Molecular Phylogeny and Ultrastructure of Caliculium glossobalani n. gen. et sp. (Apicomplexa) from a Pacific Glossobalanus minutus (Hemichordata) Confounds the Relationships Between Marine and Terrestrial Gregarines

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a “trophozoite”, that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU rDNA sequence derived from two single‐cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors.     

Ultrastructure de la Grégarine Callynthrochlamys phronimae Frenzel; Étude Comparée de son Noyau avec Celui de Thalicola salpae (Frenzel) (Eugregarina)

SYNOPSIS. The structure of the gregarine Callynthrochlamys phronimae has been studied with the electron microscope. The cuticular complex is not different from those previously described in other species of gregarines. It is composed of 2 layers of different thickness delimited by 3 unit membranes and constitutes series of oblique folds at the surface of the deutomerite. Longitudinal rods of dense material surrounded by a slight pellicle are seen under the cuticle. Pinocytic vacuoles are present under the surface of the gregarine. Cytoplasmic organelles include mitochondria, Golgi complexes, endoplasmic reticulum, vacuoles and dense bodies from different sizes. There is a connection between the different features of the cytoplasm in the protomerite and deutomerite and the corresponding cuticular organization.
A characteristic feature of the species is the peculiar differentiation of the nuclear membrane. The nucleus is surrounded by a typical double membrane of which the inner one has a dense fibrillar layer apposed to it. In mature trophozoites, tubular expansions without inner layer arise from the double membrane; the same type of nuclear membrane occurs in another species, Thalicola salpae.     

Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae): the mode of survival in the gut of Enchytraeus albidus (Annelida, Oligochaeta, Enchytraeidae) is close to that of the coccidian genus Cryptosporidium

A selenid gregarine Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae), harboring the gut lumen of the oligochaete Enchytraeus albidus, was studied by light and electron microscopy. The trophozoite of Ditrypanocystis sp. is attached to the gut wall with its apical end to be anchored eventually between enterocytes in the crypts. Simultaneously, between the surfaces of the parasite and the host cell a peculiar contact is formed made of membranous channels and vesicles of unknown origin, the host cell surface in the contact area lacking cilia. The trophozoite becomes progressively enclosed within a parasitophorous vacuole made of layers of fused ciliar membranes of enterocytes. The fused cilia may be a source of membranes lining channels and vesicles of the contact area. Such a mode of parasitophorous arrangements has never been described before for gregarines, however, it bears a some likeness with that of the coccidian genus Cryptosporidium (similarity and differences being discussed). With regard to some molecular phylogeny constructions, claiming the "sister" relationship between gregarines and the coccidian genus Cryptosporidium (Carreno et al., 1999; Leander et al., 2003), this common feature in host-parasite relationships enabled us to put forward an idea of a possible evolutionary route from extracellularity of gregarines to intracellularity of coccidia, as exemplified by species of Cryptosporidium.     

Characterization of a coelomic gregarine parasite from Thitarodes pui (Lepidoptera: Hepialidae) in the Tibetan Plateau

Thitarodes pui, one of the host species of entomopathogenic fungus Ophiocordyceps sinensis, has great economic importance in the Tibetan Plateau. We report here, for the first time, a gregarine parasite found in the coelom of 7th instar and adults of T. pui. Gregarine gamonts (ovoid, ～15×8μm) underwent syzygy to produce reproductive gametocysts in T. pui larval hemolymph. All infected T. pui carried 2-17 mature gametocysts filled with numerous oocysts (lemon-shaped, 17.17±0.73×6.49±0.4μm). Transmission electron microscopy showed that these oocysts contained vacuoles of various sizes and amylopectin granules in the cytoplasm; scanning electron microscopy revealed a number of small bumps all over the surface of these oocysts. Small subunit ribosomal DNA sequence analysis showed a close relationship between the gregarine and the species of Ascogregarina (Eugregarinorida: Lecudinidae). Internal transcribed spacers and 5.8S ribosomal DNA from this gregarine exhibited 76% highest sequence identity with that from Ascogregarina culicis Ross.     

