Gregarina Dufour 1828

Introduction

Gregarines are a diverse group of apicomplexan parasites that inhabit the intestines, coeloms and reproductive vesicles of marine, freshwater and terrestrial invertebrates. Approximately 250 genera and 1650 species have been described so far (Clopton 2000; Hausmann et al. 2003; Levine 1976, 1977, 1988). Some ancestral characters found in gregarines (e.g. extracellular feeding stages and a monoxenous life-cycle) have given the group a reputation of being “primitive”. Although some lineages of gregarines have retained characters inferred to be ancestral for the group, and perhaps apicomplexans as a whole, most gregarines represent highly derived parasites with novel ultrastructural and behavioural adaptations (Leander 2008). Many marine gregarines, for instance, have become giants among single-celled organisms and have evolved diverse patterns of surface structures (Fig. 1).

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Fig 1. Scanning electron micrographs of different marine gregarines; from left to right: Selenidium, surface of Selenidium (high magnification), Lecudina, surface of Pterospora (high magnification), Lankesteria and surface of Lankesteria (high magnification). (© Sonja I. Rueckert/Brian S. Leander)

Comparative gregarine cell morphology placed in a modern phylogenetic context helps clarify the earliest stages of apicomplexan evolution and demonstrates novel ways in which these parasites have solved fundamental biological problems, such as locomotion (nematode-like bending movements, gliding motility and peristalsis), host-dependent nutrition (myzocytosis, nutrition absorption via the surface) and reproduction. The overall biodiversity and phylogenetic relationships of gregarines are very poorly understood and only a small minority of the (predicted) total number of extant gregarine species have been described so far. Less than 1% of known invertebrates have been examined for gregarine infection (Clopton 2000). A compounding issue is that only a tiny number of described gregarine species have been studied at the molecular level, which makes species delimitation (using molecular barcodes) and inferences about broader phylogenetic relationships among gregarines very difficult.

Characteristics

Gregarines are single-celled apicomplexan parasites of invertebrates; some gregarines species might be commensals. The group is characterised by the following general features:

• Apical complex in the sporozoite stage; lost in the trophozoite stage in eugregarines and neogregarines
  
• Attachment to host via a mucron (in aseptate gregarines) or an epimerite (in septate gregarines); some gregarines (e.g. urosporidians) float freely within extracellular body cavities (e.g. coelom)
  
• Relatively large spindle-shaped cells, compared to other apicomplexans and eukaryotes in general (some over 850 µm in length)
• Tophozoite cell shapes very diverse in dfferent species
  
• Monoxenous life-cycle, requiring only one host (nearly all species)
• Inhabit extracellular body cavities of invertebrates such as the intestines, coeloms and reproductive vesicles
  
• Diverse modes of locomotion (e.g. gliding, bending and peristalsis)
• Different modes of feeding (e.g. myzocytosis and surface mediated nutrition)
• Most gregarines with longitudinal epicytic folds; crenulations in urosporidians
• Paraglycogen storage products give gregarine trophozoites a brownish colour
• Mitochondria with tubular cristae, often distributed near the cell periphery
  
• Syzygy—the process in which two mature trophozoites pair up before the formation of a gametocyst
  
• Large conspicuous nucleus (and nucleolus) in the trophozoites

Hosts

Gregarines inhabit different body spaces within the invertebrate host, such as the intestines, coeloms and reproductive vesicles. Most of the species are known to be host specific: Selenidium spp. infect polychaetes and sipunculids; Lankesteria spp. infect the intestines of ascidians; Monocystis spp. infect the reproductive vesicles of earthworms; neogregarinida infect the cuticle of insects, such as butterflies (Ophryocystis on the Monarch Butterfly); septate gregarines infect the intestines of marine and terrestrial arthropods.

