Gregarines of the Genus Lecudina (Protozoa,Apicomplexa) from Pacific Ocean Polychaetes*

The gregarine Lecudina longissima Hoshide, 1944 is described from the intestine of the marine polychaete Lumbrineris zonata (Johnson, 1901) from Santa Catalina Island, Calif. L. catalinensis n. sp. is described from the intestine of L. inflata Moore, 1911 from the same island. L. pherusae sp. n. and L. zimmeri sp. n. are described from the intestine of the marine polychaete Pherusa capulata (Moore, 1909) off Santa Catalina Island.     

Revision and Checklist of the Species (Other Than Lecudina) of the Aseptate Gregarine Family Lecudinidae*

SYNOPSIS. A checklist is given of the 89 named species of the gregarine family Lecudininae, exclusive of the 42 named species of the genus Lecudina (phylum Apicomplexa. class Sporozoea, subclass Gregarinia, order Eugregarinida, suborder Aseptatina). The list includes also the synonyms, host names, locations in hosts, known geographic distributions of the species, as well as key references. Another list is given of synonyms, lapsi calami, nomina nuda, etc., associated with the genera. A new genus, Paraophioidina g. n., with type species, Paraophioidina haeckeli (Mingazzini, 1891) and a new species, Lankesteria ormieresi sp. n., are described. There are also new combinations in the genera Bhatiella, Ancora, Monocystella, Ascocystis, and Paraophioidina.     

Calcium signaling in closely related protozoan groups (Alveolata): non-parasitic ciliates (Paramecium, Tetrahymena) vs. parasitic Apicomplexa (Plasmodium, Toxoplasma)

The importance of Ca2+-signaling for many subcellular processes is well established in higher eukaryotes, whereas information about protozoa is restricted. Recent genome analyses have stimulated such work also with Alveolates, such as ciliates (Paramecium, Tetrahymena) and their pathogenic close relatives, the Apicomplexa (Plasmodium, Toxoplasma). Here we compare Ca2+ signaling in the two closely related groups. Acidic Ca2+ stores have been characterized in detail in Apicomplexa, but hardly in ciliates. Two-pore channels engaged in Ca2+-release from acidic stores in higher eukaryotes have not been stingently characterized in either group. Both groups are endowed with plasma membrane- and endoplasmic reticulum-type Ca2+-ATPases (PMCA, SERCA), respectively. Only recently was it possible to identify in Paramecium a number of homologs of ryanodine and inositol 1,3,4-trisphosphate receptors (RyR, IP3R) and to localize them to widely different organelles participating in vesicle trafficking. For Apicomplexa, physiological experiments suggest the presence of related channels although their identity remains elusive. In Paramecium, IP3Rs are constitutively active in the contractile vacuole complex; RyR-related channels in alveolar sacs are activated during exocytosis stimulation, whereas in the parasites the homologous structure (inner membrane complex) may no longer function as a Ca2+ store. Scrutinized comparison of the two closely related protozoan phyla may stimulate further work and elucidate adaptation to parasitic life. See also "Conclusions" section.     

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.     

Molecular phylogeny and surface morphology of marine aseptate gregarines (Apicomplexa): Selenidium spp. and Lecudina spp

Many aseptate gregarines from marine invertebrate hosts are thought to have retained several plesiomorphic characteristics and are instrumental in understanding the early evolution of intracellular parasitism in apicomplexans and the phylogenetic position of cryptosporidians. We sequenced the small-subunit (SSU) ribosomal RNA genes from 2 archigregarines, Selenidium terebellae and Selenidium vivax, and 2 morphotypes of the marine eugregarine Lecudina polymorpha. We also used scanning electron microscopy to investigate the surface morphology of trophozoites from Lecudina tuzetae, Monocystis agilis, the 2 species of Selenidium, and the 2 morphotypes of L. polymorpha. The SSU ribosomal DNA sequences from S. vivax and L. polymorpha had long branch lengths characteristic of other gregarine sequences. However, the sequence from S. terebellae was not exceptionally divergent and consistently emerged as 1 of the earliest 'true' gregarines in phylogenetic analyses. Statistical support for the sister relationship between Cryptosporidium spp. and gregarines was significantly bolstered in analyses including the sequence from S. terebellae but excluding the longest branches in the alignment. Eugregarines formed a monophyletic group with the neogregarine Ophryocystis, suggesting that trophozoites with elaborate cortex folds and gliding motility evolved only once. The trophozoites from the 2 species of Selenidium shared novel transverse striations but differed from one another in overall cell morphologies and writhing behavior.     

