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)

Abstract

Background: 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.

Keywords: Apicomplexa; Environmental DNA sequences; Marine gregarines; Phylogeny; SSU and LSU rDNA; Taxonomy; Ultrastructure.

Figure 1. Strategy used to obtain contigs of the ribosomal operon from the samples of A. sagittata Roscoff 2009 and A. sagittata WSBS 2006.

Upper part: schematic ribosomal operon with approximate positions of the forward and reverse primers. Lower part: the amplified fragments of ribosomal DNA aligned with the ribosomal operon (above). Numbers indicate the length of the overlapping regions. Roman numerals denote the amplified fragments.

Figure 2. Light (A) and scanning electron microscopy (B–G) of Ancora sagittata.

(A and B) General view of the gregarine; (C) Epicyte; (D) View of the gregarine from the apical pole of the cell; (E) Epicytic folds at the base of the lateral projections (lp); (F, G) Apical pole of Ancora sagittata (arrows) with (F) and without the apical papilla (G). lp, lateral projections of the cell.

Figure 3. Transmission electron microscopy of Ancora sagittata.

(A–C) Cross sections in the middle of the cell show epicytic folds (ef) with fibrils (f) inside, internal lamina (il), circular cortical filaments (cf), and granules of amylopectin (ap); (D) Cross section through the top of the epicytic fold reveals a structure of the pellicle consisting of the plasma membrane (pm) and the internal membrane complex (imc) with rippled dense structures (= apical arcs, aa) and an electron dense plate (arrow); (E) Cross section through the epicytic folds of the frontal zone of the cell with electron dense globules inside the folds; (F) Cross section at the level of the lateral projections: a micropore (mp) and circular cortical filaments (cf) are visible; (G) Tangential section of the cortex in the posterior region of the trophozoite reveals circular cortical filaments (cf).

Figure 4. Transmission electron microscopy of the attachment apparatus of Ancora sagittata.

(A) Longitudinal section of the gregarine forebody embedded in a host cell shows a large frontal vacuole (fv) and amylopectin granules (ap) within the attachment organelle and the main part of the cell; the black arrows indicate the base of the contact zone (circular groove, see B); (B) Longitudinal section through the base of the contact zone between the gregarine and host cell under a higher magnification: gregarine cell forms a circular groove (black arrow) pinching the host cell; the rear wall of the frontal vacuole (double arrow) arises from this area; parallel filaments (fi) arise from the groove zone backward; the white arrow indicates the terminus of the internal membrane complex (imc) of the pellicle; pm is the plasma membrane of the gregarine cell.

Figure 5. Diagram of the contact between the gregarine and the host cell as inferred from TEM micrographs.

Abbreviations are the same as in Fig. 4.

Figure 6. Predicted secondary structures of ITS2 transcripts of two Ancora sagittata ribotypes demonstrating differences between them.

(A) Ribotype 1; (B) Ribotype 2. Nucleotide substitutions and insertions in the ribotype 2 are highlighted in gray. Nucleotides involved in compensatory base changes are encircled.

Figure 7. Bayesian inference tree of alveolates calculated by using the GTR+Г+I model from the dataset of 114 SSU rDNA sequences (1,570 sites).

Numbers at the nodes indicate Bayesian posterior probabilities/ML bootstrap percentage. Black dots on the branches indicate Bayesian posterior probabilities and bootstrap percentages of at least 0.95% and 95%, respectively. The newly obtained sequences of Ancora sagittata are highlighted in by a black rectangle. Asterisks indicate aseptate gregarines within the “septate” clade; arrows indicate neogregarines.

Figure 8. Bayesian inference tree of Ancora sagittata and related sequences obtained by using the GTR+Г+I model from the dataset of 52 SSU rDNA sequences (1,709 sites).

Numbers at the nodes indicate Bayesian posterior probabilities/ML bootstrap percentage. Black dots on the branches indicate Bayesian posterior probabilities and bootstrap percentages of at least 0.95% and 95%, respectively. The newly obtained sequences of Ancora sagittata are highlighted by black rectangles. Black triangles indicate clusters of near-identical sequences (identity of 99% or more), each of which was represented by a single representative.

Figure 9. Bayesian inference trees of the alveolates obtained by using the GTR+Г+I model and 50 sequences.

(A) LSU rDNA dataset (2,911 sites); (B) Ribosomal operon dataset (4,636 sites). Numbers at the nodes indicate Bayesian posterior probabilities/ML bootstrap percentages. Black dots on the branches indicate Bayesian posterior probabilities and bootstrap percentages of at least 95% and 95%, respectively. The newly obtained sequences of Ancora sagittata are highlighted by black rectangles. Accession numbers in (B) are arranged in following order: SSU rDNA, 5.8S (if available), LSU rDNA. The sequences of Babesia bigemina were obtained from the Sanger Institute genome project (https://www.sanger.ac.uk/Projects/B_bigemina/). Asterisks mark partial LSU rDNA sequences of small size (300–700 bp).

Figure 10. Comparison of the attachment organelles of archigregarines Selenidium spp. (A–E) with septate and aseptate eugregarines (F–I).

