Epicytic Folds Research Articles

Evidence for Host Jumping and Diversification of Marine Cephaloidophorid Gregarines (Apicomplexa) Between Two Distantly Related Animals, viz., Crustaceans and Salps

This study examined the evolutionary history and diversity of marine gregarine parasites of pelagic zooplankton, and highlighted a unique example of a host-jumping event of cephaloidophorid gregarines between two distantly related host groups, crustaceans and chordates. Candacia bipinnata Giesbrecht, 1889, a free-living calanoid copepod, and a salp, Salpa fusiformis Cuvier, 1804, were collected on oceanic research cruises in 2018 and 2019, in the West Pacific aboard TRV SEISUI MARU and TOYOSHIO MARU, respectively. A molecular phylogeny based on 18S rDNA nested the gregarine parasite from S. fusiformis among cephaloidophorids, within a clade exclusively comprised of gregarines from crustaceans. The relationship between these groups was underpinned with ultrastructural data including the presence of a septum, and similarities in the apices of the epicytic folds. Subsequently, it was concluded to establish a new combination, Cephaloidophora cf. flava n. comb (Ex. Thalicola flava) and transfer the other two members of the Thalicola (also parasites of salps) to the Cephaloidophora. This study also attempted to ascertain the origin of cephaloidophorids in S. fusiformis. However, the relationship between Cephaloidophora bipinnatae n. sp., and C. cf. flava n. comb. had only modest support.

Molecular Phylogeny of Marine Gregarines (Apicomplexa) from the Sea of Japan and the Northwest Pacific Including the Description of Three Novel Species of Selenidium and Trollidium akkeshiense n. gen. n. sp.

This study set out to bolster morphological and molecular datasets of marine gregarine apicomplexans. Gregarines were sampled from the Sea of Japan and Northwest Pacific from cirratuliform polychaetes (Acrocirridae, Cirratulidae, and Flabelligeridae), as well as sipunculids. Trophozoites (feeding stages) were gathered for identification using light microscopy, scanning electron microscopy, and transmission electron microscopy. Cells were also collected for molecular phylogenetic analysis using 18S rDNA and 28S rDNA. As a result, three new species of Selenidium, S. planusae n. sp., S. validusae n. sp., and S. pyroidea n. sp. were described, and additional morphological and genetic data were gathered for an existing species, S. orientale; and Trollidium was established as a new genus. Trollidium akkeshiense n. gen. n. sp. possessed a unique, unsymmetrical organization of microtubules running the longitudinal length of one side of the trophozoite, corresponding to a zig-zag pattern of epicytic (surface) folds, and a flicking pattern of movement. Phylogenetic analyses of 18S rDNA and 28S rDNA showed that these portions of the ribosomal operon are able to resolve some relationships among Selenidium, while other lineages including Trollidium akkeshiense n. gen. n. sp. appeared to be highly influenced by long branch attraction. High evolutionary rates along the ribosomal operon of gregarines may hinder this marker from resolving deeper nodes among early apicomplexans.

Gregarines (Apicomplexa, Gregarinasina) in psocids (Insecta, Psocoptera) including a new species description and their potential use as pest control agents

Gregarine apicomplexans are unicellular organisms that infect invertebrate hosts in marine, freshwater and terrestrial habitats. The largest group of invertebrates infested on land is the insects. The insect order Psocoptera (booklice) has recently gained wider interest due to specimens occurring in stored food products and therefore being considered pest organisms. Biological control agents are often used to eliminate pest organisms. In this study we examined the psocid Dorypteryx domestica, an invasive psocid species that is spreading all over the world. We were able to isolate and describe a new gregarine species (Enterocystis dorypterygis sp. n.) infecting D. domestica. The trophozoites are panduri- or pyriform and their association/syzygy is caudo-frontal. The surface is inscribed by longitudinal epicytic folds covering the complete cell. Phylogenetic analyses of the SSU rDNA gene revealed an only weakly supported relationship with two Gregarina species G. ormieri and G. basiconstrictonea, both from tenebrionid beetles. Gregarines have been proposed to have some potential as biological control agents for several insects. Identifying the gregarine species infecting pest organisms like psocids is a first step and prerequisite for the probable utilization of these parasites as biological control agents in the future.

