Apicomplexa: Eimeriorina)
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ScholarsArchive@OSU Parasitol Res (2012) 111:1331–1342 DOI 10.1007/s00436-012-2969-8 ORIGINAL PAPER Phylogeny of fish-infecting Calyptospora species (Apicomplexa: Eimeriorina) Christopher M. Whipps & John W. Fournie & David A. Morrison & Carlos Azevedo & Edilson Matos & Per Thebo & Michael L. Kent Received: 28 November 2011 /Accepted: 9 May 2012 /Published online: 29 May 2012 # Springer-Verlag 2012 Abstract There are numerous species of apicomplexans that experimental transmission studies, it may be difficult to dem- infect poikilothermic vertebrates, such as fishes, and possess onstrate whether all Calyptospora species are heteroxenous. unique morphological features that provide insight into the Other distinct morphological characteristics of Calyptospora evolution of this important phylum of parasites. Here, the such as an incomplete sporocyst suture, an apical opening for relationship of the fish-infecting Calyptospora species to other sporozoite release, a thin veil surrounding sporocysts supported coccidians was investigated based on DNA sequence analysis. by sporopodia, and a lack of Stieda and sub-Stieda bodies Genetic data from the small subunit ribosomal DNA region of suggest there may be adequate features to delineate these taxa. the genome were obtained for three of the five nominal species Even without life cycle data for all species, the morphology and in the genus Calyptospora. Phylogenetic analyses supported a genetic data provide a means to reliably classify Calyptospora monophyletic lineage sister to a group composed of mostly species. Placement in either the Calyptosporidae or Eimeriidae Eimeria species. The monophyly of Calyptospora species sup- is discussed, along with issues relating to the phylogeny of the ports the validity of the family Calyptosporidae, but the sister genus Goussia. relationship to Eimeria species might also suggest the Eimer- iidae be expanded to encompass Calyptospora. The validity of the family Calyptosporidae has been questioned because it is Introduction delineated from the Eimeriidae largely based on life cycle characteristics and sporocyst morphology. In general, Eimeria Members of the phylum Apicomplexa are parasites of a vast species have a homoxenous life cycle, whereas the type species range of invertebrate and vertebrate animal taxa (Levine of Calyptospora is heteroxenous. In the absence of 1988). Research has tended to focus on species that infect C. M. Whipps (*) C. Azevedo SUNY‐ESF, State University of New York, College of Environmental Department of Cell Biology, Institute of Biomedical Sciences, Science and Forestry, Environmental and Forest Biology, University of Porto, 246 Illick Hall, 1 Forestry Drive, Porto, Portugal Syracuse,NY13210,USA e-mail: [email protected] C. Azevedo Zoology Department, College of Science, King Saud University, J. W. Fournie 11451 Riyadh, Saudi Arabia Gulf Ecology Division, National Health and Environmental Effects Research Laboratory, E. Matos U.S. Environmental Protection Agency, Laboratório de Pesquisa Carlos Azevedo, 1 Sabine Island Dr., Universidade Federal Rural da Amazônia, Gulf Breeze, FL 32561, USA Belém, PA, Brazil D. A. Morrison : P. Thebo M. L. Kent Section for Parasitology (SWEPAR), Center for Fish Disease Research, Department of Microbiology, Department of Biomedical Sciences and Veterinary Public Health, Oregon State University, Swedish University of Agricultural Sciences, 220 Nash Hall, 75189 Uppsala, Sweden Corvallis, OR 97331-3404, USA 1332 Parasitol Res (2012) 111:1331–1342 and cause disease in birds and mammals, where potential grass shrimp, Palaemonetes pugio (Fournie and Overtstreet control is possible and incentive for action is strong. In other 1983). Sporozoites develop in the invertebrate digestive tract vertebrate hosts, such as fishes, there is much more of a and do occur intracellularly in basal cells (Fournie et al. 2000). limited understanding of apicomplexan diversity with only a A two-host life cycle, however, has yet to be demonstrated for few hundred described species, mostly Eimeria and Goussia other species of Calyptospora,weakeningthestatusofthe species (Lom and Dyková 1992). The morphological diver- family Calyptosporidae based primarily on that characteristic. sity represented in other genera of fish-infecting Apicom- Fournie et al. (1985) did observe numerous motile sporozoites plexa may provide clues to understanding the evolution of free in the intestinal contents of experimentally infected this important phylum. Palaemonetes kadiakensis and suggested that benthic crusta- Calyptospora species are apicomplexan parasites of tele- ceans are likely candidates for intermediate hosts in Calypto- ost fishes. Five