The Fine Structure of Rhynchocystis pilosa (Sporozoa, Eugregarinida)

SYNOPSIS. The trophozoite of Rhynchocystis pilosa obtained from the seminal vesicles of the earthworm Lumbricus terrestris was studied by light and electron microscopy. The trophozoite's cortical organization is particularly interesting because of its unusual evaginations and associated fibrillar structures. The pellicle is formed by 2 concentric membranes elevated into 60–70 alternating primary and secondary ridges extending posteriad. Numerous long ‘hairs’ or cytopilia originate along the primary ridges and each contains a system of fibrils originating from an underlying longitudinal myoneme. Longitudinal rows of pores lie between adjacent pollicular ridges. Three systems of fibrils lie in the cortex of the trophozoite. A longitudinal myoneme consisting of 12–18 fibrils lies below each primary pellicular ridge. Circular myonemes lie below the pellicle in a parallel array along the length of the organism. Each myoneme consists of 4–8 fibrils structurally similar to those of the longitudinal myonemes. Pairs of fine filaments also lie in the inner pellicular membrane along the apex of each ridge. The trophozoite's anterior end is modified as an attachment organelle consisting of 30–35 delicate pellicular folds which originate at the base of an anterior papilla. The folds extend approximately 15 μ posteriad where they become continuous with the primary pellicular ridges. The nucleus lies in the cytoplasm near the posterior level of the attachment organelle and is surrounded by a double membrane perforated by numerous pores. The cytoplasm contains numerous small vesicles which may be found in dense aggregations. These aggregations often occur in proximity to Golgi complexes and certain membrane-bound bodies. Mitochondria are abundant in the cytoplasm as are large, ovoid paraglycogen bodies. Occasionally layers of granular membranes are arranged parallel to the surface of the paraglycogen bodies but also occur thruout the cytoplasm.     

Species boundaries in gregarine apicomplexan parasites: a case study-comparison of morphometric and molecular variability in Lecudina cf. tuzetae (Eugregarinorida, Lecudinidae)

Trophozoites of gregarine apicomplexans are large feeding cells with diverse morphologies that have played a prominent role in gregarine systematics. The range of variability in trophozoite shapes and sizes can be very high even within a single species depending on developmental stages and host environmental conditions; this makes the delimitation of different species of gregarines based on morphological criteria alone very difficult. Accordingly, comparisons of morphological variability and molecular variability in gregarines are necessary to provide a pragmatic framework for establishing species boundaries within this diverse and poorly understood group of parasites. We investigated the morphological and molecular variability present in the gregarine Lecudina cf. tuzetae from the intestines of Nereis vexillosa (Polychaeta) collected in two different locations in Canada. Three distinct morphotypes of trophozoites were identified and the small subunit (SSU) rDNA was sequenced either from multicell isolates of the same morphotype or from single cells. The aim of this investigation was to determine whether the different morphotypes and localities reflected phylogenetic relatedness as inferred from the SSU rDNA sequence data. Phylogenetic analyses of the SSU rDNA demonstrated that the new sequences did not cluster according to morphotype or locality and instead were intermingled within a strongly supported clade. A comparison of 1,657 bp from 45 new sequences demonstrated divergences between 0% and 3.9%. These data suggest that it is necessary to acquire both morphological and molecular data in order to effectively delimit the "clouds" of variation associated with each gregarine species and to unambiguously reidentify these species in the future.     

