, equal amounts of RNA (ng) were and resulting in the increase of transcriptomic information in taken from all samples (3 replicates  3 sampling points) of each protozoan parasites (reviewed by Gomez et al., 2010 and group (control and treatment) and subsequently pooled for con- references therein; Ehrenkaufer et al., 2013; Walker et al., 2015). structing each library. These two pools (control and treatment) Transcriptome analysis is crucial for interrogating the functional were sequenced at the Wellcome Trust Centre for Human Genetics, elements of the genome and reveals the dynamic molecular Oxford. The two samples were barcoded and sequenced in two lanes constitution of cells and tissues (Wang et al., 2009), which can on the Illumina HiSeq 2000 instrument using a 100 base pair (bp) help to understand infectivity processes. Next-generation tran- paired-end (PE) reads strategy. Both samples were run in both lanes, scription profiling techniques, like RNA sequencing (RNA-seq), thus obtaining two technical-sequencing replicates per sample. can be used to unravel the transcriptomic landscape in a detailed Residual adaptor sequences and low quality bases were trimmed and accurate way. using Trimmomatic (Bolger et al., 2014) (version 0.32). Reads where The main aim of the present study was to gain an in depth both pairs had a length >36 bp post-filtering were retained. knowledge about the P. olseni transcriptome at the trophozoite For the present study samples were pooled, all samples of both stage, as well as to obtain a preliminary view of gene expression groups being independently stored for further refined analyses to of the host-parasite interaction. RNA was extracted from tropho- be carried out using a Perkinsus-specific oligo-microarray. zoites in vitro exposed to Manila clam (R. philippinarum) plasma and their appropriate controls, and sequenced on an Illumina 2.3. Transcriptome re-construction HiSeq 2000 platform. To our knowledge, this is the first transcrip- tomic study in P. olseni, providing invaluable genomic information Given the absence of a reference genome or transcriptome for for further studies and also a glimpse of the molecular basis of P. olseni,ade novo transcriptome assembly was carried out. Two Perkinsus adaptation, infectivity and virulence. recommended de Bruijn graph assemblers were used: Trinity 24 Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33

(version trinityrnaseq_r2013_08_14, Grabherr et al., 2011) and immunopathology function. To identify sequences putatively ABySS (version 1.3.7, Simpson et al., 2009). Trinity was run with linked to genes and pathways of parasite therapeutic interest, a a k-mer size of 25 and default settings, and ABySS with a 64 k-mer manual search on the transcriptome annotation was performed size, scaffolding and contig options on, and remaining parameters since the KEGG database contains canonical pathway but does by default. Resultant contigs from each bioinformatics strategy were not cover organism-specific metabolic routes (Kanehisa et al., further assembled using CAP3 (http://seq.cs.iastate.edu./)(Huang 2012). and Madan, 1999) with an overlapping identity percentage and minimum overlapping length parameters set to >85% and 50 bp, 3. Results and discussion respectively, in order to obtain highly reliable contigs and avoid redundancy. Both de novo assemblies were evaluated for chi- 3.1. Transcriptome sequencing and quality control maerism. Briefly, assembled sequences were first blasted against NCBI nucleotide collection (nt) database with cut-off E-value of Illumina sequencing generated a total of 67.8 million 100 bp PE 0.01 and maximum target sequence length of 100 bp to facilitate reads (31.3 million PE reads from the unstimulated trophozoites detection of chimaerism. Then, assembled transcripts with more and 36.5 million from the stimulated ones). After trimming, 64.7 than one significant BLASTn hit were blasted against NCBI non- million high quality PE reads (95.4%) were retained for reconstruct- redundant protein database (nr) using BLASTx. Trans-chimaeras ing the de novo transcriptome representing P. olseni trophozoites were identified using the BLASTx output following the steps and exposed to Manila clam plasma. RNA-seq data has been deposited parameters recommended by Yang and Smith (2013). in the SRA database under accession number SRP060596.

