Dispersal and invasive stages of Urospora eugregarines (Apicomplexa) from brown bodies of a polychaete host.

Urosporid eugregarines (Apicomplexa: Urosporidae) are unicellular eukaryotic parasites inhabiting the coelom or the intestine of marine invertebrates such as annelids, molluscs, nemerteans, and echinoderms. Despite the availability of published morphological and phylogenetical analyses of coelomic gregarines, their long-term survival in the host body cavity and dispersal routes into the marine environment remain unclear. Here, we focus on Urospora gametocysts and oocysts with sporozoites, which were found viable inside the so-called brown bodies floating in the body cavity of the polychaete Travisia forbesii. Brown bodies form as a result of host defence where coelomocytes encapsulate dead host cells and foreign objects including potential pathogens. We hypothesise the long-term persistence of Urospora eugregarines in brown bodies through evasion of the host immunity and outline possible pathways for their egress into the marine environment, applicable as dispersal routes for other parasites as well. Unique features revealed by detailed ultrastructural analysis of detected eugregarine stages include asynchronous sporogony, a massive sporozoite secretion apparatus, as well as the presence of free (possibly autoinfective) sporozoites within the gametocyst. The assignment to the genus Urospora and the complete identity with U. ovalis and U. travisiae were confirmed by analysing 18S rDNA sequences obtained from isolated gametocysts. The 18S rDNA phylogeny confirmed the affiliation of Urosporidae to Lecudinoidea and the grouping of all Urospora sequences with Difficilina from nemerteans and environmental sequences from the Artic region. We also enriched the Apicomplexa set by partial 28S rDNA sequences of two Urospora species enabling more complex phylogenetic analyses prospectively.

Evidence from the resurrected family Polyrhabdinidae Kamm, 1922 (Apicomplexa: Gregarinomorpha) supports the epimerite, an attachment organelle, as a major eugregarine innovation.

BACKGROUND: Gregarines are a major group of apicomplexan parasites of invertebrates. The gregarine classification is largely incomplete because it relies primarily on light microscopy, while electron microscopy and molecular data in the group are fragmentary and often do not overlap. A key characteristic in gregarine taxonomy is the structure and function of their attachment organelles (AOs). AOs have been commonly classified as "mucrons" or "epimerites" based on their association with other cellular traits such as septation. An alternative proposal focused on the AOs structure, functional role, and developmental fate has recently restricted the terms "mucron" to archigregarines and "epimerite" to eugregarines. METHODS: Light microscopy and scanning and transmission electron microscopy, molecular phylogenetic analyses of ribosomal RNA genes. RESULTS: We obtained the first data on fine morphology of aseptate eugregarines Polyrhabdina pygospionis and Polyrhabdina cf. spionis, the type species. We demonstrate that their AOs differ from the mucron in archigregarines and represent an epimerite structurally resembling that in other eugregarines examined using electron microscopy. We then used the concatenated ribosomal operon DNA sequences (SSU, 5.8S, and LSU rDNA) of P. pygospionis to explore the phylogeny of eugregarines with a resolution superior to SSU rDNA alone. The obtained phylogenies show that the Polyrhabdina clade represents an independent, deep-branching family in the Ancoroidea clade within eugregarines. Combined, these results lend strong support to the hypothesis that the epimerite is a synapomorphic innovation of eugregarines. Based on these findings, we resurrect the family Polyrhabdinidae Kamm, 1922 and erect and diagnose the family Trollidiidae fam. n. within the superfamily Ancoroidea Simdyanov et al., 2017. Additionally, we re-describe the characteristics of P. pygospionis, emend the diagnoses of the genus Polyrhabdina, the family Polyrhabdinidae, and the superfamily Ancoroidea.

Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis.

The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

Eudiplozoon nipponicum: morphofunctional adaptations of diplozoid monogeneans for confronting their host.