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Fig. 2. Light micrographs of a few marine invertebrates known to be infected with intestinal gregarines; from left to right: Thelepus (Polychaetes), Lumbrinereis (Polychaetes), Phascolosoma (Sipuncula), Cnemidocarpa (Urochordata) and Balanus (Crustacea). (© Sonja I. Rueckert/Brian S. Leander)

Classification

Fig. 3. Sketch of the morphology of an aseptate (a) and a septate (b) gregarine © Sonja I. Rueckert

Traditionally, three general categories have been used to lump gregarines based on habitat, host range and morphological features of the trophozoites: archigregarines, eugregarines and neogregarines.

As the term archigregarines implies, some members of the group (e.g. Selenidium) have retained several ancestral features, such as exclusive presence in marine habitats and the presence of intestinal trophozoites that are similar in overall morphology to the infective sporozoites. They also have 0-50 longitudinal epicytic folds, bundles of microtubules beneath the cell surface and nematode-like bending behaviour (compare Grassé 1953, Schrével 1971a, b).

Eugregarines are found in marine, freshwater and terrestrial habitats and possess large trophozoites that are significantly different in morphology and behaviour from the sporozoite stage. The trophozoites have lost the apical complex and instead possess a mucron (or an epimerite). The surface is inscribed by 90-300 epicytic folds, resulting in stiff cells that are capable of gliding motility. Intestinal eugregarines are separated into septate (mostly terrestrial, like Gregarina) and aspetate (mostly marine like the Lecudinidae and Urosporidae) gregarines, depending on whether the trophozoite cell is superficially marked by a transverse septum (Fig. 3). Urosporidians (e.g. Pterospora) are aseptate eugregarines that infect the coelomic spaces of marine polychaetes. Monocystids are aseptate eugregarines that infect the reproductive vesicles of terrestrial annelids and tend to branch closely with neogregarines in phylogenetic trees.

Neogregarines are found in terrestrial hosts (e.g. insects); they have reduced trophozoite stages and, like monocystids, tend to infect host tissues other than the intestines (Leander 2008).

General Life Cycle

The transmission of gregarines to new hosts usually takes place by oral ingestion of oocysts in both aquatic and terrestrial environments. Some gregarine oocysts might be transmitted with host gametes during copulation (e.g. Monocystis, see Fig. 5). In either case, four or more sporozoites (depending on the species) equipped with an apical complex eventually escape from the oocysts (Fig. 4, steps 8 and 1), find their way to the appropriate body cavity and penetrate the host cells. The sporozoites emerge from a host cell, begin to feed and develop into larger trophozoites (Fig. 4, step 2). Some gregarines have sporozoites and trophozoites that are capable of asexual replication, a process called schizogony (or merogony); most gregarines appear to lack schizogony in their life-cycles (or at least this process has yet to be observed).

Two mature trophozoites eventually pair up in a process called syzygy and develop into gamonts (Fig. 4, step 3). The orientation of gamonts during syzygy differs depending on the species (e.g. side-to-side and head-to-tail). A gametocyst wall forms around each pair of gamonts (Fig. 3, step 4), which then begin to divide into hundreds of gametes (Fig. 4, step 5). This process is called gametogeny (Fig. 4, process B). Pairs of gametes fuse and form zygotes (Fig. 4, step 6), each of which becomes surrounded by an oocyst wall (Fig. 4, step 7). Within the oocyst, meiosis occurs to yield four or more spindle-shaped sporozoites (Fig. 4, process A = sporogony) (Kuriyama et al. 2005). Hundreds of oocysts accumulate within each gametocyst, and are usually released via host feces or via host death and decay.

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Fig. 4.  Illustration of the general life cycle of gregarines: A. Spindle-shaped sporozoites are produced in the oocyst via meiosis (sporogony); B. Inside the gametocyst gamonts divide into hundreds of gametes (gametogeny); see text above for a detailed description. © Sonja I. Rueckert (modified after Vivier & Desportes 1990)

