Journal of Invertebrate Pathology 135 (2016) 22–33 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip 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 Â 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,