Pavla Bartošová-Sojková

Classification and Phylogenetics of Myxozoa

Myxozoans evolved as an endoparasitic radiation of cnidarians exploiting invertebrate and vertebrate (primarily fish) hosts in freshwater and marine environments. Currently, there are some 2,200 nominal species classified to 64 genera and 17 families. These groupings are mainly based on spore morphology. This chapter provides an updated spore-based taxonomic key that takes into account the recent recognition of the cnidarian origin of myxozoans as well as important revisions at generic, family and suborder levels over the last decade. A list of generic synonyms is also reviewed here. Myxozoan molecular phylogenies largely disagree with traditional spore-based classification systems, probably due to extreme plasticity in myxospore morphologies that has resulted in extensive convergence. Molecular phylogenies of myxozoans (based on all existing SSU sequences and those available for species with known actinospore-producing stages) resolve the malacosporeans, the freshwater myxosporeans, the marine myxosporeans and the sphaerosporid lineages. Within these clades species can be resolved according to definitive host type and, partially, according to host environment. Numerous exceptions are indicative of several recursions of species into freshwater or marine environments. Further resolution within clades identifies groups of species according to tissue tropism in their vertebrate hosts. We suggest future studies of myxozoan systematics to address persisting taxonomic and phylogenetic discrepancies and make recommendations for describing taxa in the absence of sequence data or when sequence and morphological data are incongruent.

Evolutionary origin of Ceratonova shasta and phylogeny of the marine myxosporean lineage.

In order to clarify the phylogenetic relationships among the main marine myxosporean clades including newly established Ceratonova clade and scrutinizing their evolutionary origins, we performed large-scale phylogenetic analysis of all myxosporean species from the marine myxosporean lineage based on three gene analyses and statistical topology tests. Furthermore, we obtained new molecular data for Ceratonova shasta, C. gasterostea, eight Ceratomyxa species and one Myxodavisia species. We described five new species: Ceratomyxa ayami n. sp., C. leatherjacketi n. sp., C. synaphobranchi n. sp., C. verudaensis n. sp. and Myxodavisia bulani n. sp.; two of these formed a new, basal Ceratomyxa subclade. We identified that the Ceratomyxa clade is basal to all other marine myxosporean lineages, and Kudoa with Enteromyxum are the most recently branching clades. Topologies were least stable at the nodes connecting the marine urinary clade, the marine gall bladder clade and the Ceratonova clade. Bayesian inference analysis of SSU rDNA and the statistical tree topology tests suggested that Ceratonova is closely related to the Enteromyxum and Kudoa clades, which represent a large group of histozoic species. A close relationship between Ceratomyxa and Ceratonova was not supported, despite their similar myxospore morphologies. Overall, the site of sporulation in the vertebrate host is a more accurate predictor of phylogenetic relationships than the morphology of the myxospore.

Hidden diversity and evolutionary trends in malacosporean parasites (Cnidaria: Myxozoa) identified using molecular phylogenetics.

Malacosporeans represent a small fraction of myxozoan biodiversity with only two genera and three species described. They cycle between bryozoans and freshwater fish. In this study, we (i) microscopically examine and screen different freshwater/marine fish species from various geographic locations and habitats for the presence of malacosporeans using PCR; (ii) study the morphology, prevalence, host species/habitat preference and distribution of malacosporeans; (iii) perform small subunit/large subunit rDNA and Elongation factor 2 based phylogenetic analyses of newly gathered data, together with all available malacosporean data in GenBank; and (iv) investigate the evolutionary trends of malacosporeans by mapping the morphology of bryozoan-related stages, host species, habitat and geographic data on the small subunit rDNA-based phylogenetic tree. We reveal a high prevalence and diversity of malacosporeans in several fish hosts in European freshwater habitats by adding five new species of Buddenbrockia and Tetracapsuloides from cyprinid and perciform fishes. Comprehensive phylogenetic analyses revealed that, apart from Buddenbrockia and Tetracapsuloides clades, a novel malacosporean lineage (likely a new genus) exists. The fish host species spectrum was extended for Buddenbrockia plumatellae and Buddenbrockia sp. 2. Co-infections of up to three malacosporean species were found in individual fish. The significant increase in malacosporean species richness revealed in the present study points to a hidden biodiversity in this parasite group. This is most probably due to the cryptic nature of malacosporean sporogonic and presporogonic stages and mostly asymptomatic infections in the fish hosts. The potential existence of malacosporean life cycles in the marine environment as well as the evolution of worm- and sac-like morphology is discussed. This study improves the understanding of the biodiversity, prevalence, distribution, habitat and host preference of malacosporeans and unveils their evolutionary trends.