2.4. Functional annotation 3.2. De novo transcriptome assembly: performance evaluation The de novo assembled P. olseni transcriptome was annotated against NCBI nr database. A BLASTx search was conducted for all Trinity+CAP3 rendered a total of 47,590 unique transcripts with P. olseni sequences and the first three hits with a minimum align- a minimum length of 201 bp and N50 of 1891 bp, while ABySS ment length of 100 bp were considered. In order to discard any +CAP3 generated an assembly of 10,702 unique transcripts with exogenous mRNA contamination, annotated transcripts with cut- minimum length of 72 bp and N50 of 1743 bp (Table 1). The Trin- off E-value <1.0EÀ20 from Bacteria, Mollusca and Chordata were ity+CAP3 assembly also rendered a higher number of unique tran- further inspected. Protein sequences to which those transcripts scripts >500 bp (31,640) as compared to ABySS+CAP3 (8358). The were annotated were blasted against NCBI Alveolata (taxid: size distribution of transcripts shows that the modal-group length 33630) nr database (the closest related taxon to Perkinsus) using was 270 bp for the Trinity+CAP3 assembly and 125 bp for the BLASTp. The protein sequences without significant matches ABySS+CAP3 assembly (Fig. 1). The post-assembly evaluations (<1.EÀ20) against NCBI Alveolata (taxid: 33630) database were showed 5884 BLASTn hits (12.36%), 0.54% chimaeras and 1.9 considered as possible contaminants and were discarded from redundancy in the Trinity+CAP3 assembly, while 2537 BLASTn hits our P. olseni transcriptome. Finally, unique selected transcripts (23.71%), 2.25% chimaeras and 1.1 redundancy in the ABySS+CAP3 were further characterized through functional annotation using assembly (Table 2). A higher number of reconstructed full-length Gene Ontology (GO) terms (Ashburner et al., 2000) and Kyoto reference transcripts and a larger number of annotated splicing Encyclopedia of Genes and Genomes (KEGG) pathways running patterns were reported in the Schizosaccharomyces pombe tran- Blast2GO Version 2.7 software (Conesa et al., 2005) with default scriptome assembled by Trinity (Grabherr et al., 2011), and the parameters. Trinity+CAP3-blast assembly strategy has also been suggested as the best method for reducing chimaerism (Yang and Smith, 2.5. Differential expression 2013) and increasing assembly continuity (Duan et al., 2012). In contrast, ABySS has been recommended for generating better con- A preliminary evaluation of differentially expressed genes tiguous assemblies, as for whole genome assembling (Liu et al., (DEGs) between stimulated and control trophozoites was carried 2013). Since there is currently no consensus on the best assembler out using variation between technical replicates as statistical error. for transcriptome reconstruction, the trade-off between maximiz- All reads from both control and treatment samples were first ing transcript number with lower length-range and, at the same aligned against the reconstructed de novo P. olseni transcriptome time, minimizing chimaerism vs maximizing blast hits and mini- using RSEM (version 1.2.17) (http://deweylab.biostat.wisc.edu/ mizing redundancy pointed towards Trinity+CAP3 as the best rsem/) and read counts for each transcript were obtained. After- strategy for reconstructing the P. olseni transcriptome. Finally, wards, differential expression between infected and control sam- the P. olseni de novo transcriptome (25.3 Mb) is very close to the ples was calculated using EdgeR and resulting P-values were full length genome estimation for P. olseni by Marques et al. corrected for false discovery rate (FDR). DEGs were defined as (2012) (around 28 Mb), suggesting certain redundancy and/or an those showing a log2 fold change (FC) between control and treat- important amount of splice variants in our transcriptome, along ment higher/lower than ±1 and at least an average of 10 reads per with a rather compact genome. million. P. olseni DEGs putatively involved in host-parasite interaction 3.3. Transcriptome annotation were selected on the grounds of GO terms relevant to pathogenesis and immune system. Additionally, a number of sequences were BLASTx search against NCBI non-redundant (nr) protein data- chosen as immunopathology-related genes by checking the pres- base resulted in 23,700 annotated transcripts (49.8%) correspond- ence of one or more of the following key words in their annotation ing to 9959 unique proteins. Among them, a small proportion description, as reported in previous works (Joseph et al., 2010; (652 sequences; 6.5%) was annotated to Bacteria (174), Mollusca Piña-Vázquez et al., 2012; Soudant et al., 2013; Pales Espinosa (163) and Chordata (315) taxons. A subsequent BLASTp search et al., 2014): virulence, pathogen, host, oxidation, antioxidation, resulted in 457 (out of 652 sequences) showing significant hits homeostasis, apoptosis, stress, inhibition, invasion, proteolysis, (<1.0EÀ20) with the Alveolata (taxid: 33630) protein database. It modification or adaptation. Afterwards, all selected sequences is unclear why some sequences were significantly annotated to were manually checked to remove those with ambiguous such distant taxa, however contamination likelihood in the Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33 25