BACKGROUND: Monogeneans, in general, show a range of unique adaptations to a parasitic lifestyle, making this group enormously diverse. Due to their unique biological properties, diplozoid monogeneans represent an attractive model group for various investigations on diverse biological interactions. However, despite numerous studies, there are still gaps in our knowledge of diplozoid biology and morphofunctional adaptations. RESULTS: In this study, we provide a comprehensive microscopic analysis of systems/structures involved in niche searching, sensing and self-protection against the host environment, and excretory/secretory processes in Eudiplozoon nipponicum. Freeze-etching enabled us to detect syncytium organisational features not visible by TEM alone, such as the presence of a membrane subjacent to the apical plasma membrane (separated by a dense protein layer) and a lack of basal plasma membrane. We located several types of secretory/excretory vesicles and bodies, including those attached to the superficial membranes of the tegument. Giant unicellular glands were seen accumulating predominantly in the apical forebody and hindbody haptor region. Muscle layer organisation differed from that generally described, with the outer circular and inner longitudinal muscles being basket-like interwoven by diagonal muscles with additional perpendicular muscles anchored to the tegument. Abundant muscles within the tegumentary ridges were detected, which presumably assist in fixing the parasite between the gill lamellae. Freeze-etching, alongside transmission electron and confocal microscopy with tubulin labelling, enabled visualisation of the protonephridia and nervous system, including the peripheral network and receptor innervation. Three types of receptor were identified: 1) uniciliated sensory endings with a subtle (or missing) tegumentary rim, 2) obviously raised uniciliated receptors with a prominent tegumentary rim (packed with massive innervation and muscles) and 3) non-ciliated papillae (restricted to the hindbody lateral region). CONCLUSIONS: This study points to specific morphofunctional adaptations that have evolved in diplozoid monogeneans to confront their fish host. We clearly demonstrate that the combination of different microscopic techniques is beneficial and can reveal hidden differences, even in much-studied model organisms such as E. nipponicum.

Motility and cytoskeletal organisation in the archigregarine Selenidium pygospionis (Apicomplexa): observations on native and experimentally affected parasites.

Representatives of Apicomplexa perform various kinds of movements that are linked to the different stages of their life cycle. Ancestral apicomplexan lineages, including gregarines, represent organisms suitable for research into the evolution and diversification of motility within the group. The vermiform trophozoites and gamonts of the archigregarine Selenidium pygospionis perform a very active type of bending motility. Experimental assays and subsequent light, electron, and confocal microscopic analyses demonstrated the fundamental role of the cytoskeletal proteins actin and tubulin in S. pygospionis motility and allowed us to compare the mechanism of its movement to the gliding machinery (the so-called glideosome concept) described in apicomplexan zoites. Actin-modifying drugs caused a reduction in the movement speed (cytochalasin D) or stopped the motility of archigregarines completely (jasplakinolide). Microtubule-disrupting drugs (oryzalin and colchicine) had an even more noticeable effect on archigregarine motility. The fading and disappearance of microtubules were documented in ultrathin sections, along with the formation of α-tubulin clusters visible after the immunofluorescent labelling of drug-treated archigregarines. The obtained data indicate that subpellicular microtubules most likely constitute the main motor structure involved in S. pygospionis bending motility, while actin has rather a supportive function.

Fine structure and Molecular Phylogenetic Position of Two Marine Gregarines, Selenidium pygospionis sp. n. and S. pherusae sp. n., with Notes on the Phylogeny of Archigregarinida (Apicomplexa).

Archigregarines are a key group for understanding the early evolution of Apicomplexa. Here we report morphological, ultrastructural, and molecular phylogenetic evidence from two archigregarine species: Selenidium pygospionis sp. n. and S. pherusae sp. n. They exhibited typical features of archigregarines. Additionally, an axial row of vacuoles of a presumably nutrient distribution system was revealed in S. pygospionis. Intracellular stages of S. pygospionis found in the host intestinal epithelium may point to the initial intracellular localization in the course of parasite development. Available archigregarine SSU (18S) rDNA sequences formed four major lineages fitting the taxonomical affiliations of their hosts, but not the morphological or biological features used for the taxonomical revision by Levine (1971). Consequently, the genus Selenidioides Levine, 1971 should be abolished. The branching order of these lineages was unresolved; topology tests rejected neither para- nor monophyly of archigregarines. We provided phylogenies based on LSU (28S) rDNA and near-complete ribosomal operon (concatenated SSU, 5.8S, LSU rDNAs) sequences including S. pygospionis sequences. Although being preliminary, they nevertheless revealed the monophyly of gregarines previously challenged by many molecular phylogenetic studies. Despite their molecular-phylogenetic heterogeneity, archigregarines exhibit an extremely conservative plesiomorphic structure; their ultrastructural key features appear to be symplesiomorphies rather than synapomorphies.