Chronologie des Modifications Ultrastructurales au Cours de la Croissance de Gregarina blaberae*

SYNOPSIS. In an ultrastructural study the development of the sporozoite as well as the growth and development of the trophozoite of Gregarina blaberae were followed in the course of experimental infections of larvae of the cockroach Blaberus craniifer. The spectacular growth involved the transformation within 18 days of the sporozoite, measuring 15 × 1 μm, to a cephaline—trophozoite affixed to the intestinal epithelium—of 250 μm length and 65 μm diameter. The sporozoite's ultrastructure is not different from that of sporozoites of other Sporozoa studied to date—the conoid and dense bodies are present. The pellicle consists of 3 membranes, but there are some interruptions in the internal membrane complex. The first dictyosomes are formed from the nuclear envelope. The migration of the nucleus and of the dense bodies, followed by the regression of all the structures characteristic of the sporozoites, and the establishment of a cortical zone that comes to cover the epimerite, take place within 48 hr after infection and mark the transformation of the sporozoite into the trophozoite. Development of the cephaline involves the formation of the epicytic folds, which occurs at the base of the deutomerite, starting on the 3rd day of development. A regular system of longitudinal or epicytic folds is formed over the entire surface of the gregarine. On the 4th and 5th days of development, a vacuolar system and a chondriome become differentiated in the epimerite, while a fibrillar septum separates the protomerite from the deutomerite. The next stage, starting on the 6th day, is characterized by distribution of polysaccharide reserves between these 2 segments. The model studied allows us to determine the role of the epimerite in the parasite's nutrition, as well as the development of the chondriome and of the cortical membranes in the course of the vegetative growth phase of the cephaline gregarine.     

Comparative surface morphology of marine coelomic gregarines (Apicomplexa, Urosporidae): Pterospora floridiensis and Pterospora schizosoma

Two species in the aseptate gregarine genus Pterospora from the Pacific and Gulf coasts were analyzed by scanning electron microscopy, which revealed characteristics not reported in other gregarines. The gamonts of these species had branching trunks that ended in terminal digits, and both species moved by cytoplasmic streaming and peristalsis. Pterospora floridiensis had surface pits and tracts of parallel ridges that bended and connected with one another. Pterospora schizosoma had irregular-shaped surface swellings that were usually arranged in rosette patterns. These unique surface features have not been reported for other gregarines, and are strikingly different from the surface features of many septate and aseptate gregarines that inhabit the intestinal lumena of their hosts and move by gliding. The correlation of Pterospora's unique pellicular features to the habitat and cytoplasmic streaming characteristic of the genus may be significant, and may reflect an adaptation for development in coelomic environments.     

Molecular Characterization of Gregarines from Sand Flies (Diptera: Psychodidae) and Description of Psychodiella n. g. (Apicomplexa: Gregarinida)

ABSTRACT. Sand fly and mosquito gregarines have been lumped for a long time in the single genus Ascogregarina and on the basis of their morphological characters and the lack of merogony been placed into the eugregarine family Lecudinidae. Phylogenetic analyses performed in this study clearly demonstrated paraphyly of the current genus Ascogregarina and revealed disparate phylogenetic positions of gregarines parasitizing mosquitoes and gregarines retrieved from sand flies. Therefore, we reclassified the genus Ascogregarina and created a new genus Psychodiella to accommodate gregarines from sand flies. The genus Psychodiella is distinguished from all other related gregarine genera by the characteristic localization of oocysts in accessory glands of female hosts, distinctive nucleotide sequences of the small subunit rDNA, and host specificity to flies belonging to the subfamily Phlebotominae. The genus comprises three described species: the type species for the new genus— Psychodiella chagasi ( Adler and Mayrink 1961 ) n. comb., Psychodiella mackiei ( Shortt and Swaminath 1927 ) n. comb., and Psychodiella saraviae ( Ostrovska, Warburg, and Montoya-Lerma 1990 ) n. comb. Its creation is additionally supported by sequencing data from other gregarine species originating from the sand fly Phlebotomus sergenti . In the evolutionary context, both genera of gregarines from mosquitoes ( Ascogregarina ) and sand flies ( Psychodiella ) have a close relationship to neogregarines; the genera represent clades distinct from the other previously sequenced gregarines.     