Table 1 Trinity+CAP3 and AbySS+CAP3 assembly statistics.

Assemblies k-mer Unique N50 length (bp) N90 length (bp) No. of transcripts Average transcript Transcript length transcripts assembled (length) > 500 bp length (bp) range (bp) Trinity+CAP3 25 47,590 1891 597 31,640 1,243.36 201–14,187 ABySS+CAP3 64 10,702 1743 773 8358 1,290.73 72–14,136

Alveolata database and/or horizontal gene transfer, as reported in prokaryotes and eukaryotes (Soucy et al., 2015), could explain this circumstance. Finally 9764 unique protein sequences representing 23,505 transcripts were retained for further analysis (Supplemen- tary Table S1). The drop-off from total annotated transcripts to unique anno- tated sequences (from 23,505 to 9764) indicates that there is a notable redundancy (58.5%) in the P. olseni transcriptome and/or notable alternative splicing. Such high level of gene repetition has been reported in other eukaryotic parasites like Entamoeba histolyt- ica and E. invadens (Ehrenkaufer et al., 2013) and can also be linked to low annotation profiles of parasite databases and to the conser- vative approaches followed for transcriptome assembling to mini- mize chimaerism. Among the annotated sequences, 92.5% matched to Chromalveolata, and the remaining to Animalia (4.5%), Bacteria (1.2%), Plantae (0.7%), Fungi (0.5%) and others (0.5%) (Fig. 2A). In accordance with our results, Joseph et al. (2010) reported most significant hits of P. marinus ESTs to Chroma- lveolata, and to a minor extent to metazoan/fungi, viridiplantae and bacteria. Among chromalveolate (Fig. 2B), the most represented taxon was the genus Perkinsus itself (97.1%), which was also shown in the top-hit species distribution (9003 hits; Fig. 3). A total of 4495 sequences (46%) were annotated as well-known proteins. A high number of unknown proteins (5269) was expected since parasite protein databases are far from complete. Similar results were obtained in Plasmodium falciparum where 60% of the transcripts were annotated as hypothetical proteins (Gardner et al., 2002).

