Ultrastructure and 28S rDNA Phylogeny of Two Gregarines: Cephaloidophora cf. communis and Heliospora cf. longissima with Remarks on Gregarine Morphology and Phylogenetic Analysis

18S 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. longissima gamonts are tricystid and uniseptate, similar to other eugregarines

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

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)

International audienceBackground: Gregarines 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.Methods Scanning 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 Discussion: We 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

Morphology of <i>Eleutheroschizon duboscqi</i> gamonts.

<p><b>A.</b> Attached gamont. SEM. <b>B.</b> Macrogamont with a large central nucleus. TEM. <b>C.</b> Microgamont with several nuclei. TEM. <b>D.</b> Macrogamont enclosed by host tissue. TEM, RR. <b>E.</b> The PS tail of the macrogamont shown in D. Note the pores and the mucosubstances present in their surroundings. TEM, RR. <b>F.</b> High magnification of the caudal PS part with the tail showing numerous pores. SEM. <b>G.</b> Detailed view of the tail and gamont caudal part. TEM, RR. <b>H.</b> Upper view of an individual with a ruptured PS. SEM. <b>I.</b> The caudal region of a naked individual. SEM. <b>J.</b> High magnification of the interface between the parasite and PS in the area of the tail. TEM, RR. <b>K.</b> Gamont with two tails at the PS. SEM. <i>a—</i>parasite amylopectin, <i>arrow</i>—PS, <i>asterisk</i>—space between the parasite and the PS, <i>black arrowhead—</i>parasite plasma membrane, <i>black double/paired arrowheads</i>—parasite cytomembranes, <i>c</i>—parasite cytoplasm, <i>db—</i>parasite dense bodies, <i>fa</i>—attachment fascicles, <i>g</i>—glycocalyx, <i>h—</i>host cell, <i>l</i>—attachment lobe, <i>ld—</i>parasite lipid droplets, <i>m</i>—parasite mitochondria, <i>mc</i>—host microcilia, <i>n—</i>parasite nucleus, <i>p</i>—parasite, <i>po</i>—pore, <i>s</i>—mucosubstances, <i>t</i>—tail of the PS, <i>v</i>—vesicles, <i>white arrowhead</i>—base of the PS.</p

Fine structure of <i>Eleutheroschizon duboscqi</i> mature trophozoites.

<p><b>A.</b> Mature trophozoite transforming into a gamont stage. TEM. <b>B.</b> Detailed view of the annular joint point and a well-developed fascicle of filaments. TEM. <b>C.</b> The view of mitochondria and a micropore (white circle) at the attachment site. The inset shows the micropore in detail. TEM. <b>D.</b> Higher magnification of the caudal region. TEM <b>E.</b> Two partially detached, mature trophozoites. SEM. <b>F.</b> The attachment site of a partially detached, mature trophozoite with well-developed fascicles and short filaments. SEM. <b>G.</b> Diagonal section of the apical part of a mature trophozoite. TEM. <b>H-I.</b> Craters left after detachment of mature trophozoites with well-developed attachment fascicles. Flat holes organised in one circle correspond to the developing lobes. SEM. <b>J.</b> A crater left after a trophozoite of more advanced stage as indicated by the presence of one circle of deep holes corresponding to well-developed lobes and one extra lobe starting the formation of a second circle. SEM. <i>a—</i>parasite amylopectin, <i>black arrow</i>—PS, <i>black arrowhead—</i>parasite plasma membrane, <i>black asterisk</i>—space between the parasite and PS, <i>black double/paired arrowheads</i>—parasite cytomembranes, <i>c</i>—parasite cytoplasm, <i>er</i>—parasite endoplasmic reticulum, <i>fa</i>—attachment fascicles, <i>fh—</i>holes in the host tissue left after the fascicles of the detached parasite, <i>fi—</i>short attachment filaments, <i>g</i>—glycocalyx, <i>h—</i>host cell, <i>l</i>—attachment lobe, <i>lh</i>—holes in the host tissue left after the lobes of the detached parasite, <i>m</i>—parasite mitochondria, <i>mv</i>—host microvilli, <i>n—</i>parasite nucleus, <i>p</i>—parasite, <i>sf—</i>parasite subpellicular filaments, <i>white arrow</i>—host cell plasma membrane, <i>white arrowhead</i>—dense band, <i>white asterisk</i>—empty attachment site, <i>white double arrowhead</i>—base of the PS.</p

Immunolocalisation of <i>Eleutheroschizon duboscqi</i> cytoskeletal proteins.