(A) Drawing of the apical part of a Selenidium hollandei cell; (B) Ultrastructure of the apical part of an S. orientale cell, a longitudinal section; (C) The frontal region of the mucron under a higher magnification; (D) Mucron of the gamont (syzygy partner) of S. pennatum, a longitudinal section; (E) Predicted myzocytotic feeding in Selenidium; the mucron is embedded in the host cell and contains well-developed apical complex consisting of the conoid (co), polar ring (pr) giving rise to subpellicular microtubules (smt), rhoptries (rh) with rhoptry ducts (rd), and a large mucronal vacuole (mv); the tegument of the mucron comprises a trimembrane pellicle (pe) consisting of the plasma membrane (pm) and internal membrane complex, IMC (imc), with the exception of a small region in front of the conoid, a “cytostome site,” where the IMC is absent and only single plasma membrane is present; the cytostome is intermittently opened in this region to myzocytosis: at first, food comes through the duct (temporary cytopharynx) in the newly formed mucronal vacuole (mv), which then becomes a food vacuole (fv) and is transported into the cell along microtubules (mt) for digestion; the parasite-host contact is mediated by the septate cell junction (scj) with a characteristic wide gap between the plasma membranes (pm and hm, respectively). The mucron with the apical complex persists for a long time into the syzygy; the mucronal food vacuole is absent because the syzygy is a non-feeding stage (D). (F) Development of trophozoite of the septate gregarine Gregarina blaberae (scheme): (i), epimerite (ep) develops as a bulb in front of the apical complex consisting of the conoid and axial organelle (ao), which is likely a homologue of mucronal vacuole (also see (Giii)); the IMC terminates near the apical part of conoid (similarly to mature Selenidium), therefore the developing epimerite is covered only by a single plasma membrane, not by the pellicle; (ii–vi), the apical complex disappears, the epimerite is growing; a large flattened frontal vacuole (frv) arising from the layer of membrane alveoli (ma) of endoplasmic-reticulum (er) origin, numerous mitochondria (m), granules of storage carbohydrate amylopectin (sc), lipid drops (ld), and vacuoles (v) are present in the epimerite cytoplasm; (vi), finally, protomerite (p) and deutomerite (d) are separated by the septum (s). (G) Comparison of developing attachment organelles in the youngest trophozoites of the aseptate gregarine Lecudina sp. from the polychaete Cirriformia (Syn. Audouinia) tentaculata: ((i) and (ii); (ii) shows the details of the cell junction marked by the rectangle in (i)) and G. blaberae ((iii), the magnified fragment of (Fi)): both organelles develop ahead of the conoid in the same way and are covered by a single plasma membrane; the cell junction (cj) between the parasite and host cells is, unlike Selenidium, formed by two closely adjacent plasma membranes (parasite and host); an electron-dense fibrillar zone adjoins the cell junction in the gregarine cell (arrow); the cell junction is bordered by the circular groove (cg) pinching a small portion of the host cell; the IMC terminates (it) at the apical part of the conoid. (H) Comparison of the “mucron” of a well-developed trophozoite of the same Lecudina sp. ((i) and (ii); (ii) is the magnified fragment of (i) marked by the rectangle) and underdeveloped epimerite (ep) of a growing trophozoite of G. blaberae (iii), the same stage as in (Fiv): the IMC terminates (ie) at the base of the attachment organelle (it marks the former apex of the sporozoite mucron), the cell junction consists of two closely adjacent plasma membranes bordered by the circular groove (cg) pinching a small portion of the host cell, a large flattened frontal vacuole (frv) with fibrillar content develops just beneath the region of cell junction. (I) Comparison of the developing epimerite of an older trophozoite of G. blaberae ((i), stage (vi) from (F), magnified) and the attachment organelle of Lecudina (Syn. Cygnicollum) lankesteri (ii); (m), mitochondria. (J) A trophozoite and mature gamonts of L. lankesteri: losing of the epimerite. (A) is reprinted from: Schrével, 1968 (© 1968 Société Française de Microscopie Electronique, Paris), with permission from the Journal de Microscopie et Biology Cellulaire published by Société Française de Microscopie Electronique, Paris; (B, C, and E) are reprinted from: Simdyanov & Kuvardina, 2007 (© 2007 Elsevier), with permission from Elsevier (D) is reprinted from: Kuvardina & Simdyanov, 2002 (© 2002 by Russia, Protistology), with permission from the journal Protistology (Apr 19, 2017); (F, Giii, Hiii, and Ii) are reprinted from: Tronchin & Schrével, 1977 (© 1977 Society of Protozoologists, © John Wiley and Sons), with permission from John Wiley and Sons (Gi, Gii, and Hi) are reprinted from: Ouassi & Porchet-Henneré (1978), with permission from Elsevier #RP016388; (Iii and J) are reprinted from: Desportes & Théodoridès, 1986 (© 1986 Elsevier), with permission from Elsevier #RP016388.

Figure 11. Comparison of archigregarine (A–B) and eugregarine (C–F) cell organization with their main diagnostic characteristics (candidate synapomorphies).

(A and C) Cross sections of the cortex of a typical representatives showing regularly arranged longitudinal subpellicular microtubules (smt) in archigregarine longitudinal folds vs. ripple dense structures (apical arcs (aa)) and 12-nm filaments (apical filaments (af)) closely adjacent to the inner membrane complex (imc) of the pellicle within the tops of eugregarine epicytic crests; typically, internal lamina (il) forms links in the bases of the epicytic crests; pm, plasma membrane. (B) Archigregarine trophozoite showing a mucron (mu) with an apical complex (conoid (co) and rhoptries (rh)) and mucronal food vacuole (mv) performing myzocytosis (the cell junction type between the host and parasite cells is septate junction); the cytoplasm is rich in microneme-like organelles (mo). (D) Formation of the epimerite (ep) in eugregarines: a protuberance of the gregarine cell emerging ahead of the degrading apical complex. (E) Epimerite (so-called “mucron”) of some aseptate gregarines Lecudina spp. without the apical complex and with a large flat frontal vacuole and microtubules in the base. (F) Epimerite of septate gregarines with the same structures and with mitochondria. In eugregarines, the cell junction between the host and parasite is formed by two closely adjacent plasma membranes and there is no myzocytosis (or perhaps only in the earliest developmental stages before the reduction of the apical complex).