3.4. Functional categorization of P. olseni transcriptome

A total of 6701 sequences (28.5% of the 23,505 annotated tran- scripts) had at least one associated GO term (level 2). Metabolic process (3318 sequences) and Cellular process (3062) were the most represented BP terms (Fig. 4A). Several GO terms related to Fig. 1. Size distribution of transcripts assembled by Trinity+CAP3 (A) and by ABySS life cycle and host-pathogen interaction were identified, including +CAP3 (B). those associated with signalling, growth, reproduction, develop- ment, biological adhesion and immune system process (Fig. 4A). Within the CC category (Fig. 4B), the GO term Cell with 1721 Table 2 sequences was the most represented followed by Organelle Performance evaluation of two different assemblies using BLASTn searches against (1235), Membrane (1006) and Macromolecular Complex (880) NCBI. terms. Catalytic activity (3199 sequences) and Binding (3101) asso- Database Evaluation criteria Assemblies ciated with about 90% transcripts were the most represented terms Trinity ABySS in the MF category (Fig. 4C). Some transcripts were also associated +CAP3 +CAP3 with GO terms related to significant biological functions such as NCBI nucleotide No. of assembled transcripts with 5884 2537 antioxidation, transcription factor and transducer activities collection (nt) BLAST hits (Fig. 4C). GO classification description of P. olseni transcriptome is % of annotated transcripts with 99.6 99.6 similar to that reported for P. marinus (Joseph et al., 2010) and À BLAST hits <1.00E 10 for P. olseni DEGs exposed to R. decussatus haemolymph (Ascenso % of annotated transcripts with 64.4 67.8 BLAST hits (>80%) et al., 2007). Mean alignment length (bp) 861.2 1106.9 KEGG analysis showed 105 pathways related to metabolism of % of chimaerism 0.5 2.3 nucleotides (346 sequences), carbohydrates (313 sequences), a Level of redundancy 1.9 1.1 amino acids (242 sequences) or energy metabolism (171 a The number of assembled unique transcripts with blast hits divided by the sequences). Several genes were associated with signalling path- number of unique accession numbers to which transcripts blasted. ways including mTOR signalling, phosphatidylinositol signalling 26 Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33

Fig. 2. Distribution of annotated sequences among taxonomic groups. A: Major taxonomic domain; others include Excavata, Amoebozoa, Choanoflagellatea, Filasterea, Rhodophyta, Crenarchaeota, Euryarchaeota, Rhizaria and Virus. B: Chromalveolata; others include Cryptophyta, Haptophyta, Chromerida. C: Animalia; others include Nematoda, Platyhelminthes, Nemertea, Hydrozoa, Cnidaria, Parazoa, Rotifera, Echinodermata, Phoronida, Hemichordata, Sipuncula.

or T cell receptor signalling, and these will be described more thor- 3.6. Genes related to host-parasite interactions: pathogenicity and oughly in the following sections. response to immunity

A number of genes of the P. olseni transcriptome (See supple- 3.5. Differential expressed genes (DEGs): a preliminary analysis mentary Table S2) were selected and grouped into broader cate- gories for a more appropriate discussion considering their Comparison of gene expression in the P. olseni stimulated vs associated GO terms related to immunopathology and their func- control groups revealed 1699 DEGs, of which 1157 (679 up- tional roles implicated in host-pathogen interaction. regulated and 478 down-regulated) were annotated and associated with specific GO categories (Fig. 5). Among DEGs, the top 15 up- and 15 down-regulated annotated genes included many related 3.6.1. Cell communication, signal transduction and signalling to binding, response to stimulus, oxidoreductase activity, transport Molecular crosstalk between parasite and host cells is crucial for and proteolysis (Tables 3 and 4). Regulation of some of these genes host-parasite interaction. Parasites alter host defences through has also been linked to adaptation, cell proliferation and invasion interfering with host signalling pathways (Hakimi and Cannella, processes of parasites (Que and Reed, 2000; Morgan et al., 2001; 2011). In the present transcriptome, several genes (e.g. adenylate Ruiz et al., 2001; Goel et al., 2003; Dou and Carruthers, 2011; Ali guanylate cyclise, tripartite motif-containing protein 59 (TRIM59)) and Nozaki, 2013; Long et al., 2014). More details will be discussed playing a role in cell communication, signalling and signal transduc- in the following section. tion were up- and/or down-regulated. Salmon et al. (2012) reported Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33 27