Specific Examples of Gregarine Life Cycles

Monocystis

Fig. 5 illustrates the life cycle of Monocystis agilis. The infection starts with the hermaphroditic earthworm host ingesting an oocyst (1, 2) that releases sporozoites (3, 4) within the intestines (green). The sporozoite is motile and penetrates the intestinal wall (5) and enters the dorsal blood vessel (6). The sporozoite enters the seminal vesicles (yellow) via the (dark red) dorsoventral hearts (7). The sporozoites feed on the host's developing spermatocytes (8) in the wall of the seminal vesicle. The gregarines then move into the lumen of the vesicle where they mature into trophozoites. Each ball of sperm within the seminal vesicle contains a young trophozoite (9). These trophozoites are covered by remnants of the sperm cells and often superficially take on the appearance of a ciliated eukaryotic cell. After consuming the sperm ball, the now mature trophozoites pair up in syzygy (10). The trophozoites develop into gamonts, and a gametocyst wall forms around each pair (11). Each of the two gamonts undergoes multiple nuclear divisions to produce many nuclei developing into gametes. Two gametes each fuse and form zygotes, which are surrounded by oocyst walls (12). Within this oocyst, the diploid zygote undergoes meiosis and then mitosis to produce eight haploid daughter cells by a process known as sporogony. The gametocysts, or oocysts if they have been released, leave the earthworm through the male genital pore and are liberated into the soil (13, 14). Infection of a new host occurs by oral ingestion of an oocyst (or perhaps through the process of mutual cross fertilization during host sexual reproduction).

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Fig. 5. An illustration of the Monocystis life cycle. The infection starts with an earthworm ingesting an oocyst, and the parasites then develop within the host's seminal vesicle; see text above for a detailed description. © Sonja I. Rueckert (modified after Olsen 1974)

Ophryocystis elektroscirrha

The life cycle of Ophryocystis elektroscirrha is closely correlated with that of its host the monarch butterfly (Danaus plexippus). Parasite spores are transmitted maternally during oviposition, when infected females scatter spores over eggs and plant (i.e. milkweed) surfaces (McLaughlin & Myers 1970). Spores can also be transferred horizontally between adults during mating or other incidental contact. However, the ingestion of the spores by larvae is necessary to start a new infection. Within the larval gut, the spores lyse and release sporozoites that penetrate the intestinal wall, enter the hypoderm and undergo two vegetative, asexual replications.  During this time, the monarch larvae pass through five developmental instars (compare Altizer & Oberhauser 1999). After pupation of the host, the parasite completes a sexual reproduction cycle, which ends with the formation of dormant spores on the scales of the developing adult butterfly (McLaughlin & Myers 1970). The density of the spores is highest on the abdomen, but spores can also be found on the wings, head and thorax (Leong et al. 1992).

Heavily infected adults have difficulty emerging from the pupal cases and expanding their wings; butterflies with low parasite infections appear to be normal (McLaughlin & Myers 1970, Leong et al. 1992). Ophryocystis elektroscirrha compromises the fitness of monarch butterflies by affecting survival to eclosion, adult size, lifespan, and male reproductive success. These effects are dependent on the parasite load of the butterflies and are mainly restricted to heavily infected individuals (see Altizer & Oberhauser 1999).

Motility

Gregarines have adopted different modes of motility. The trophozoites of Selenidium species, for example, are able to bend and coil; see movies:

• View Selenidium terebellae movement (from the intestines of a spaghetti worm, Thelepus)
• View Selenidium vivax movement (from the intestines of a peanut worm, Phascolosoma) (Leander 2006)

One or more layers of microtubules are known to be responsible for these nematode-like movements (Leander 2007, Schrével 1971a, Stebbings et al. 1974). Within the eugregarines, the number of epicytic folds has increased in some lineages (revisit the SEM of Lecudina in Fig. 1) leading to relatively stiff cells and a distinctive gliding motility; see movie:

• View Lecudina sp. gliding (from the intestines of the pile worm Nereis) (Leander 2008).