A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarine Ancora sagittata (Leuckart, 1860) Labbé, 1899 (Apicomplexa: Eugregarinida).

BackgroundGregarines are a group of early branching Apicomplexa parasitizing invertebrate animals. Despite their wide distribution and relevance to the understanding the phylogenesis of apicomplexans, gregarines remain understudied: light microscopy data are insufficient for classification, and electron microscopy and molecular data are fragmentary and overlap only partially.MethodsScanning and transmission electron microscopy, PCR, DNA cloning and sequencing (Sanger and NGS), molecular phylogenetic analyses using ribosomal RNA genes (18S (SSU), 5.8S, and 28S (LSU) ribosomal DNAs (rDNAs)).Results and DiscussionWe present the results of an ultrastructural and molecular phylogenetic study on the marine gregarine Ancora sagittata from the polychaete Capitella capitata followed by evolutionary and taxonomic synthesis of the morphological and molecular phylogenetic evidence on eugregarines. The ultrastructure of Ancora sagittata generally corresponds to that of other eugregarines, but reveals some differences in epicytic folds (crests) and attachment apparatus to gregarines in the family Lecudinidae, where Ancora sagittata has been classified. Molecular phylogenetic trees based on SSU (18S) rDNA reveal several robust clades (superfamilies) of eugregarines, including Ancoroidea superfam. nov., which comprises two families (Ancoridae fam. nov. and Polyplicariidae) and branches separately from the Lecudinidae; thus, all representatives of Ancoroidea are here officially removed from the Lecudinidae. Analysis of sequence data also points to possible cryptic species within Ancora sagittata and the inclusion of numerous environmental sequences from anoxic habitats within the Ancoroidea. LSU (28S) rDNA phylogenies, unlike the analysis of SSU rDNA alone, recover a well-supported monophyly of the gregarines involved (eugregarines), although this conclusion is currently limited by sparse taxon sampling and the presence of fast-evolving sequences in some species. Comparative morphological analyses of gregarine teguments and attachment organelles lead us to revise their terminology. The terms “longitudinal folds” and “mucron” are restricted to archigregarines, whereas the terms “epicystic crests” and “epimerite” are proposed to describe the candidate synapomorphies of eugregarines, which, consequently, are considered as a monophyletic group. Abolishing the suborders Aseptata and Septata, incorporating neogregarines into the Eugregarinida, and treating the major molecular phylogenetic lineages of eugregarines as superfamilies appear as the best way of reconciling recent morphological and molecular evidence. Accordingly, the diagnosis of the order Eugregarinida Léger, 1900 is updated.

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).

Morphology and molecular phylogeny of the marine gregarine parasite Selenidium oshoroense n. sp. (Gregarina, Apicomplexa) isolated from a Northwest Pacific Hydroides ezoensis Okuda 1934 (Serpulidae, Polychaeta)