Fig. 3. Top-hit species distribution of the BLASTx hits against the nr protein database with a cutoff E-value of 1.0EÀ3. that transmembrane receptor–like adenylate cyclases in Try- phosphatase were differentially expressed, as also reported in panosoma brucei inhibit host innate immune response. The TRIM P. marinus (Wright et al., 2002; Schott et al., 2006; Soudant et al., family of RING finger domain-containing proteins modulates 2013; Pales Espinosa et al., 2014). Peroxiredoxin II and V and pattern-recognition receptor (PRR) signalling pathways and thus SOD have been detected in the cell proteome of P. olseni regulates the host’s innate immune response (Kawai and Akira, (Fernández-Boo et al., 2014, 2015), and antioxidant activity linked 2011). Down-regulation of TRIM59 in the stimulated Perkinsus to peroxidases has also been reported in this species (Araujo et al., trophozoite might be due to either induction of apoptosis by clam 2013). The antioxidant network may have a significant role in haemocyte-derived signals or to a parasite mimicry strategy to cir- parasite virulence by limiting the oxidative environment produced cumvent the host’s interferon activity. Genes encoding kinases, by the host as a defence mechanism (Piacenza et al., 2009). We also which are involved in cell-cycle regulation and signalling pathways observed differential expression of iron-sulfur (Fe-S) assembly in other apicomplexa parasites (Billker et al., 2009; Lourido et al., protein, 2Fe-2S ferredoxin, and pyruvate:ferredoxin oxidoreduc- 2010), were also found to be differentially expressed (calcium- and tase NADPH-cytochrome involved in oxidative molecular balance. calmodulin-dependent kinase, mitogen-activated protein kinase). An association between the availability of intracellular Fe (II) in P. olseni and its proliferation and virulence has been reported 3.6.2. Adhesion, GPI anchor formation and transport (Elandalloussi et al., 2003; Leite et al., 2008; Araujo et al., 2013). Several genes (e.g. MSP, clathrin heavy chain) were associated Genes linked to ubiquitination process (e.g. ubiquitin- with GO terms like cell adhesion, glycosylphosphatidylinositol conjugating enzyme e2 and TRIM59) were regulated in P. olseni. (GPI) anchor biosynthetic process and transport. Some of them Ubiquitin and ubiquitin-like proteins are involved in many differ- were differentially expressed, as previously reported in P. marinus ent processes like cell cycle regulation, cell death, endocytosis, stimulated with oyster pallial mucus, an activator of parasite viru- autophagy, DNA repair (Al-Hakim et al., 2010), and initiation and lence (Pales Espinosa et al., 2014). Some of these genes are regulation of the innate immune response (Bhoj and Chen, 2009; involved in parasite invasion (Horn and McCulloch, 2011; Malynn and Ma, 2010). Tomavo et al., 2013; Hull and Dlamini, 2014). Genes such as lipopolysaccharide-induced TNF factor, cyclophilin, macrophage migration inhibitory factor (MIF), 3.6.3. Antioxidation, stimuli-response and cell homeostasis interferon-induced guanylate-binding protein, which have Like other parasites, P. olseni needs to reorganize its physiology, immunosuppressive function (Pales Espinosa et al., 2014), were metabolic processes and virulence mechanism for invading and regulated in the stimulated P. olseni. In parasites, MIF-like proteins adapting to host tissues. In the present study, antioxidants such are likely related to survival, immunomodulation and pro- as superoxide dismutases (SOD), glutathione peroxidase, peroxire- inflammatory responses, and host cell apoptosis (Jang et al., doxin 5, thioredoxin domain-containing protein, and prostatic acid 2011; Miller et al., 2012). 28 Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33

Fig. 4. Level 2 Gene Ontology (GO) assignment for the P. olseni transcriptome. A: Biological Process (BP), B: Cellular Component (CC), and C: Molecular Function (MF).

Genes encoding chaperons/heat shock proteins (HSPs) related reported (Pales Espinosa et al., 2014; Fernández-Boo et al., 2015). to stress response were also regulated in P. olseni. In response to Molecular chaperones in parasites are thought to play a role in stress conditions, expression of HSPs in Perkinsus species has been cell-environment communication (Botha et al., 2007). Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33 29

Fig. 5. Differentially expressed genes in P. olseni trophozoite in vitro stimulated with clam-plasma.