<p><b>A-B.</b> Actin labelling with a medium intensity in a trophozoite (PFA fixation). CLSM, IFA (A) and CLSM in a combination with transmission LM, IFA/DAPI (B). B represents a single median optical section. <b>C.</b> Actin labelling in a macrogamont treated with 30 μM JAS for 7 hours (PFA fixation). Note the increased accumulation of parasite actin (FITC) organised in longitudinal bands exhibiting strong fluorescence and strong F-actin (TRITC) labelling with a diffuse character. CLSM, IFA/phalloidin-TRITC. <b>D.</b> A gamont exhibiting a more diffuse actin (FITC) labelling of medium intensity after treatment with 10 μM cytochalasin D for 9 hours (PFA fixation). The F-actin (TRITC) labelling of the parasite did not change significantly. CLSM, IFA/phalloidin-TRITC. <b>E.</b> Very strong myosin (TRITC) labelling restricted to the PS and host tissue (PFA fixation). CLSM, IFA/DAPI. <b>F.</b> Strong spectrin (FITC) labelling of the PS in a macrogamont (PFA fixation). CLSM, IFA/DAPI. Single median optical section. <b>G.</b> Labelling of α-tubulin (FITC) of strong intensity in a young microgamont (PFA fixation). CLSM, IFA/DAPI. <b>H-I.</b> A trophozoite (fixed in ice-cold methanol) exhibiting a labelling of medium intensity for α-tubulin (FITC) and very strong intensity for myosin (TRITC). CLSM, IFA/DAPI. <b>J.</b> Labelling of α-tubulin (FITC) and myosin (TRITC) in an early trophozoite treated for 7 hours with 10 μM oryzalin (fixed in ice-cold methanol). The fluorescence signals for both antibodies did not change significantly. CLSM, IFA. <b>K-L.</b> Localisation of α-tubulin (FITC) and myosin (TRITC) in an individual (probably a young microgamont) treated with 30 μM oryzalin for 3 hours (fixed in ice-cold methanol). The fluorescence signal for tubulin became very weak, while it remained very strong for myosin. CLSM, IFA/DAPI. <b>M.</b> Co-localisation of α-tubulin (FITC) and F-actin (TRITC) in a macrogamont treated for 7 hours with 10 μM oryzalin (PFA fixation). CLSM, IFA/phalloidin-TRITC. <b>N-O.</b> Labelling of α-tubulin (FITC) and F-actin (TRITC) in a maturing trophozoite treated for 3 hours with 30 μM oryzalin (PFA fixation). CLSM, IFA/phalloidin-TRITC/DAPI. In both the preparations (M-O), there was almost no fluorescence signal for α-tubulin, while the F-actin labelled with a strong intensity. <i>arrow</i>—tail of the PS, <i>asterisk</i>—parasite attachment site, <i>black arrowhead</i>—PS, <i>h—</i>host tissue, <i>n—</i>parasite nucleus, <i>white arrowhead</i>—parasite pellicle.</p

Architecture of attachment site of <i>Eleutheroschizon duboscqi</i> gamonts.

<p><b>A.</b> Host intestinal tissue with a detached gamont revealing its attachment site at the base of PS. SEM. <b>B.</b> Detail of the gamont attachment site. SEM. <b>C.</b> Detailed view of the fascicles of long filaments alternating with short filaments, organised in ring. SEM. <b>D.</b> A detail of attachment fascicles. SEM. <b>E.</b> Host intestinal tissue with an attached parasite and a crater left after detached ones. SEM. <b>F.</b> A detail of crater left after gamont with well-developed attachment fascicles and two circles of lobes. SEM. <b>G.</b> Host epithelium showing the crater left after the parasite detached. TEM. <b>H.</b> A detail of the PS membrane remains covering the crater. TEM. <i>arrow</i>—PS, <i>fa</i>—attachment fascicle of filaments, <i>fh—</i>holes in the host tissue left after the fascicles of the detached parasite, <i>fi—</i>short attachment filaments, <i>l</i>—attachment lobe, <i>lh</i>—holes in the host tissue left after the lobes of the detached parasite, <i>mv</i>—microvilli and cilia of the host enterocyte, <i>p</i>—parasite, <i>white arrowhead</i>—dense band, <i>white asterisk</i>—empty attachment site, <i>white double arrowhead</i>—base of the PS.</p

Fluorescent visualisation of an <i>Eleutheroschizon duboscqi</i> parasitophorous sac.

