Protist, Vol. 166, 659–676, December 2015 http://www.elsevier.de/protis Published online date 30 September 2015 ORIGINAL PAPER Phylogeny, Morphology, and Metabolic and Invasive Capabilities of Epicellular Fish Coccidium Goussia janae a b b,c,d Sunil Kumar Dogga , Pavla Bartosová-Sojkovᡠ, Julius Lukesˇ , and a,1 Dominique Soldati-Favre a Department of Microbiology and Molecular Medicine, University of Geneva. CMU, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland b ˇ Institute of Parasitology, Biology Centre, Branisovskᡠ31, Ceské Budejoviceˇ (Budweis), Czech Republic c Faculty of Science, University of South Bohemia, Branisovskᡠ1645/31A, ˇ Ceské Budejoviceˇ (Budweis), Czech Republic d Canadian Institute for Advanced Research, 180 Dundas St W, Toronto, ON M5G 1Z8, Canada Submitted June 29, 2015; Accepted September 15, 2015 Monitoring Editor: Frank Seeber To fill the knowledge gap on the biology of the fish coccidian Goussia janae, RNA extracted from exogenously sporulated oocysts was sequenced. Analysis by Trinity and Trinotate pipelines showed that 84.6% of assembled transcripts share the highest similarity with Toxoplasma gondii and Neospora caninum. Phylogenetic and interpretive analyses from RNA-seq data provide novel insight into the metabolic capabilities, composition of the invasive machinery and the phylogenetic relationships of this parasite of cold-blooded vertebrates with other coccidians. This allows re-evaluation of the phy- logenetic position of G. janae and sheds light on the emergence of the highly successful obligatory intracellularity of apicomplexan parasites. G. janae possesses a partial glideosome and along with it, the metabolic capabilities and adaptions of G. janae might provide cues as to how apicomplexans adjusted to extra- or intra-cytoplasmic niches and also to become obligate intracellular parasites. Unlike the similarly localized epicellular Cryptosporidium spp., G. janae lacks the feeder organelle necessary for directly scavenging nutrients from the host. Transcriptome analysis indicates that G. janae pos- sesses metabolic capabilities comparable to T. gondii. Additionally, this enteric coccidium might also access host cell nutrients given the presence of a recently identified gene encoding the molecular sieve at the parasitophorous vacuole membrane. © 2015 Elsevier GmbH. All rights reserved. Key words: Apicomplexa; Coccidia; Goussia janae; phylogeny; ultrastructure; invasion; central carbon metabolism. Introduction 1 Members of the phylum Apicomplexa share dis- Corresponding author; fax +41-22-3795702 e-mail [email protected] (D. Soldati-Favre). tinctive morphological traits, the presence of a http://dx.doi.org/10.1016/j.protis.2015.09.003 1434-4610/© 2015 Elsevier GmbH. All rights reserved. 660 S.K. Dogga et al. set of unique organelles at the apical end being epithelial cells, where they are covered by very their prominent unifying feature. This apical com- closely apposed enterocyte and parasitophorous plex includes the secretory organelles called the vacuole membranes (PVMs) (Lukesˇ and Stary´ rhoptries and micronemes, the apical polar ring, 1992; Molnár 1996). The more frequent “monopo- and the conoid. The rhoptries and micronemes are dial” subtype of the epicellular interaction means unique secretory organelles that are associated that the coccidium forms a single zone of attach- with motility, adhesion, invasion and establish- ment with the host cell cytoplasm, whereas the ment of a parasitophorous vacuole. The conoid so-called “spider-like” subtype describes stages is a cone-shaped structure believed to play a positioned above the microvillar region, connected mechanical role in the invasion of host cells. The to the host cell only through several narrow zones of parasites possess flattened membranous vesicles attachment (Barto soˇ vá-Sojková et al. 2015; Lukesˇ called alveoli, closely apposed beneath the plasma and Stary´ 1992). membrane. The composite structure of the para- In the 18S rDNA-based phylogeny, some piscine site membrane and the inner membrane complex coccidians (e.g. G. janae, G. ameliae, G. pan- (IMC) is associated with a number of cytoskele- nonica, G. szekelyi) form basal lineages of tal elements, including actin, myosin, microtubules, the species-rich eimeriorinid clade, or of its and a network of intermediate filament-like pro- sarcocystid subclade, which encompass econom- teins. The IMC plays a structural role and is an ically important parasites of both homeothermic important scaffold element during cytokinesis, in and poikilothermic vertebrate hosts (Barto soˇ vá- addition to being involved in motility and invasion Sojková et al. 2015; Jirku˚ et al. 2002; Lovy and of the host cell (Harding and Meissner 2014). Most Friend 2015; Molnár et al. 