In this study, we describe a novel marine gregarine parasite, Selenidium oshoroense n. sp., isolated from Hydroides ezoensis Okuda 1934 (Serpulidae, Polychaeta). Trophozoites (feeding stages) of S. oshoroense n. sp. were isolated from the gut of H. ezoensis collected from the intertidal shore near Oshoro, Hokkaido, Japan, and prepared for analysis with scanning and transmission electron microscopy (SEM and TEM), and molecular phylogenetic analysis using 18S rDNA. Trophozoites of S. oshoroense n. sp. were, on average, 120 μm long and 13 μm wide. Observation of the cells under SEM and TEM revealed 23 longitudinally running epicytic folds on the surface of the cells. Peduncles, multimembrane-bound whorls, and inclusions were also observed and were indicative of surface-mediated nutrition. Myzocytotic feeding at the apical end of the cell was also indicated in sections of host gut tissue that were viewed under TEM, suggesting that myzocytosis could be a shared feature among this clade of Selenidium from tube-forming polychaetes. Molecular phylogenetic analysis of the 18S rDNA grouped S. oshoroense n. sp. within a clade of Selenidium from tube-forming polychaetes, sister to S. serpulae. The two 18S rDNA sequences generated from separate isolates of S. oshoroense n. sp. had a similarity of 0.997, but were 0.965 and 0.966 similar to S. serpulae. S. oshoroense n. sp. was differentiated from, S. serpulae, based on the absence of transverse striations on the surface of the epicytic folds and difference in ecological niche (host). Morphological differences were supported in phylogenetic analysis which grouped S. oshoroense n. sp. isolates, to the exclusion of S. serpulae.

Morphology and Molecular Phylogeny of Coelomic Gregarines (Apicomplexa) with Different Types of Motility: Urospora ovalis and U. travisiae from the Polychaete Travisia forbesii

Urosporids (Apicomplexa: Urosporidae) are eugregarines that parasitise marine invertebrates, such as annelids, molluscs, nemerteans and echinoderms, inhabiting their coelom and intestine. Urosporids exhibit considerable morphological plasticity, which correlates with their different modes of motility and variations in structure of their cortical zone, according to the localisation within the host. The gregarines Urospora ovalis and U. travisiae from the marine polychaete Travisia forbesii were investigated with an emphasis on their general morphology and phylogenetic position. Solitary ovoid trophozoites and syzygies of U. ovalis were located free in the host coelom and showed metabolic activity, a non-progressive movement with periodic changes of the cell shape. Solitary trophozoites of U. travisiae, attached to the host tissue or free floating in the coelom, were V-shaped. Detached trophozoites demonstrated gliding motility, a progressive movement without observable cell body changes. In both gregarines, the cortex formed numerous epicytic folds, but superfolds appeared exclusively on the surface of U. ovalis during metabolic activity. SSU rDNA sequences obtained from U. ovalis and U. travisiae revealed that they belong to the Lecudinoidea clade; however, they are not affiliated with other coelomic urosporids (Pterospora spp. and Lithocystis spp.), but surprisingly with intestinal lecudinids (Difficilina spp.) parasitising nemerteans.

Thiriotia euchirellae n. sp., a new gregarine species (Apicomplexa: Eugregarinorida) from the mesopelagic copepod Euchirella rostrata in Sagami Bay, Japan

A new gregarine species, Thiriotia euchirellae n. sp. (Apicomplexa: Eugregarinorida), is described from the gut lumen of a mesopelagic copepod, Euchirella rostrata (Calanoida: Aetideidae), collected in Sagami Bay, Japan. Thiriotia euchirellae n. sp. has an elongated and cylindrical body with an average length of 174 μm, and is distinguished from its congeners by its shorter and thicker body, the rounded anterior end with thicker folds than for the rest of the body, and the posterior end covered by epicytic folds. Molecular phylogenetic analysis based on nuclear 18S rDNA placed T. euchirellae n. sp. in the clade of eugregarines infecting marine crustaceans, and in the same subclade as T. pugettiae. Thiriotia euchirellae n. sp. infected 95 % of the E. rostrata population, but was not detected in the other mesopelagic copepod species examined in the present study. Euchirella rostrata has an elongated gut with a swollen and sigmoidal anterior part and a gradually narrowing posterior part connected through a narrow path. Thiriotia euchirellae n. sp. were mostly observed in the posterior part of the gut. The high infection rate of E. rostrata by T. euchirellae n. sp. and the characteristic gut morphology of the host suggest a specific host–parasite relationship between these species.