The Three Cortical Membranes of the Gregarines (Parasitic Protozoa). III. Comparative Studies of the Membrane Proteins Among Different Sporozoan Species During Their Vegetative Phase1

ABSTRACT. The cortical membrane proteins of three gregarine and one coccidian species were compared using sodium dodecyl sulfate/polyacrylamide-gel electrophoresis. About 30 proteins were identified in the ghosts of Gregarina blaberae and G. garnhami and 20 in G. rhyparobiae ghosts and Sarcocystis tenella pellicles. No protein with the electrophoretic mobility of muscular actin was present in the ghosts of the sporozoan species under study. Each species possessed a characteristic electrophoretic pattern; no protein was present simultaneously in the four sporozoan species and only one protein band with a similar electrophoretic mobility was found in the three gregarine species (52 Kd protein). Two G. garnhami subpopulations living in Locusta migratoria and Schistocerca gregaria exhibited the same ghost protein pattern. Thus, large differences were observed between species and not within species, and the protein electrophoretic analysis appears to be a powerful tool for taxonomic investigations in gregarines. Gregarina blaberae and G. garnhami glycoconjugates were compared after periodate/Schiff staining of the polyacrylamide gel slabs. Several glycoconjugates were reported to belong to the cytoplasmic fraction; and, in view of cytochemical and ultrastructural data, a contribution of these glycocomponents to the secretion of a mucus is discussed.     

Ultrastructure of Infection, Development and Gametocyst Formation of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in Its Mosquito Host, Aedes albopictus (Diptera: Culicidae)

ABSTRACT. The life history of the protozoan parasite Ascogregarina taiwanensis in mosquito larvae ( Aedes albopictus , collected in southern Taiwan) was shown to consist of two consecutive stages—intracellular and extracellular. Light microscopy showed that most trophozoites moved into the Malpighian tubules and developed into giant trophozoites during the first day pupa. The locomotion may be associated with bristle-like ridges of the trophozoite. The stage for sexual reproduction, i.e. the gamete, was then formed by segmentation of the giant trophozoite and twisting off the anucleate extremities of the body. Sexual reproduction occurred via fertilization by fusion of two resulting gametes, presumably two opposed sexes. The fused gametes finally generate the formation of the gametocyst, within which oocysts develop by budding from the cytoplasmic mass. This type of sexual reproduction has not been reported previously in any gregarine protozoa. We here proposed it as a new hypothesis for further elucidation of the protozoan reproduction.     

Septate Gregarines From Reticulitermes Flavipes and Reticulitermes Virginicus (Isoptera: Rhinotermitidae)

ABSTRACT. The gregarine parasites of Reticulitermes virginicus and Reticulitermes flavipes begin their development as trophozoites attached to the midgut epithelium by a small button-shaped epimerite. the epimerite is lost when the parasite becomes free-living in the gut lumen as a solitary gamont. Syzygy is late and was not observed. When full-grown, gamonts enter the hemocoel and fuse in pairs to form large gametocysts that are attached to the midgut of the termite by a duct. Thousands of sporocysts are formed within the original gametocyst. the mature sporocysts are released into the lumen of the midgut through the connecting duct. They are then passed out with the feces. These gregarines are believed to be identical to Gregarina termitis Leidy which was described from a single gamont and later erroneously placed in the genus Hirmocystis by Henry.     

An Ultrastructural Study of Eimeria iroquoina Molnar & Fernando, 1974 in Experimentally Infected Fathead Minnows (Pimephales promelas,Cyprinidae) 3. Merogony1