2012; Whipps et al. apicomplexans also have another unique structure, 2012). a chloroplast-like organelle called the apicoplast, Due to their early-branching position within a derived non-photosynthetic plastid (van Dooren coccidians, G. janae and the related piscine coc- and Striepen 2013). cidia may share numerous ancestral features. The genus Goussia Labbé, 1896 (Apicomplexa, Indeed, two-valved sporocysts of Goussia spp. Eimeriorina) accommodates piscine and amphib- with a simple longitudinal suture seem to rep- ian coccidia with thin-walled oocysts which lack resent the ancestral state of the Sarcocystidae micropyle and contain four dizoic bivalved sporo- + Eimeriidae + Calyptosporidae clade, from which cysts. Although members of the genus Goussia radiations into an array of excystation structures are mostly encountered in the intestine, infections occurred (Jirku˚ et al. 2002). These include the of other organs, such as kidney and spleen are four-valved structures of sarcocystids, hemivalved quite common. Most species have homoxenous arrangements with a thin membrane-covered life cycles, but a more complex cycle involving oblong apical opening of the sporocyst wall of fish as definitive hosts and tubificids and other calyptosporids, and finally the univalved sporocysts invertebrates as vectors and/or paratenic hosts was containing Stieda and sub-Stieda bodies of eimeri- experimentally proven in a few cases (Dyková and ids (Whipps et al. 2012). A similar scenario can be Lom 1981; Lom and Dyková 1992; Fournie and envisioned for the evolution of host-parasite interac- Overstreet 1983; Steinhagen and Körting 1990). tions, with the ancestral epicellular location retained Goussia janae was described from the intestine in G. janae and related species parasitizing cold- of dace Leuciscus leuciscus and chub Squalius blooded vertebrates, followed by a radiation of cephalus. It is a causative agent of heavy infec- coccidians with various derived intracellular pos- tions manifested by microvillar atrophy of epithelial itions. However, the basal position of G. janae and cells and subsequent formation of multiple sec- its relatives may be affected by long-branch attrac- ondary mucosal folds (Lukesˇ and Dyková 1990). tion and/or on phylogenies based on just 18S rRNA The parasite has a strict seasonal occurrence, with gene (Jirku˚ et al. 2009). Hence, to resolve the immature oocysts being shed in spring from its fish above-mentioned uncertainties, it is important to host in feces or massively in casts. Sporulation extend the phylogenetic analyses by inclusion of ◦ is exogenous and at 10 C takes about 48 hours protein-coding genes. (Lukesˇ and Dyková 1990; Lukesˇ and Stary´ 1992). As every host and host tissue represent a G. janae development occurs inside the intestinal nutritionally different environment, development of epithelium, yet in a characteristic epicellular loca- specific adaptations related to feeding behav- tion (also known as extracytoplasmic). This means ior is a genuine part of the parasitic lifestyle. that merogonial, gamogonial and early sporogonial Indeed, when compared with the intracellular Plas- stages are confined to the microvillar region of the modium spp., the genetic analyses of epicellular Epicellular Fish Coccidium Goussia janae 661 Cryptosporidium parvum and C. hominis have As revealed by transmission electron microscopy, revealed the presence of several novel classes of all merogonial, gamogonial, and early sporogo- cell-surface and secreted proteins with potential nial stages were intracellular, localized within the roles in host-parasite interactions and pathogene- PVM squeezed between the enterocytic mem- sis (Abrahamsen et al. 2004; Mazurie et al. 2013; brane and cytoplasm, in what is termed the Xu et al. 2004). However, a more extensive com- epicellular localization. The earliest developmen- parative analysis has been hindered by the lack of tal stages observed were small dense granules genomic and/or transcriptomic data from an epicel- and microneme-containing oval meronts, wedged lular coccidium. among the microvilli of the enterocyte (Fig. 1G). In order to rectify this situation, we have per- Mature meronts had already developed typi- formed morphological investigation of all life cycle cal apicomplexan features such as rhoptries, stages of G. janae, and obtained the transcrip- micronemes, and dense granules (Fig. 1F, J). tome of its sporulated phase which was selected Merozoites were usually formed by ectomerogony because of the easy accessibility of a huge number following multiple nuclear divisions and invagina- of oocysts from host’s casts minimally contami- tions of meront’s cytoplasm (Fig. 1E). nated by host tissue. The collected data was used Early developmental stages assumed