3.6.4. Programmed cell death processes and cell proliferation our P. olseni transcriptome. Serine-type endopeptidase and hydro- Although many genes in the present P. olseni transcriptome lase activities have been previously reported in P. marinus (Brown were associated with GO terms linked to apoptosis regulation, et al., 2005; Joseph et al., 2010; Pales Espinosa et al., 2014). autophagy, phagocytosis, cell proliferation and cell migration, only Cysteine-type cathepsins are involved in host-protein degradation, a few were detected as differentially expressed in this preliminary host cell invasion, growth and survival of parasites (Que and Reed, study. Some examples are: gene encoding autophagy 8i putative 2000; Joseph et al., 2010; Dou and Carruthers, 2011) and their role protein (ATG8i), which is involved in autophagic processes in yeast in parasitic infection processes has also been documented (Saffer (Kirisako et al., 1999; Kabeya et al., 2000), and liver stage antigen et al., 1989; Long et al., 2014). The turnover of proteases and pro- (LSA), which has been expressed during the pre-erythrocytic liver tease inhibitors is likely linked to the suicide-inhibition mecha- stage of P. falciparum (Guerin-Marchand et al., 1987). Pales nism of serpin family inhibitors (Gubb et al., 2010). The role of Espinosa et al. (2014) reported regulation of genes with apoptotic serpins in host-parasite interactions is well known (Prevot et al., and anti-apoptotic properties in P. marinus exposed to Crassostrea 2006; Molehin et al., 2012). virginica pallial mucus. In host-parasite interaction, host-cell There were numerous genes in our study linked to adhesion, autophagy could be a survival strategy, while apoptosis leading transport, signalling pathways, apoptosis, and proteases secretion to death may help the parasite to minimize antigen exposure to (See supplementary Table S2) which showed no modulation. A loss the host immune system (Reece et al., 2011). of virulence and pathogenicity in Perkinsus species has been In relation to critical roles of phosphorylation in survival, inva- observed in in vitro culture (Volety and Chu, 1994; Bushek and sion and host cell remodelling (Yokoyama et al., 1998; Billker et al., Allen, 1996; Ford et al., 2002). Such absence of regulation in many 2009; Fentress et al., 2010; Leykauf et al., 2010), some serine/ther- key genes needs further in vivo molecular study on gene expression onine phosphatases (e.g. protein phosphatise,dual specific phos- in both parasite (P. olseni) and host (clam) taking the different phatases (DSPs)) were detected in this P. olseni transcriptome, infection stages into account. some of them differentially expressed. Modulation of serine/thre- onine kinases has been reported in P. marinus exposed to oyster pallial mucus (Pales Espinosa et al., 2014). 3.7. Genes involved in pathways of therapeutic interest

3.7.1. DOXP/MEP pathway: Isoprenoid biosynthesis 3.6.5. Proteases and protease inhibitors The presence of seven genes involved in the 1-deoxy-D- In perkinsosis, proteases play a role suppressing host defence xylulose-5-phosphate (DOXP)/methylerythritol phosphate (MEP) factors (Garreis et al., 1996; La Peyre et al., 1996; Tall et al., pathway (Table 5) in this P. olseni transcriptome suggests the 1999). Several genes involved in peptidase activity, hydrolase existence of the isoprenoid biosynthesis pathway in P. olseni,as activity and endopeptidase inhibitor activity were identified in previously implied in P. marinus (Matsuzaki et al., 2006, 2008; 30 Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33

Table 3 Table 4 Top 15 annotated up-regulated genes in P. olseni-trophozoite in vitro exposed to Top 15 annotated down-regulated genes in P. olseni-trophozoite in vitro exposed to Manila clam-plasma. Manila clam-plasma.