Ultrastructure and 28S rDNA phylogeny of two gregarines: Cephaloidophora cf. communis and Heliospora cf. longissima with remarks on gregarine morphology and phylogenetic analysis

S rRNA gene sequences (SSU rDNA) in gregarines are problematic for phylogenetic analysis, mainly due to artifacts related to long branch attraction (LBA). In this study, we sequenced 18S rRNA (SSU rRNA), 5.8S rRNA, and 28S rRNA (LSU rRNA) genes of two gregarine species from crustacean hosts (gregarine superfamily Cephaloidophoroidea): Cephaloidophora cf. communis from a marine cirripedian Balanus balanus (White Sea), and Heliospora cf. longissima from the freshwater amphipods, Eulimnogammarus verrucosus and E. vittatus (Lake Baikal). Phylogenetic analyses of SSU rDNA sequences failed to produce a robust tree topology, for a limited taxon sample (31 operational taxonomic units (OTU), based on 1,604 sites), while LSU (2,869 sites), and concatenated dataset based on SSU, 5.8S, and LSU (4,627 sites) produced more consistent tree topologies for the same taxon sample. Analyses testing for LBA-influence were negative, therefore we suggested that the main reason of the failed topologies in SSU rDNA analyses is insufficient data (insufficient taxon sampling and limited molecular data), rather than LBA. Possible advantages of Bayesian analyses, compared to Maximum Likelihood, and usage of LSU rDNA within the context of apicomplexan phylogenetics were discussed. One of the advantages of LSU is likely its lower rate of evolution in long-branching apicomplexans (e.g., gregarines), relative to other (non-long-branching) apicomplexans, compared to SSU rDNA. Ultrastructure of the epicytic folds was studied. There are 3 to 5 apical arcs (also known as rippled dense structures) and 2 to 5 apical filaments in the tops of the folds. This small number of the apical structures fits into morphological diversity of the epicyte in other Cephaloidophoroidea, but this is not a synapomorphy of the group because this was also detected in several unrelated gregarines. C. cf. communis was found to contain a septum between the epimerite and the protomerite, which has not been reported in other gregarines. More exact terminology, which takes into account number of body sections and septa, is proposed for morphological descriptions of trophozoites and free mature gamonts of gregarines. In accordance with this, C. cf. communis gamonts are tricystid and biseptate, whereas H. cf. longis­ sima gamonts are tricystid and uniseptate, similar to other eugregarines.

Molecular systematics of marine gregarine apicomplexans from Pacific tunicates, with descriptions of five novel species of Lankesteria.

The eugregarines are a group of apicomplexan parasites that mostly infect the intestines of invertebrates. The high level of morphological variation found within and among species of eugregarines makes it difficult to find consistent and reliable traits that unite even closely related lineages. Based mostly on traits observed with light microscopy, the majority of described eugregarines from marine invertebrates has been classified into a single group, the Lecudinidae. Our understanding of the overall diversity and phylogenetic relationships of lecudinids is very poor, mainly because only a modest amount of exploratory research has been done on the group and very few species of lecudinids have been characterized at the molecular phylogenetic level. In an attempt to understand the diversity of marine gregarines better, we surveyed lecudinids that infect the intestines of Pacific ascidians (i.e. sea squirts) using ultrastructural and molecular phylogenetic approaches; currently, these species fall within one genus, Lankesteria. We collected lecudinid gregarines from six ascidian host species, and our data demonstrated that each host was infected by a different species of Lankesteria: (i) Lankesteria hesperidiiformis sp. nov., isolated from Distaplia occidentalis, (ii) Lankesteria metandrocarpae sp. nov., isolated from Metandrocarpa taylori, (iii) Lankesteria halocynthiae sp. nov., isolated from Halocynthia aurantium, (iv) Lankesteria herdmaniae sp. nov., isolated from Herdmania momus, (v) Lankesteria cf. ritterellae, isolated from Ritterella rubra, and (vi) Lankesteria didemni sp. nov., isolated from Didemnum vexillum. Visualization of the trophozoites with scanning electron microscopy showed that four of these species were covered with epicytic folds, whereas two of the species were covered with a dense pattern of epicytic knobs. The molecular phylogenetic data suggested that species of Lankesteria with surface knobs form a clade that is nested within a paraphyletic assemblage species of Lankesteria with epicytic folds.