Particularities of mitochondrial structure in parasitic protists (Apicomplexa and Kinetoplastida)

Without mitochondria, eukaryotic cells would depend entirely on anaerobic glycolysis for ATP generation. This also holds true for protists, both free-living and parasitic. Parasitic protists include agents of human and animal diseases that have a huge impact on world populations. In the phylum Apicomplexa, several species of Plasmodium cause malaria, whereas Toxoplasma gondii is a cosmopolite parasite found on all continents. Flagellates of the order Kinetoplastida include the genera Leishmania and Trypanosoma causative agents of human leishmaniasis and (depending on the species) African trypanosomiasis and Chagas disease. Although clearly distinct in many aspects, the members of these two groups bear a single and usually well developed mitochondrion. The single mitochondrion of Apicomplexa has a dense matrix and many cristae with a circular profile. The organelle is even more peculiar in the order Kinetoplastida, exhibiting a condensed network of DNA at a specific position, always close to the flagellar basal body. This arrangement is known as Kinetoplast and the name of the order derived from it. Kinetoplastids also bear glycosomes, peroxisomes that concentrate enzymes of the glycolytic cycle. Mitochondrial volume and activity is maximum when glycosomal is low and vice versa. In both Apicomplexa and trypanosomatids, mitochondria show particularities that are absent in other eukaryotic organisms. These peculiar features make them an attractive target for therapeutic drugs for the diseases they cause.     

Formation and diversity of parasitophorous vacuoles in parasitic protozoa. The Coccidia (Sporozoa, Apicomplexa)

Data on parasitophorous vacuole (PV) formation in host cells (HC) harbouring different intracellular protozoan parasites have been reviewed and critically analysed, with special reference to the main representatives of the Coccidia. The vacuole membrane (PVM) is the interface between host and parasite, playing a role in nutrient acquisition by the parasite from the HC. The PV phenomenon is regarded as a generalized HC response to the introduction of alien bodies (microorganisms), which eventually reflects the evolutionary established host-parasite relationships at cellular, subcellular and molecular levels. Special attention has been paid to the existing morpho-functional diversity of the PVs within the same genera and species of parasites, and even at different stages of the parasite life cycle. The PVM is generally considered to derive from the HC plasmalemma, whose biochemical composition undergoes significant changes as the intravacuolar parasite grows. The original HC proteins are selectively excluded from the PVM, while those of the parasite are incorporated. As the result, the changed PVM becomes not fusigenic for HC lysosomes. For Toxoplasma gondii and other cyst-forming coccidia (Isospora, Sarcocystis), a definite correlation has been noticed between the extent of rhoptry and dense granule secrets released by a zoite during HC internalization, on the one hand, and the pattern of the PV that forms, on the other one. In T. gondii, tachyzoites, known to discharge abundant secrets, commonly force the development of PVs limited with a single unit membrane and equipped with a tubulovesicular network in the lumen. Unlike, bradyzoites known to be deficient in secretory materials trigger the formation of PVs with a three-membrane lining composed of the changed invaginated plasmalemma in addition to two membranes of endoplasmic reticulum. The two different types of PV harbour, respectively, exoenteric and enteric stages of T. gondii, the latter being confined to the cat intestine only. Unlike, all endogenous stages of the classic intestinal coccidia (Eimeria spp.) develop within PVs limited with a single membrane, with some invaginations extending into the PV lumen. Unusual PV patterns are characteristic of the extracytoplasmic eimerian coccidia (Cryptosporidium, Epieimeria) and adeleid haemogreagarines (Karyolysus). In cyst-forming coccidia, the PVM is actively involved in tissue cyst wall formation, thus protecting the encysted parasites from recognition by the host immune system. All this strongly suggests that the PV is far from being an indifferent membraneous vesicle containing a parasite, but represents a metabolically active compartment in infected cells. Since all the coccidia are obligate intracellular parasites, the mode of their intimate interaction with the HC, largely accomplished via the PV and its membrane, is vital for their survival as biological species.     