Fathead minnows, Pimephales promelas, raised from eggs in the laboratory, were experimentally infected with oocysts of Eimeria iroquoina from either P. promelas or the common shiner, Notropis cornutus. Within intestinal epithelial cells, trophozoites thought to be derived from the sporozoites contained a prominent electron-dense refractile body. Merozoites dedifferentiated into trophic forms by losing components of their apical complex and pellicle. The inner membrane components of the pellicle appeared discontinuous, and the micronemes became enclosed within vacuoles. Prior to merozoite formation, multinucleate meronts were limited by a single membrane. Golgi complexes were associated with the nuclei of this stage. Merozoites were formed by ectomerogony in one generation and by endomerogony in the final generation. In both forms of merogony the final nuclear division was coupled with the onset of differentiation of the merozoites and featured eccentric mitotic spindles associated with centrocones located within the nuclear envelope and with the precursors of the apical complex. A Golgi complex was closely associated with the nucleus and apical tip of the forming merozoite. Unlike other Eimeria species, the complete pellicle of the merozoites of the final asexual generation of E. iroquoina was formed within the cytoplasm of the meront, without association with the limiting membrane, thus, all pellicular components are synthesized de novo. The inner membranes of the pellicle initially appeared as longitudinal strips, each of which was associated with a pair of the 22–24 subpellicular microtubules. Mature meronts of the final asexual generation averaged 9 μm in diameter and produced 13–16 merozoites. With the exception of the internal completion of the pellicle of the final generation merozoites, the basic processes of merogony in fish Eimeria species are similar to those recorded in terrestrial hosts.     

Molecular Phylogenetic Positions and Ultrastructure of Marine Gregarines (Apicomplexa) Cuspisella ishikariensis n. gen., n. sp. and Loxomorpha cf. harmothoe from Western Pacific scaleworms (Polynoidae)

Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts.     

The Ultra‐Structural Similarities between Cryptosporidium parvum and the Gregarines

Using a transmission electron microscopy‐based approach, this study details the striking similarities between Cryptosporidium parvum and the gregarines during in vitro axenic development at high ultra‐structural resolution. C. parvum zoites displayed three unusual regions within uninucleated parasites: epimerite‐like, protomerite‐like, and the cell body; these regions exhibited a high degree of morphological similarity to gregarine‐like trophozoites. The presence of a mucron‐like bulging structure at the side of the free ovoid gregarine‐like zoites was observed after 2 h of cultivation. An irregular pattern of epicytic‐like folds were found to cover the surface of the parasites 24 h postcultivation. Some extracellular stages were paired in laterocaudal or side‐side syzygy, with the presence of a fusion zone between some of these zoites. The present findings are in agreement with phylogenetic studies that have proposed a sister relationship with gregarines. Cryptosporidium appears to exhibit tremendous variety in cell structure depending on the surrounding environment, thereby mimicking the “primitive” gregarines in terms of the co‐evolution strategy between the parasites and their environments. Given this degree of similarity, different aspects of the evolutionary biology of Cryptosporidium need to be examined, considering the knowledge gained from the study of gregarines.     

The response of Gregarina niphandrodes (Apicomplexa: Eugregarinida: Septatina) to host starvation in Tenebrio molitor (Coleoptera: Tenebrionidae) adults.

Numerous studies of host starvation have emphasized pathological effects of parasites on their insect host, but little attention has been focused on the effects of host starvation on the parasites. This study addressed the possibility that parasite life-cycle events could be manipulated by withholding food from the host. The system used was Gregarina niphandrodes (Apicomplexa: Eugregarinida) in Tenebrio molitor (Coleoptera: Tenebrionidae) adults. Gregarine gametocyst formation and shedding ceased after 1 day in starved beetles but continued in fed controls. There were no statistically significant differences between total lengths of associated (3 of 5 trials) or unassociated (5 of 5 trials) gregarines found between experimental and control groups, but average numbers of the 2 life cycle events were generally higher in fed hosts than in starved ones. If infected, fed control beetles continued to form gametocysts throughout the 7-day trial periods, and gametocysts could be observed in the gut. Starved experimental beetles had no gametocysts in their guts. Refeeding of starved beetles after 4 days resulted in resumption of gametocyst formation and shedding. The studies demonstrated that the gregarine life cycle could be stopped and then started at the gametocyst formation stage like an off/on switch, simply by withholding food from, then refeeding, the host.     