Putative gene GO terms Log2 Fold Putative gene GO terms Log2 change Fold (FC) change (FC) Bidirectional sugar P:cellular response to salicylic acid 13.49 transporter sweet16- stimulus; P:sucrose transport; Bidirectional sugar transporter P:cellular response to salicylic À13.96 like P:cellular response to osmotic sweet16-like acid stimulus; P:sucrose stress; P:cellular response to transport; P:cellular response abscisic acid stimulus to osmotic stress; P:cellular Low affinity potassium P:potassium ion transmembrane 13.03 response to abscisic acid transport system transport stimulus protein kup Far upstream element-binding F:RNA binding À12.20 Alpha beta domain protein P:menaquinone biosynthetic 11.71 Nucleotide pyrophosphatase F:hydrolase activity À10.94 process; F:transferase activity; F: Phospho-2-dehydro-3- F:3-deoxy-7- À10.37 hydrolase activity deoxyheptonate aldolase, phosphoheptulonate synthase Protein BMH2, putative P:Ras protein signal transduction; 10.79 Phe-sensitive, putative activity; P:aromatic amino acid F:phosphoserine binding; P: family biosynthetic process negative regulation of ubiquitin- Dipeptidyl peptidase 2 F:RNA binding; P:proteolysis; À9.89 protein ligase activity involved in F:serine-type peptidase mitotic cell cycle activity; F:carboxypeptidase Lish motif-containing – 10.59 activity protein Glutathione synthetase P:single-organism metabolic À9.81 Gag/pol/env polyprotein, F:nucleic acid binding; P:DNA 10.55 process; F:ligase activity; putative integration P:cellular metabolic process ATP-dependent RNA P:regulation of gene expression; F: 9.98 Gibberellin 3-beta P:oxidation-reduction process; À9.77 helicase ded-1, putative ATP-dependent helicase activity; F:oxidoreductase activity P:cellular response to stimulus; P: Membrane protein – À9.58 apoptotic process Calcium ion binding C:cytosol; C:nucleus; À9.57 Peptide synthetase F:catalytic activity 9.90 F:calcium ion binding Transcription factor iws-1, P:transcription, DNA-templated; C: 9.88 TAF9 rna polymerase tata box P:positive regulation of growth À9.43 putative nucleus; F:DNA binding binding protein -associated rate; F:adenylate kinase Sodium/potassium- C:integral component of 9.74 factor isoform 2 family activity; C:Cajal body; C: transporting ATPase membrane; F:ATP binding; protein nucleolus; F:protein binding; alpha chain, putative F:cation-transporting ATPase P:phosphorylation activity; P:nucleobase-containing Cathepsin L F:oxidoreductase activity; P: À9.04 compound metabolic process proteolysis; F:cysteine-type Conserved oligomeric C:Golgi transport complex 9.69 peptidase activity Golgi complex Pre-mrna-splicing factor F:nucleic acid binding; F: À9.01 component, putative nucleotide binding Neural polypyrimidine P:RNA processing; F:nucleic acid 9.64 Gag/pol/env polyprotein, F:nucleic acid binding; P:DNA À8.31 tract binding protein, binding; F:nucleotide binding putative integration putative Ferroportin family C:integral component of À7.31 Dimethylaniline F:nucleotide binding; 9.59 membrane; F:iron ion monooxygenase, F:monooxygenase activity; transmembrane transporter putative P:cellular response to stimulus activity; P:iron ion NAD transhydrogenase C:integral component of 9.48 transmembrane transport subunit alpha membrane; F:NAD(P)+ Gamma-tubulin complex C:microtubule organizing À6.18 transhydrogenase (AB-specific) component center; C:spindle pole; P: activity; P:oxidation-reduction microtubule nucleation process 3-deoxy-7- F:3-deoxy-7-phosphoheptulonate 9.32 phosphoheptulonate synthase activity; P:aromatic amino synthase acid family biosynthetic process host immune function and contribute to parasite virulence (Catisti et al., 2000; Kubata et al., 2000; Chu et al., 2004).