Sporogonic Development of the Gregarine Ascogregarina taiwanensis (Lien and Levine) (Apicomplexa: Lecudinidae) in Its Natural Host Aedes albopictus (Skuse) (Diptera: Culicidae)

ABSTRACT. Sexual reproduction of Ascogregarina taiwanensis occurred in pupal Malpighian tubules of its natural host Aedes albopictus , resulting in the formation of gametocysts within which oocysts developed. Sporogony proceeded in each newly formed unsporulated oocyst; eight sporozoites were formed after completion of nuclear divisions followed by the cytokinesis. Developing oocysts were separated by gradient centrifugation on percoll based on different buoyant densities. The slender sporozoite had a typical apical complex composed of a coiled conoid, polar rings, rhoptries with ductules, subpellicular microtubules and micronemes. An apical cavity was seen in the gland-like rhoptries. Mitochondria of gregarines were not seen in any stage during the sporogony. Howeever, amylopectin granules were frequently seen in the cytoplasm. These starch-related granules became scant when the sporozoite was formed. We assumed they were associated with the energy source. Since the apical complex was only present in the sporozoite stage, it was most likely related to the invasion of host epithelial cells of the midgut during the early phase of infection.     

Cochleomeritus emersoni sp. n. (Protozoa,Apicomplexa), a Lecudinid Gregarine from the Pacific Ocean Polychaete Diopatra ornata Moore, 1911*

The gregarine Cochleomeritus emersoni sp. n. is described from the intestine of the marine polychaete Diopatra ornata from the Pacific Ocean off Point Hueneme, California.     

Morphology and Phylogenetic Position of Two New Gregarine Species (Apicomplexa: Eugregarinorida) Parasitizing the Lubber Grasshopper Taeniopoda centurio (Drury, 1770) (Insecta: Orthoptera: Romaleidae) in Mexico

Eugregarines are understudied apicomplexan parasites of invertebrates inhabiting marine, freshwater, and terrestrial environments. Most currently known terrestrial eugregarines have been described parasitizing the gut from less than 1% of total insect diversity, with a high likelihood that the remaining insect species are infected. Eugregarine diversity in orthopterans (grasshoppers, locusts, katydids, and crickets) is still little known. We carried out a survey of the eugregarines parasitizing the Mexican lubber grasshopper, Taeniopoda centurio, an endemic species to the northwest of Mexico. We described two new eugregarine species from the gut of the host: Amoebogregarina taeniopoda n. sp. and Quadruspinospora mexicana n. sp. Both species are morphologically dissimilar in their life‐cycle stages. Our SSU rDNA phylogenetic analysis showed that both species are phylogenetically distant to each other, even though they parasitize the same host. Amoebogregarina taeniopoda n. sp. clustered within the clade Gregarinoidea, being closely related to Amoebogregarina nigra from the grasshopper Melanoplus differentialis. Quadruspinospora mexicana n. sp. clustered within the clade Actinocephaloidea and grouped with Prismatospora evansi, a parasite from dragonfly naiads. Amoebogregarina taeniopoda n. sp. and Q. mexicana n. sp. represent the first record of eugregarines found to infect a species of the family Romaleidae.     

Heterogeneous distribution of the cAMP receptor protein RII in the nervous system: evidence for its intracellular accumulation on microtubules, microtubule-organizing centers, and in the area of the Golgi complex

The cellular and subcellular distribution of the regulatory subunit RII of cAMP-dependent protein kinase was studied by light and electron microscopy immunocytochemistry in tissue sections from rat brain and in primary cultures of brain cells. RII immunoreactivity was present in most neurons, although at variable concentration. In addition, RII was also detectable in other cell types including glia, neuroepithelial cells, and cells of mesenchymal origin. In the cell cytoplasm, RII immunoreactivity was concentrated at certain sites. An accumulation of RII immunoreactivity was found in all RII-positive cells at the Golgi area, precisely at a region directly adjacent to one of the two major faces of the Golgi complex. RII was also highly concentrated in some microtubule-rich cell processes such as cilia and neuronal dendrites, but was below detectability in most axons. In neurons, its concentration in dendrites is consistent with the previously demonstrated high affinity interaction between RII and the dendritic microtubule-associated protein 2. In addition, RII was accumulated at basal bodies of cilia and at centrosomes, i.e., sites known to act as microtubule organizers. RII-labeled centrosomes, however, were visible only in cells where the Golgi complex had a pericentrosomal organization, and not in cells where the Golgi complex was perinuclear such as in neurons and glia in situ. We hypothesize that centrosomal RII is bound to the pericentriolar microtubule-organizing material and that this material remains associated with the trans region of the Golgi complex when the latter is no longer associated with the centrosome. Our results suggest a key but not obligatory role of cAMP in the Golgi-centrosomal area, the headquarters of cell polarity, mobility and intracellular traffic, and in the function of a subpopulation of microtubules.     