<p><b>A.</b> Macrogamont stained with Evans blue. CLSM (lower) and CLSM in a combination with transmission LM (upper two). <b>B-E.</b> Trophozoites (B-D) and a gamont (E) stained with Evans blue. CLSM, output image not coloured. <b>F-H.</b> Localisation of F-actin in trophozoites. One circle of lobes is visible in the attachment site of the trophozoite shown in G. CLSM in a combination with transmission LM (F) and CLSM (G, H), phalloidin-TRITC/DAPI. <b>I.</b> F-actin labelling of a putative young microgamont with two primary nuclei. CLSM, phalloidin-TRITC/DAPI. <b>J.</b> F-actin in a microgamont with numerous nuclei. CLSM, phalloidin-TRITC/DAPI. <b>K-M.</b> F-actin in a putative macrogamont. CLSM in a combination with transmission LM (K) and CLSM (L, M), phalloidin-TRITC/DAPI. <b>N-P.</b> F-actin in a macrogamont equipped with attachment lobes organised in two circles. CLSM, phalloidin-TRITC. The intensity of signal for F-actin shown in F-P was strong for PS and medium for parasites. <b>Q.</b> Labelling of F-actin in an individual treated for 9 hours with 10 μM JAS showing the very strong labelling of PS. Individual optical sections also revealed a slightly increased F-actin labelling of the parasite. CLSM, phalloidin-TRITC/DAPI. <b>R.</b> Treatment with 30 μM JAS for 7 hours resulted in further increase of F-actin labelling in the PS, parasite and host tissue. The individual with several nuclei corresponds to the microgamont stage. CLSM, phalloidin-TRITC/DAPI. <b>S.</b> Visualisation of F-actin in an individual (putative young microgamont with two primary nuclei) treated for 9 hours with 10 μM cytochalasin D. Note the strong labelling of PS in contrast to the parasite and host tissue exhibiting only very weak signal. CLSM, phalloidin-TRITC/DAPI. <b>T.</b> Very weak F-actin labelling in a specimen treated for 7 hours with 30 μM cytochalasin D. CLSM, phalloidin-TRITC/DAPI. A-L, N-O, Q-T are composite views created by flattening a series of optical sections, while M and P represent single median optical sections. All samples were fixed in PFA. <i>arrow</i>—tail of the PS, <i>asterisk</i>—parasite attachment site, <i>black arrowhead</i>—PS, <i>h—</i>host tissue, <i>n—</i>parasite nucleus, <i>white arrowhead</i>—parasite pellicle.</p

Fine structure of a parasitophorous sac and pellicle in <i>Eleutheroschizon duboscqi</i> gamonts.

<p><b>A.</b> Attached gamonts. SEM. <b>B.</b> Cross-sectioned gamont showing its surface organised in 12 broad folds and shallow grooves corresponding to the regularly arranged interruptions of subpellicular filaments. TEM. <b>C.</b> Longitudinal section showing the organisation of PS, gamont pellicle and the subpellicular layer of filaments that is repeatedly interrupted in areas corresponding to the localisation of micropores. TEM, RR. <b>D.</b> Superficial section of the PS and the gamont pellicle. The channel-like structures located in the space between the PS and parasite correspond to the folding of the PS observed under SEM. TEM, RR. <b>E.</b> Tangential section of the gamont surface underlined with subpellicular layer of filaments. TEM, RR. <b>F.</b> Diagonal section of the gamont surface revealing mitochondria connected with micropores. TEM, RR. <b>G.</b> Cross-section of pellicle showing the subpellicular filaments interrupted in the micropore area. TEM, RR. <b>H.</b> Almost longitudinal section of pellicle with interrupted subpellicular filaments. TEM, RR. <b>I.</b> Pellicle with continuous cytomembranes. TEM. <b>J-K.</b> Re-building of the parasite IMC indicated by the discontinuous cytomembranes and numerous vesicles located between the parasite plasma membrane and the subpellicular layer of the filaments. TEM, RR. <i>a—</i>parasite amylopectin, <i>arrow</i>—PS, <i>asterisk</i>—space between the parasite and PS, <i>black arrowhead—</i>parasite plasma membrane, <i>black double/paired arrowheads</i>—parasite cytomembranes, <i>db—</i>parasite dense bodies, <i>er</i>—parasite endoplasmic reticulum, <i>g</i>—glycocalyx, <i>h—</i>host tissue, <i>ld</i>—parasite lipid droplet, <i>m</i>—parasite mitochondria, <i>p</i>—parasite, <i>sf—</i>parasite subpellicular filaments, <i>v—</i>vesicles, <i>white arrowheads</i>—channel-like structures. Micropores are indicated by white circles, interruptions of subpellicular filaments—by white asterisks.</p