Grauvogel et al., 2007; Joseph et al., 2010). The DOXP/MEP path- way has been suggested as a drug target (e.g. fosmidomycin, Fos) 3.7.3. Folate biosynthesis against apicomplexan parasites (Jomaa et al., 1999; Surolia et al., KEGG analysis revealed the presence of the folate biosynthesis 2004). pathway in our P. olseni transcriptome with 6 transcripts encoding for 5 enzymes (Table 5). The existence of a folate pathway had been previously suggested in this species since pyrimethamine, 3.7.2. Lipid synthesis pathway an inhibitor of the folate pathway, inhibits P. olseni proliferation Lipids and phospholipids are energy reserves and structural (Elandalloussi et al., 2005a). Folate metabolism is an important tar- components required for growth, development and life cycle com- get of antiprotozoal agents (Elandalloussi et al., 2005b). pletion of parasites (Lund and Chu, 2002; Krishnegowda and Gowda, 2003; Bisanz et al., 2006). Several genes involved in the fatty acid synthase type II (FAS II) pathway (Table 5; Seeber and 3.7.4. De novo pyrimidine biosynthesis Soldati-Favre, 2010) and the arachidonic acid (AA) synthesis path- We identified several genes involved in the biosynthesis of way were identified in the present transcriptome. FAS II and AA pyrimidines (Table 5). In pathogenic parasites, pyrimidine biosyn- pathways have been previously reported in P. marinus (Chu et al., thesis has significant roles in growth, cell proliferation, adaptation 2004; Lund et al., 2005; Stelter et al., 2007; Joseph et al., 2010). to cell stress and expression of virulence factors (Fox and Bzik, AA is an essential polyunsaturated fatty acid which acts as precur- 2002; Hegewald et al., 2013; De Gontijo et al., 2014) and it is a sor for the synthesis of prostaglandins, which are deleterious to potential anti-parasite target for drug development. Abul Farah Md. Hasanuzzaman et al. / Journal of Invertebrate Pathology 135 (2016) 22–33 31

Table 5 Conflict of interest Genes identified in pathways of therapeutic interest.

Pathways Putative genes The authors wish to disclose that there are no known conflicts DOXP/MEP pathway: 1-deoxy-d-xylulose-5-phosphate synthase of interest associated with this publication. Isoprenoid ((DXP synthase); 1-deoxy-d-xylulose 5- biosynthesis phosphate reductoisomerase (DXR/IspC), 2-c- Acknowledgments methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), 4-diphosphocytidyl- 2c-methyl-d-erythritol kinase (IspE), 2-C- This investigation was funded by the Ministerio de Educación y methyl-D-erythritol 2,4-cyclodiphosphate Ciencia of the Spanish Government (Project AGL2012-37981). The synthase (ME-CPP synthase, IspF), 4-hydroxy-3- first author would like to acknowledge the PhD scholarship methylbut-2-en-1-yl diphosphate synthase (HMB-PP synthase, IspG), 4-hydroxy-3- awarded by the EXPERTS III Consortium of the European Commu- methylbut-2-enyl diphosphate reductase, nity Mobility Programme ‘‘Erasmus Mundus Action 2, Strand 1” putative (HMB-PP reductase (LytB, IspH) (EMA2). Diego Robledo was supported by a FPU fellowship from Lipid synthesis Acetyl-CoA carboxylase, lipoic acid synthase, the Ministerio de Educación, Cultura y Deporte of the Spanish pantothenate kinase, delta9-elongating activity Government. We would like to acknowledge the support of the protein, delta5-desaturase, delta -fatty-acid desaturase-like, and delta 12 fatty acid Centro de Supercomputación de Galicia (CESGA) in the completion desaturase of this work. The authors are also grateful to Lucía Insua for provid- Folate biosynthesis gtp cyclohydrolase, folylpolyglutamate ing technical assistance. synthase, dihydropteroate synthase, dihydrofolate reductase-thymidylate synthase, and diphosphokinase Appendix A. 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