Comparative Ultrastructure and Molecular Phylogeny of Selenidium melongena n. sp. and S. terebellae Ray 1930 Demonstrate Niche Partitioning in Marine Gregarine Parasites (Apicomplexa)

Gregarine apicomplexans are a diverse group of single-celled parasites that have feeding stages (trophozoites) and gamonts that generally inhabit the extracellular spaces of invertebrate hosts living in marine, freshwater, and terrestrial environments. Inferences about the evolutionary morphology of gregarine apicomplexans are being incrementally refined by molecular phylogenetic data, which suggest that several traits associated with the feeding cells of gregarines arose by convergent evolution. The study reported here supports these inferences by showing how molecular data reveals traits that are phylogenetically misleading within the context of comparative morphology alone. We examined the ultrastructure and molecular phylogenetic positions of two gregarine species isolated from the spaghetti worm Thelepus japonicus: Selenidium terebellaeRay 1930 and S. melongena n. sp. The ultrastructural traits of S. terebellae were very similar to other species of Selenidium sensu stricto, such as having vermiform trophozoites with an apical complex, few epicytic folds, and a dense array of microtubules underlying the trilayered pellicle. By contrast, S. melongena n. sp. lacked a comparably discrete assembly of subpellicular microtubules, instead employing a system of fibrils beneath the cell surface that supported a relatively dense array of helically arranged epicytic folds. Molecular phylogenetic analyses of small subunit rDNA sequences derived from single-cell PCR unexpectedly demonstrated that these two gregarines are close sister species. The ultrastructural differences between these two species were consistent with the fact that S. terebellae infects the inner lining of the host intestines, and S. melongena n. sp. primarily inhabits the coelom, infecting the outside wall of the host intestine. Altogether, these data demonstrate a compelling case of niche partitioning and associated morphological divergence in marine gregarine apicomplexans.

Discovery of a Diverse Clade of Gregarine Apicomplexans (Apicomplexa: Eugregarinorida) from Pacific Eunicid and Onuphid Polychaetes, Including Descriptions of Paralecudina n. gen., Trichotokara japonica n. sp., and T. eunicae n. sp.

Marine gregarines are poorly understood apicomplexan parasites with large trophozoites that inhabit the body cavities of marine invertebrates. Two novel species of gregarines were discovered in polychaete hosts collected in Canada and Japan. The trophozoites of Trichotokara japonica n. sp. were oval to rhomboidal shaped, and covered with longitudinal epicytic folds with a density of six to eight folds/micron. The nucleus was situated in the middle of the cell, and the mucron was elongated and covered with hair-like projections; antler-like projections also extended from the anterior tip of the mucron. The distinctively large trophozoites of Trichotokara eunicae n. sp. lacked an elongated mucron and had a tadpole-like cell shape consisting of a bulbous anterior region and a tapered tail-like posterior region. The cell surface was covered with longitudinal epicytic folds with a density of three to five folds/micron. Small subunit (SSU) rDNA sequences of both species were very divergent and formed a strongly supported clade with the recently described species Trichotokara nothriae and an environmental sequence (AB275074). This phylogenetic context combined with the morphological features of T. eunicae n. sp. required us to amend the description for Trichotokara. The sister clade to the Trichotokara clade consisted of environmental sequences and Lecudina polymorpha, which also possesses densely packed epicyctic folds (3-5 folds/micron) and a prominently elongated mucron. This improved morphological and molecular phylogenetic context justified the establishment of Paralecudina (ex. Lecudina) polymorpha n. gen. et comb.