Approaches for Characterising Myxozoan Species

Myxozoan species and genera have been defined traditionally using morphological characteristics of spores and developmental stages, host preference and tissue specificity. Use of morphology is, however, limited in some taxa by a lack of reliable characters or ambiguities in their origin. For instance, morphological variation can characterise spores of closely-related species while similar spore morphologies in distantly-related species reflect convergence. Therefore, morphological traits are accompanied routinely by DNA sequences in the identification of myxozoan species. DNA sequencing methods have inherent limitations, too, which include co-amplification of host, inability to distinguish mixed infections and PCR and primer biases for some taxa over others. By combining several approaches, myxozoan researchers are revealing novel diversity and demonstrating that strains and morphologically cryptic species characterise many taxa. Extensive geographic sampling of economically significant myxozoans, including Myxobolus cerebralis, Kudoa thyrsites, Ceratonova shasta and Tetracapsuloides bryosalmonae, has demonstrated intraspecific genetic variation, strains linked with geographic localities, and species complexes. Application of new approaches to myxozoan classification is expected to facilitate reassessment of existing taxa, discovery of new taxa and further resolution of cryptic species.

Adaptive Radiation and Evolution Within the Myxozoa

Myxozoans are endoparasites characterized by extensive morphological simplification and complex life cycles. Their definitive hosts are bryozoans—in the case of the more primitive and species-poor Malacosporea, or annelids—in the case of the more derived and speciose Myxosporea. This chapter reviews patterns of adaptive radiation within the Myxozoa and explores the drivers that may have promoted evolutionary change. Topics covered include: multiple transitions between worm-like and sac-like forms in the Malacosporea; undersampling that likely limits our appreciation of malacosporean diversity; and multiple shifts between marine and freshwater environments in the Myxosporea. We also examine morphological simplification that is observed in stages that produce spores and associated changes in the size of these sporogonic stages. This contrasts with the evolution of morphological complexity in spores. Features proposed to be involved in diversification include the acquisition of hardened spores and plasmodia, a high diversity of definitive hosts and invasion of novel hosts and host environments in the Myxosporea. The evolution towards higher complexity in spores can, in some cases, be linked with increasing transmission success. Convergence in spore form suggests that certain morphologies are highly adaptive. The significance of many features of spores, however, remains poorly known.

Trypsin- and Chymotrypsin-like serine proteases in schistosoma mansoni-- 'the undiscovered country'.

Background Blood flukes (Schistosoma spp.) are parasites that can survive for years or decades in the vasculature of permissive mammalian hosts, including humans. Proteolytic enzymes (proteases) are crucial for successful parasitism, including aspects of invasion, maturation and reproduction. Most attention has focused on the ‘cercarial elastase’ serine proteases that facilitate skin invasion by infective schistosome larvae, and the cysteine and aspartic proteases that worms use to digest the blood meal. Apart from the cercarial elastases, information regarding other S. mansoni serine proteases (SmSPs) is limited. To address this, we investigated SmSPs using genomic, transcriptomic, phylogenetic and functional proteomic approaches. Methodology/Principal Findings Genes encoding five distinct SmSPs, termed SmSP1 - SmSP5, some of which comprise disparate protein domains, were retrieved from the S. mansoni genome database and annotated. Reverse transcription quantitative PCR (RT- qPCR) in various schistosome developmental stages indicated complex expression patterns for SmSPs, including their constituent protein domains. SmSP2 stood apart as being massively expressed in schistosomula and adult stages. Phylogenetic analysis segregated SmSPs into diverse clusters of family S1 proteases. SmSP1 to SmSP4 are trypsin-like proteases, whereas SmSP5 is chymotrypsin-like. In agreement, trypsin-like activities were shown to predominate in eggs, schistosomula and adults using peptidyl fluorogenic substrates. SmSP5 is particularly novel in the phylogenetics of family S1 schistosome proteases, as it is part of a cluster of sequences that fill a gap between the highly divergent cercarial elastases and other family S1 proteases. Conclusions/Significance Our series of post-genomics analyses clarifies the complexity of schistosome family S1 serine proteases and highlights their interrelationships, including the cercarial elastases and, not least, the identification of a ‘missing-link’ protease cluster, represented by SmSP5. A framework is now in place to guide the characterization of individual proteases, their stage-specific expression and their contributions to parasitism, in particular, their possible modulation of host physiology.