Effect of jasplakinolide and cytochalasin D on cortical elements involved in the gliding motility of the eugregarine Gregarina garnhami (Apicomplexa).

Since apicomplexans represent exclusively parasitic unicellular organisms with medical and economic impacts, the principles of their motility have been studied intensively. By contrast, the movement in apicomplexan basal groups, such as gregarines, remains to be elucidated. The present study focuses on Gregarina garnhami parasitising the digestive tract of the locust Schistocerca gregaria, and investigates the involvement of cytoskeletal elements (the ectoplasmic network and myonemes) and the secretion of mucosubstances during eugregarine gliding motility. Combined microscopic analyses were used to verify the role of actin filaments and membranes' organisation in G. garnhami motility. A freeze-etching analysis of membranes revealed the size, density, and arrangement of intramembranous particles along with the distribution and size of pores and ducts. Experimental assays using actin-modifying drugs (jasplakinolide, cytochalasin D) confirmed that actin most likely plays a role in cell motility, principally in its filamentous form (=F-actin). Myonemes, localised in the border between the ectoplasm and endoplasm, correspond to the concentric bundles of F-actin. Microscopic analyses confirmed that changes in gamonts motility corresponding to the changes in the organisation and density of myonemes and the ectoplasmic network in drug-treated cells, suggesting that these structures might serve as contractile elements facilitating gliding motility in G. garnhami.

First Ultrastructural and Molecular Phylogenetic Evidence from the Blastogregarines, an Early Branching Lineage of Plesiomorphic Apicomplexa.

Blastogregarines are poorly studied parasites of polychaetes superficially resembling gregarines, but lacking syzygy and gametocyst stages in the life cycle. Furthermore, their permanent multinuclearity and gametogenesis by means of budding considerably distinguish them from other parasitic Apicomplexa such as coccidians and hematozoans. The affiliation of blastogregarines has been uncertain: different authors considered them highly modified gregarines, an intermediate apicomplexan lineage between gregarines and coccidians, or an isolated group of eukaryotes altogether. Here, we report the ultrastructure of two blastogregarine species, Siedleckia nematoides and Chattonaria mesnili, and provide the first molecular data on their phylogeny based on SSU, 5.8S, and LSU rDNA sequences. Morphological analysis reveals that blastogregarines possess both gregarine and coccidian features. Several traits shared with archigregarines likely represent the ancestral states of the corresponding cell structures for parasitic apicomplexans: a distinctive tegument structure and myzocytotic feeding with a well-developed apical complex. Unlike gregarines but similar to coccidians however, the nuclei of male blastogregarine gametes are associated with two kinetosomes. Molecular phylogenetic analyses reveal that blastogregarines are an independent, early diverging lineage of apicomplexans. Overall, the morphological and molecular evidence congruently suggests that blastogregarines represent a separate class of Apicomplexa.

Motility in blastogregarines (Apicomplexa): Native and drug-induced organisation of Siedleckia nematoides cytoskeletal elements.