The enigma of eugregarine epicytic folds: where gliding motility originates?

BackgroundIn the past decades, many studies focused on the cell motility of apicomplexan invasive stages as they represent a potential target for chemotherapeutic intervention. Gregarines (Conoidasida, Gregarinasina) are a heterogeneous group that parasitize invertebrates and urochordates, and are thought to be an early branching lineage of Apicomplexa. As characteristic of apicomplexan zoites, gregarines are covered by a complicated pellicle, consisting of the plasma membrane and the closely apposed inner membrane complex, which is associated with a number of cytoskeletal elements. The cell cortex of eugregarines, the epicyte, is more complicated than that of other apicomplexans, as it forms various superficial structures.ResultsThe epicyte of the eugregarines, Gregarina cuneata, G. polymorpha and G. steini, analysed in the present study is organised in longitudinal folds covering the entire cell. In mature trophozoites and gamonts, each epicytic fold exhibits similar ectoplasmic structures and is built up from the plasma membrane, inner membrane complex, 12-nm filaments, rippled dense structures and basal lamina. In addition, rib-like myonemes and an ectoplasmic network are frequently observed. Under experimental conditions, eugregarines showed varied speeds and paths of simple linear gliding. In all three species, actin and myosin were associated with the pellicle, and this actomyosin complex appeared to be restricted to the lateral parts of the epicytic folds. Treatment of living gamonts with jasplakinolide and cytochalasin D confirmed that actin actively participates in gregarine gliding. Contributions to gliding of specific subcellular components are discussed.ConclusionsCell motility in gregarines and other apicomplexans share features in common, i.e. a three-layered pellicle, an actomyosin complex, and the polymerisation of actin during gliding. Although the general architecture and supramolecular organisation of the pellicle is not correlated with gliding rates of eugregarines, an increase in cytoplasmic mucus concentration is correlated. Furthermore, our data suggest that gregarines utilize several mechanisms of cell motility and that this is influenced by environmental conditions.

Description of Trichotokara nothriae n. gen. et sp. (Apicomplexa, Lecudinidae) – An intestinal gregarine of Nothria conchylega (Polychaeta, Onuphidae)

The trophozoites of a novel gregarine apicomplexan, Trichotokara nothriae n. gen. et sp., were isolated and characterized from the intestines of the onuphid tubeworm Nothria conchylega (Polychaeta), collected at 20 m depth from the North-eastern Pacific Coast. The trophozoites were 50–155 μm long with a mid-cell indentation that formed two prominent bulges (anterior bulge, 14–48 μm wide; posterior bulge, 15–55 μm wide). Scanning electron microscopy (SEM) demonstrated that approximately 400 densely packed, longitudinal epicytic folds (5 folds/μm) inscribe the surface of the trophozoites, and a prominently elongated mucron (14–60 μm long and 6–12 μm wide) was covered with hair-like projections (mean length, 1.97 μm; mean width, 0.2 μm at the base). Although a septum occurred at the junction between the cell proper and the mucron in most trophozoites, light microscopy (LM) demonstrated that the cell proper extended into the core of the mucron as a thin prolongation. A spherical nucleus (8–20 μm) was situated in the middle of the trophozoites, and gamonts underwent caudal syzygy. The small subunit (SSU) rDNA sequence and molecular phylogenetic position of T. nothriae was also characterized. The sequence from this species was the most divergent of all SSU rDNA sequences currently known from gregarines and formed a weakly supported clade with Lecudina polymorpha, which also possesses densely packed epicyctic folds (3–5 folds/μm) and a prominently elongated mucron.

Molecular systematics of marine gregarines (Apicomplexa) from North-eastern Pacific polychaetes and nemerteans, with descriptions of three novel species: Lecudina phyllochaetopteri sp. nov., Difficilina tubulani sp. nov. and Difficilina paranemertis sp. nov.