Diversification in Hawaiian long-legged flies (Diptera: Dolichopodidae: Campsicnemus): Biogeographic isolation and ecological adaptation

Flies in the genus Campsicnemus have diversified into the second-largest adaptive radiation of Diptera in the Hawaiian Islands, with 179 Hawaiian endemic species currently described. Here we present the first phylogenetic analysis of Campsicnemus, with a focus on the Hawaiian fauna. We analyzed a combination of two nuclear (CAD, EF1α) and five mitochondrial (COI, COII, 12S, 16S, ND2) loci using Bayesian and maximum likelihood approaches to generate a phylogenetic hypothesis for the genus Campsicnemus. Our sampling included a total of 84 species (6 species from Europe, 1 from North America, 7 species from French Polynesia and 70 species from the Hawaiian Islands). The phylogenies were used to estimate divergence times, reconstruct biogeographic history, and infer ancestral ecological associations within this large genus. We found strong support for a South Pacific+Hawaiian clade, as well as for a monophyletic Hawaiian lineage. Divergence time estimates suggest that Hawaiian Islands were colonized approximately 4.6 million years ago, suggesting that most of the diversity within Campsicnemus evolved since the current high islands began forming ∼5 million years ago. We also observe a novel ecotype within the Pacific Campsicnemus; a widespread obligate water-skating form that has arisen multiple times across the Pacific Islands. Together, these analyses suggest that a combination of ecological, biogeographic and temporal factors have led to the impressive diversity of long-legged flies in Hawaii and elsewhere in the Pacific.

Epicellular Apicomplexans: Parasites “On the Way In”

The Coccidia and the Cryptosporidia infect both coldand warm-blooded vertebrates, yet members of the genera Toxoplasma, Eimeria, Sarcocystis, and Cryptosporidium (which mostly parasitize the latter hosts) have received most of the attention by far because of their importance to human and veterinary health. Our knowledge about a wide array of apicomplexans found in fish, amphibians, and reptiles is thus primarily confined to the morphological description of their exogenous stages (i.e., oocysts) and sites of infection, rarely with notes on pathogenicity. The life cycles of these “neglected” parasites resemble those of apicomplexans from birds and mammals. They undergo successive multiplication by merogony (asexual divisions producing meronts with merozoites), followed by gamogony (fusion of two types of gametes resulting in an oocyst), and sporogony (asexual reproduction producing sporozoites) [1]. In the Coccidia of homeotherms, these developmental phases take place in different hosts, whereas the majority of the Coccidia of poikilotherms typically have direct life cycles. The intestine is the favored site for sporogony, but piscine coccidians are characterized by their frequent extraintestinal development with either exogenous (outside the host) or endogenous (inside the host) sporulation [1]. Coccidians of poikilotherm hosts differ from those parasitizing homeotherms by (1) thin-walled oocysts lacking micropyle (an opening of oocyst wall), (2) macrogamonts without wall-forming bodies, (3) sporocysts with excystation structures (sutures) and projections of the sporocyst wall (membranaceous veils, sporopodia), and (4) frequent epicellular localization, which is shared with some gregarines, the protococcidian Eleuteroschizon dubosqi, and cryptosporidians [1–4]. Phylogenetic analyses based on the 18S rRNA gene sequences, now available from a number of apicomplexans parasitizing cold-blooded vertebrates (e.g., members of the genera Cryptosporidium, Goussia, Acroeimeria, Eimeria, Calyptospora, and Choleoeimeria), allowed for the inference of their phylogenetic relationships. Piscine cryptosporidians represent a well-defined monophyletic group at the base of the cryptosporidian clade, whereas amphibian and reptile cryptosporidians are mixed with those infecting homeotherms. Coccidians of poikilotherm hosts constitute basal lineages of the whole eucoccidian clade, or of its eimeriid or sarcocystid subclades, with piscine coccidians representing the most basal groups (Fig 1) [2,3,5–7]. Because of their early-branching position, these parasites of poikilotherms likely possess ancestral features, the scrutiny of which may help us better understand the evolution of Apicomplexa. Indeed, the simple excystation structures of piscine, amphibian, and reptile coccidians have diverged into more complex excystation structures in eimeriids, sarcocystids, and calyptosporiids [2,3]. The

Parasite Cathepsin D-Like Peptidases and Their Relevance as Therapeutic Targets.

Inhibition of aspartic cathepsin D-like peptidases (APDs) has been often discussed as an antiparasite intervention strategy. APDs have been considered as virulence factors of Trypanosoma cruzi and Leishmania spp., and have been demonstrated to have important roles in protein trafficking mechanisms of apicomplexan parasites. APDs also initiate blood digestion as components of multienzyme proteolytic complexes in malaria, platyhelminths, nematodes, and ticks. Increasing DNA and RNA sequencing data indicate that parasites express multiple APD isoenzymes of various functions that can now be specifically evaluated using new functional-genomic and biochemical tools, from which we can further assess the potential of APDs as targets for novel effective intervention strategies against parasitic diseases that still pose an alarming threat to mankind.

Phylogeny, Morphology, and Metabolic and Invasive Capabilities of Epicellular Fish Coccidium Goussia janae

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 phylogenetic 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 possesses 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.