Intracoelomic parasitic Sporozoa in the burrowing spatangoid echinoidEchinocardium cordatum: coelomocyte reaction and formation of brown bodies

Echinocardium cordatum frequently harbours in its coelomic cavity the protozoan parasiteLithocystis schneideri. Motile stages of this gregarine (trophozoites and gamonts) may be surrounded by echinoid coelomocytes which show a peculiar transformation of their shape (i.e. each cell develops a single spine-like extension giving a pincushion aspect to the parasite). Encysted stages of the gregarine (gametocysts and sporocysts) are found mostly within brown bodies. Brown bodies are particular mesothelium-covered formations occurring usually in the coelomic cavity ofE. cordatum. It is suggested that brown bodies naturally originate from detached fragments of mesenteries.     

Molecular Phylogenetic Positions of Two New Marine Gregarines (Apicomplexa)—Paralecudina anankea n. sp. and Lecudina caspera n. sp.—from the Intestine of Lumbrineris inflata (Polychaeta) Show Patterns of Co‐evolution

Gregarine apicomplexans are unicellular parasites commonly found in the intestines and coeloms of invertebrate hosts. Traits associated with the conspicuous feeding stage of gregarines, known as the trophozoite, have been used in combination with molecular phylogenetic data for species delimitation and the reconstruction of evolutionary history. Trophozoite morphology alone is often inadequate for inferring phylogenetic relationships and delimiting species due to frequent cases of high intraspecific variation combined with relatively low interspecific variation. The current study combined morphological data with small subunit (SSU) rDNA sequences to describe and establish two novel marine gregarine species isolated from the intestine of a polychaete host Lumbrineris inflata collected in British Columbia (Canada): Paralecudina anankea n. sp. and Lecudina caspera n. sp. The sister species to the host is Lumbrineris japonica, which can be found on the opposite side of the Pacific Ocean (Japan) and contains two different species of gregarine parasites: Paralecudina polymorpha and Lecudina longissima. Molecular phylogenetic analyses placed P. anankea n. sp. as the sister species to P. polymorpha and L. caspera n. sp. as the sister species to L. longissima. This phylogenetic pattern demonstrates a co‐evolutionary history whereby speciation of the host (Lumbrineris) corresponds with simultaneous speciation of the two different lineages of intestinal gregarines (Paralecudina and Lecudina).     

The life cycle and host specificity of Psychodiella sergenti n. sp. and Ps. tobbi n. sp. (Protozoa: Apicomplexa) in sand flies Phlebotomus sergenti and Ph. tobbi (Diptera: Psychodidae)

Two new gregarines in the recently erected genus Psychodiella (formerly Ascogregarina), Psychodiella sergenti n. sp. and Psychodiella tobbi n. sp., are described based on morphology and life cycle observations conducted on larvae and adults of their natural hosts, the sand flies Phlebotomus sergenti and Phlebotomus tobbi, respectively. The phylogenetic analyses inferred from small subunit ribosomal DNA (SSU rDNA) sequences indicate the monophyly of newly described species with Psychodiella chagasi. Ps. sergenti n. sp. and Ps. tobbi n. sp. significantly differ from each other in the life cycle and in the size of life stages. The sexual development of Ps. sergenti n. sp. (syzygy, formation of gametocysts and oocysts) takes place exclusively in blood-fed Ph. sergenti females, while the sexual development of Ps. tobbi n. sp. takes place also in males and unfed females of Ph. tobbi. The susceptibility of Phlebotomus perniciosus, Phlebotomus papatasi, Ph. sergenti, Ph. tobbi, and Phlebotomus arabicus to both gregarines was examined by exposing 1st instar larvae to parasite oocysts. High host specificity was observed, as both gregarines were able to fully develop and complete regularly the life cycle only in their natural hosts. Both gregarines are considered as serious pathogens in laboratory-reared colonies of Old World sand flies.     

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.