Most eugregarine apicomplexans infecting the intestines of marine invertebrates have been described within the family Lecudinidae and the type genus Lecudina. The diversity of these parasites is vast and poorly understood and only a tiny number of species has been characterized at the molecular phylogenetic level. DNA sequences coupled with high-resolution micrographs of trophozoites provide an efficient and precise approach for delimiting gregarine lineages from one another and also facilitate our overall understanding of gregarine biodiversity. In this study, phylogenetic analyses of small subunit (SSU) rDNA sequences from five (uncultivated) gregarines isolated from polychaetes and nemerteans in the North-eastern Pacific Ocean are presented. Lecudina phyllochaetopteri sp. nov. was isolated from the intestines of the parchment tubeworm Phyllochaetopterus prolifica (Polychaeta). Lecudina longissima and Lecudina polymorpha were both isolated from the intestines of Lumbrineris japonica (Polychaeta). Difficilina tubulani sp. nov. was isolated from the nemertean Tubulanus polymorpha and Difficilina paranemertis sp. nov. was isolated from the nemertean Paranemertes peregrina. This is the first report of molecular sequence data from gregarines that infect nemerteans. The two novel species of the genus Difficilina described in this study formed a strongly supported clade in the phylogenetic analyses. This Difficilina clade formed the sister group to a robust subclade of lecudinids consisting of Lecudina longissima, Lecudina phyllochaetopteri sp. nov. (which lacked epicytic folds), Lecudina tuzetae, species of the genus Lankesteria and several sequences derived from previous environmental DNA surveys of marine biodiversity.

Molecular Phylogeny and Surface Morphology of Marine Archigregarines (Apicomplexa), Selenidium spp., Filipodium phascolosomae n. sp., and Platyproteum n. g. and comb. from North‐Eastern Pacific Peanut Worms (Sipuncula)

The trophozoites of two novel archigregarines, Selenidium pisinnus n. sp. and Filipodium phascolosomae n. sp., were described from the sipunculid Phascolosoma agassizii. The trophozoites of S. pisinnus n. sp. were relatively small (64-100 microm long and 9-25 microm wide), had rounded ends, and had about 21 epicytic folds per side. The trophozoites of F. phascolosomae n. sp. were highly irregular in shape and possessed hair-like surface projections. The trophozoites of this species were 85-142 microm long and 40-72 microm wide and possessed a distinct longitudinal ridge that extended from the mucron to the posterior end of the cell. In addition to the small subunit (SSU) rDNA sequences of these two species, we also characterized the surface morphology and SSU rDNA sequence of Selenidium orientale, isolated from the sipunculid Themiste pyroides. Molecular phylogenetic analyses demonstrated that S. pisinnus n. sp. and S. orientale formed a strongly supported clade within other Selenidium and archigregarine-like environmental sequences. Filipodium phascolosomae n. sp. formed the nearest sister lineage to the dynamic, tape-like gregarine Selenidium vivax. Overall, these data enabled us to reassess the molecular systematics of archigregarines within sipunculid hosts and make the following revisions: (1) Filipodium was transferred from the Lecudinidae (eugregarines) to the Selenidiidae (archigregarines), and (2) Platyproteum n. g. was established for Platyproteum vivax n. comb. (ex. S. vivax) in order to account for the highly divergent morphological features and better resolved phylogenetic position of this lineage.

ULTRASTRUCTURE OF THE GREGARINA NIPHANDRODES NUCLEUS THROUGH STAGES FROM UNASSOCIATED TROPHOZOITES TO GAMONTS IN SYZYGY AND THE SYZYGY JUNCTION

In Gregarina niphandrodes, an apicomplexan parasite, the sexual stage of its life cycle begins with the association of 2 gamonts. Here, we describe the ultrastructure of the syzygy junction and the nucleus during the transition from unassociated trophozoites to paired gamonts to gamonts in syzygy. Throughout this process, the folds within the syzygy junction undergo changes that correspond to changes of the epicytic folds. The nucleus goes through dramatic changes from multiple spheres of condensed chromatin in unassociated trophozoites, to mostly uncondensed chromatin in paired gamonts, to a large single sphere of condensed chromatin encasing many smaller spheres in gamonts in syzygy. These differing nuclear ultrastructures reflect the dramatic cellular and transcriptional changes associated with life cycle transitions and are indicative of the numerous cell divisions that follow.