Recent studies on motility of Apicomplexa concur with the so-called glideosome concept applied for apicomplexan zoites, describing a unique mechanism of substrate-dependent gliding motility facilitated by a conserved form of actomyosin motor and subpellicular microtubules. In contrast, the gregarines and blastogregarines exhibit different modes and mechanisms of motility, correlating with diverse modifications of their cortex. This study focuses on the motility and cytoskeleton of the blastogregarine Siedleckia nematoides Caullery et Mesnil, 1898 parasitising the polychaete Scoloplos cf. armiger (Müller, 1776). The blastogregarine moves independently on a solid substrate without any signs of gliding motility; the motility in a liquid environment (in both the attached and detached forms) rather resembles a sequence of pendular, twisting, undulation, and sometimes spasmodic movements. Despite the presence of key glideosome components such as pellicle consisting of the plasma membrane and the inner membrane complex, actin, myosin, subpellicular microtubules, micronemes and glycocalyx layer, the motility mechanism of S. nematoides differs from the glideosome machinery. Nevertheless, experimental assays using cytoskeletal probes proved that the polymerised forms of actin and tubulin play an essential role in the S. nematoides movement. Similar to Selenidium archigregarines, the subpellicular microtubules organised in several layers seem to be the leading motor structures in blastogregarine motility. The majority of the detected actin was stabilised in a polymerised form and appeared to be located beneath the inner membrane complex. The experimental data suggest the subpellicular microtubules to be associated with filamentous structures (= cross-linking protein complexes), presumably of actin nature.

Structures related to attachment and motility in the marine eugregarine Cephaloidophora cf. communis (Apicomplexa).

Gregarines represent a highly diversified group of ancestral apicomplexans, with various modes of locomotion and host-parasite interactions. The eugregarine parasite of the barnacle Balanus balanus, Cephaloidophora cf. communis, exhibits interesting organisation of its attachment apparatus along with unique motility modes. The pellicle covered gregarine is arranged into longitudinal epicytic folds. The epimerite is separated from the protomerite by a septum consisting of tubulin-rich filamentous structures and both are packed with microneme-like structures suggestive of their function in the production of adhesives important for attachment and secreted through the abundant epimerite pores. Detached trophozoites and gamonts are capable of gliding motility, enriched by jumping and rotational movements with rapid changes in gliding direction and cell flexions. Actin in its polymerised form (F-actin) is distributed throughout the entire gregarine, while myosin, detected in the cortical region of the cell, follows the pattern of the epicytic folds. Various motility modes exhibited by individuals of C. cf. communis, together with significant changes in their cell shape during locomotion, are not concordant with the gliding mechanisms generally described in apicomplexan zoites and indicate that additional structures must be involved (e.g. two 12-nm filaments; the specific dentate appearance of internal lamina inside the epicytic folds).

Protococcidian Eleutheroschizon duboscqi, an Unusual Apicomplexan Interconnecting Gregarines and Cryptosporidia.

This study focused on the attachment strategy, cell structure and the host-parasite interactions of the protococcidian Eleutheroschizon duboscqi, parasitising the polychaete Scoloplos armiger. The attached trophozoites and gamonts of E. duboscqi were detected at different development stages. The parasite develops epicellularly, covered by a host cell-derived, two-membrane parasitophorous sac forming a caudal tipped appendage. Staining with Evans blue suggests that this tail is protein-rich, supported by the presence of a fibrous substance in this area. Despite the ultrastructural evidence for long filaments in the tail, it stained only weakly for F-actin, while spectrin seemed to accumulate in this area. The attachment apparatus consists of lobes arranged in one (trophozoites) or two (gamonts) circles, crowned by a ring of filamentous fascicles. During trophozoite maturation, the internal space between the parasitophorous sac and parasite turns translucent, the parasite trilaminar pellicle seems to reorganise and is covered by a dense fibrous glycocalyx. The parasite surface is organised in broad folds with grooves in between. Micropores are situated at the bottom of the grooves. A layer of filaments organised in bands, underlying the folds and ending above the attachment fascicles, was detected just beneath the pellicle. Confocal microscopy, along with the application of cytoskeletal drugs (jasplakinolide, cytochalasin D, oryzalin) confirmed the presence of actin and tubulin polymerised forms in both the parasitophorous sac and the parasite, while myosin labelling was restricted to the sac. Despite positive tubulin labelling, no microtubules were detected in mature stages. The attachment strategy of E. duboscqi shares features with that of cryptosporidia and gregarines, i.e. the parasite itself conspicuously resembles an epicellularly located gregarine, while the parasitophorous sac develops in a similar manner to that in cryptosporidia. This study provides a re-evaluation of epicellular development in other apicomplexans and directly compares their niche with that of E. duboscqi.