species have been described to date: one spora empristica because the fish-host must apparently die occurs in estuarine fishes from coastal waters of the northern before oocysts can be dispersed. Still, the validity of the Gulf of Mexico and the southeast Atlantic, one from an Calyptosporidae based on a heteroxenous life cycle has been inland creek in southern Mississippi, and three from fresh- questioned primarily because of the lack of asexual multipli- water systems in Brazil. An undescribed species in cation in the intermediate host (Lom and Dyková 1992). the arapaima is also from Brazil (de Albuquerque and de However, for C. funduli,anintermediatehostisrequiredfor Carvalho Brasil‐Sato 2010). All described species have fish transmission (Fournie and Overtstreet 1983) and a develop- definitive hosts and primarily infect the hepatic parenchy- mental period of about 5 days in grass shrimp is necessary to ma. The impact of Calyptospora infections on fish species is become infective to killifishes. Fournie et al. (2000)subse- largely unknown, but studies suggest that these can have a quently demonstrated that sporozoites do occur within gut significant effect. Solangi and Overstreet (1980) reported basal cells and suggested that development into an infective that up to 85 % of the hepatic parenchyma can be replaced sporozoite takes place in these basal cells. by oocysts in heavily infected gulf killifish. The infection To address some of these challenges of coccidian classifica- provokes an inflammatory response by day 10 post- tion, DNA sequence analysis has become commonplace for infection (p.i.) which intensifies by day 20 p.i. during gam- investigations into the systematics of these species (Morrison ogony. By day 30, when oocyst walls are fully formed, 2009). Molecular data supplement morphological analyses and inflammatory infiltrates disappeared and many aggregates help identify taxonomically significant characteristics. For ex- were encapsulated with pigmented macrophage aggregate ample, Morrison et al. (2004) noted some phylogenetic patterns development. Fournie and Overstreet (1993) reported that with regard to host specificity and life cycle. Species within a some experimentally infected fishes with abnormal cocci- particular family or genus of coccidia tend to cluster by broad dian development displayed more extensive host responses taxonomic groups of hosts (i.e., mammals, birds, reptiles, etc.), and pathological changes than those seen in natural defini- albeit an imperfect pattern. With regard to life cycle, hetero- tive hosts. Gulf killifish (Fundulus grandis) that are infected xenous sarcocystids form a distinct lineage from homoxenous with Calyptospora funduli can be severely impacted by eimeriids, again with some exceptions; notably, the genus Iso- exposure to low temperatures. Fournie (1985) showed that spora within the Sarcocystidae is homoxenous. From the per- exposure of experimentally infected killifish to 3–4°C spective of sporocyst morphology, Jirků et al. (2002) resulted in mortalities in infected fish, whereas uninfected emphasized the importance of the excystation structures in fish survived. Bonar et al. (2006) associated a Calyptospora coccidian taxonomy. Excystation structures may be useful for sp. as a contributing cause of mortality in arapaima imported Calyptospora species which possess a unique membrane- to the USA from Brazil. covered, oblong apical opening for sporozite excystation The genus Calyptospora Overstreet, Hawkins and Fournie (Fournie et al. 1985). An incomplete suture extends posteriorly 1984 was proposed to accommodate Eimeria species of fishes from the opening to about midway on the sporocyst wall where possessing atypical characteristics. Specifically, sporocysts it becomes fused, and a prominent ridge occurs on the posterior (Fig. 1) with a posterior thickening or extension and sporopo- one third of the sporocyst wall (Hawkins et al. 1983). This is dia surrounded by a membranous veil lack Stieda and sub- reminiscent of a bivalved Goussia species, but Calyptospora is Stieda bodies and have an apical opening for sporozoite ultimately univalved like Eimeria because the suture is partially release continuing to the opposite pole first as a tapering fused. If these features represent a stage of coccidian evolution suture, then as a protruding ridge (Overstreet et al. 1984). between valved and non-valved species, then we expect this to The family Calyptosporidae was also proposed to accommo- be reflected in the phylogeny of the coccidia. Likewise, these date Calyptospora and Goussia, distinguishing these genera features alone may warrant distinct familial status. It was our from the Eimeriidae in having species with a heteroxenous life goal to estimate the