Actin and myosin in Gregarina polymorpha.

Actin and two class XIV unconventional myosins have been cloned from Gregarina polymorpha, a large protozoan parasite inhabiting the gut of the mealworm Tenebrio molitor. These proteins were most similar to their homologues expressed in the coccidian and haemosporidian Apicomplexa such as Toxoplasma and Plasmodium despite the significant morphological differences among these parasites. Both actin and G. polymorpha myosin A (GpMyoA), a 92.6-kDa protein characterized by a canonical myosin head domain and short, highly basic tail, localized to both the longitudinally-disposed surface membrane folds (epicytic folds) of the parasite as well as to the subjacent rib-like myonemes that gird the parasite cortex. G. polymorpha myosin B (GpMyoB), a 96.3-kDa myosin, localized exclusively to the epicytic folds of the parasite. Both myosins were tightly associated with the cortical cytoskeleton and were solubilized only with a combination of high salt and detergent. Both GpMyoA and GpMyoB could bind to actin in an ATP-sensitive fashion. The distribution of actin and the unconventional myosins in G. polymorpha was consistent with their proposed participation in both the rapid (1-10 microm/sec) gliding motility exhibited by the gregarines as well as the myoneme-mediated bending motions that have been observed in these parasites.

Epimerite-host epithelium relationships among eugregarines parasitizing the damselflies Enallagma civile and Ischnura verticalis.

The host-parasite interface between 2 species of damselflies and 4 species of eugregarines was examined at the ultrastructural level. Nubenocephalus nebraskensis organisms attached to the host midgut epithelium by means of a sucker-like protomerite; the space between the epicytic folds and host epithelium was filled with electron-dense material interpreted to be adhesive in nature. Actinocephalus carrilynnae organisms attached by means of the epimerite, which had no epicytic folds, and by the fluted stalk with characteristic epicytic folds: host cell and parasite membranes appeared fused at some places on the epimerite. Hoplorhynchus acanthatholius organisms attached by means of an ovoid epimerite with backward-pointing digitations; the entire epimerite was embedded in a host cell, and host cell microvilli surrounded the stalk. Steganorhynchus dunwoodyi organisms attached by means of an ovoid stalk papilla enclosed in a retractable globular sheath; the papilla was covered with epicytic folds, but the sheath was not, and the sheath had a single membrane, whereas the epicytic folds had 2 or 3 membranes. The entire apparatus was inserted between epithelial cells, and the sheath was highly folded at its surface. The ultrastructural observations suggest that actinocephalid gregarines have evolved 2 general strategies for attaching to the host epithelium, that is, suckerlike protomerites, as in the case of N. nebraskensis, and deeply embedded epimerites inserted within or between host cells, as in the other species studied.

Evidence for septate junctions in the syzygy of the protozoon Gregarina polymorpha (Protozoa, Apicomplexa)

ABSTRACT A septate junction is described in reproductive pairs of the protozoon Gregarina polymorpha, using conventional thin sections, lanthanum tracer and freeze-fracture techniques. The septate junction is established between the plasma membranes at the tips of the joined epicytic folds. It is characterized by an intercellular space of 14–17 nm traversed by septa with a repeat of 15–25 nm. Lanthanum-treated material exhibits transparent curves forming a meshwork. Freeze fracture replicas show membrane modifications in the shape of short rows of intramembranous particles on the E fracture face of the plasma membrane. The significance of the finding of such a septate junction between protozoan cells is discussed.