The epicytic folds of some eugregarines are capable of metachronal undulations. This behaviour was initially interpreted as the driving force behind gliding motility (Vivier 1968, Vavra & Small 1969). Nonetheless, a study has subsequently shown that the gliding motility is facilitated by an actinomyosin system organized beneath the longitudinal edge of each epicytic fold (Heintzelmann 2004). In addition to these two types of motility, dynamic peristalsis-like movements can be found in the urosporidians and some archigregarines; see movies:

• View Pterospora schizosoma dynamic peristalsis-like movements (a urosporidian from the coelom of a bamboo worm, Axiothella) (Landers & Leander 2005)
• View Selenidium vivax dynamic peristalsis-like movements (from the intestines of a peanut worm, Phascolosoma) (Leander 2006)

These dynamic cell behaviours are thought to facilitate the acquisition of nutrients from the surrounding medium through micropores in the cell surface of the trophozoites.

Apicoplast?

The apicoplast is a remnant, non-photosynthetic homologue to chloroplasts found in many apicomplexan parasites. This organelle was observed first in the 1960s by transmission electron microscopy, but was not identified as a plastid until a 35 kb plastid genome was characterized in 1996 (McFadden et al. 1997, Waller & McFadden 2005; Wilson et al. 1996, Wilson & Williamson 1997). The apicoplast is surrounded by four membranes and has been found in nearly all major groups of apicomplexan parasites (Waller & McFadden 2005). This organelle is inferred to be an ancient feature of the group that was acquired by secondary endosymbiosis with a red algal prey cell. However, apicoplasts appear to be absent altogether in Cryptosporidium (Zhu et al. 2000, Abrahamsen et al. 2004, Xu et al. 2004).

The only evidence for the possible existence of an apicoplast in gregarines is a transmission electron micrograph from Selenidium pendula by Schrével (1971b). Schrével (1971b) found conspicuous vacuoles that were surrounded by four membranes; these structures were unlike any other structure described in gregarines.  At that time, apicoplasts had not been discovered.  Toso & Omoto (2007) showed by a number of criteria that G. niphandrodes does not have a plastid or plastid genome. Therefore, it remains undetermined whether or not any or all gregarines possess an apicoplast.

Discussion of Phylogenetic Relationships

Most studies on the phylogenetic relationships of gregarines are based on small-subunit ribosomal DNA sequences from a few species (Carreno et al. 1999, Leander 2007, Leander et al. 2003a, b, Leander et al. 2006, Roychoudhury 2007, Rueckert & Leander 2008, Valles & Pereira 2003). Although comparative morphology indicates that gregarines are monophyletic, whether or not other apicomplexan lineages like Cryptosporidium and coccidians sensu stricto are nested within them remains to be rigorously tested with molecular phylogenetic data.

Comparative morphology and molecular phylogenetic data strongly suggest that Selenidium (and relatives) forms a paraphyletic group from within which eugregarines and some other highly derived lineages evolved (e.g. Selenidium vivax and relatives) (Leander 2008).

The Eugregarinida contains most of the known gregarine species. They have elaborate systems of epicytic folds, with an actinomyosin-based gliding apparatus. The apical complex has been lost in the trophozoites, but an attachment structure is present (e.g. a mucron or an epimerite). The phylogenetic relationships within the Eugregarinida are just beginning to be addressed with molecular phylogenetic approaches and our understanding of the group's biodiversity and interrelationhsips is still very limited:

• Archigregarines, such as Selenidium, are paraphyletic
• The monophyly and composition of the Eugregarinida are uncertain
  
• Lecudina is paraphyletic
• Lankesteria is monophyletic and is most closely related to lineages of Lecudina
• Urosporidians (e.g. Pterospora) evolved from within the Lecudinidae
  
• Septate eugregarines (e.g. Gregarina and Leidyana) that infect insects are monophyletic
• Monocystids (e.g Monocystis) form a sister group to neogregarines (e.g. Ophryocystis and Mattesia)
  
• Gregarine SSU rDNA sequences tend to be highly divergent and form long branches in molecular phylogenetic analyses
• At this point, only 25 gregarine species have a published SSU rDNA sequence
• The vast majority of gregarine species have never been studied at the molecular level

Other Names for Gregarina Dufour 1828

• Gregarines

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