Molecular Phylogeny of Marine Gregarine Parasites (Apicomplexa) from Tube‐forming Polychaetes (Sabellariidae,Cirratulidae, and Serpulidae), Including Descriptions of Two New Species of Selenidium

Selenidium is a genus of gregarine parasites that infect the intestines of marine invertebrates and have morphological, ecological, and motility traits inferred to reflect the early evolutionary history of apicomplexans. Because the overall diversity and phylogenetic position(s) of these species remain poorly understood, we performed a species discovery survey of Selenidium from tube‐forming polychaetes. This survey uncovered five different morphotypes of trophozoites (feeding stages) living within the intestines of three different polychaete hosts. We acquired small subunit (SSU) rDNA sequences from single‐cell (trophozoite) isolates, representing all five morphotypes that were also imaged with light and scanning electron microscopy. The combination of molecular, ecological, and morphological data provided evidence for four novel species of Selenidium, two of which were established in this study: Selenidium neosabellariae n. sp. and Selenidium sensimae n. sp. The trophozoites of these species differed from one another in the overall shape of the cell, the specific shape of the posterior end, the number and form of longitudinal striations, the presence/absence of transverse striations, and the position and shape of the nucleus. A fifth morphotype of Selenidium, isolated from the tube worm Dodecaceria concharum, was inferred to have been previously described as Selenidium cf. echinatum, based on general trophozoite morphology and host association. Phylogenetic analyses of the SSU rDNA sequences resulted in a robust clade of Selenidium species collected from tube‐forming polychaetes, consisting of the two new species, the two additional morphotypes, S. cf. echinatum, and four previously described species (Selenidium serpulae, Selenidium boccardiellae, Selenidium idanthyrsae, and Selenidium cf. mesnili). Genetic distances between the SSU rDNA sequences in this clade distinguished closely related and potential cryptic species of Selenidium that were otherwise very similar in trophozoite morphology.     

Comparison between Apicystis cryptica sp. n. and Apicystis bombi (Arthrogregarida,Apicomplexa): Gregarine parasites that cause fat body hypertrophism in bees

The molecular divergence, morphology and pathology of a cryptic gregarine that is related to the bee parasite Apicystis bombi Lipa and Triggiani, 1996 is described. The 18S ribosomal DNA gene sequence of the new gregarine was equally dissimilar to that of A. bombi and the closest related genus Mattesia Naville, 1930, although phylogenetic analysis supported a closer relation to A. bombi. Pronounced divergence with A. bombi was found in the ITS1 sequence (69.6% similarity) and seven protein-coding genes (nucleotide 78.05% and protein 90.2% similarity). The new gregarine was isolated from a Bombus pascuorum Scopoli, 1763 female and caused heavy hypertrophism of the fat body tissue in its host. In addition, infected cells of the hypopharyngeal gland tissue, an important excretory organ of the host, were observed. Mature oocysts were navicular in shape and contained four sporozoites, similar to A. bombi oocysts. Given these characteristics, we proposed the name Apicystis cryptica sp. n. Detections so far indicated that distribution and host species occupation of Apicystis spp. overlap at least in Europe, and that historical detections could not discriminate between them. Specific molecular assays were developed that can be implemented in future pathogen screens that aim to discriminate Apicystis spp. in bees.     

Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani

Pteridine reductase 1 (PTR1) is an essential enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. The present work is focused on the degradation of PTR1 during growth phase in Leishmania donovani. Western blot analysis with PTR1-GFP transfected promastigotes revealed that PTR1 protein was degraded in the stationary phase of growth at the time when the parasites were undergoing metacyclogenesis. Fluorescence microscopy revealed cytoplasmic localization of GFP tagged protein extending to the flagellum in these stationary phase promastigotes, implying that degradation of the protein was not by the usual multivesicular tubule lysosome (MVT) pathway. A probable destruction box of nine amino acids Q63ADLSNVAK71 and possible lysine residue K156 was identified in L. donovani PTR1 to be the site for ubiquitin conjugation. This suggests that PTR1 degradation during the stationary phase of growth is mediated by the proteasome. This leads to lower levels of H4-biopterin, which favors metacyclogenesis, and subsequently results in a highly infective stage of the parasite. Therefore, this finding has importance to identify new target molecule like the proteasome for therapeutic intervention.     

Current conception of the Sarcosporidia (Sarcocystis, Eimeriidae, Sporozoa, Apicomplexa): their morphofunctional organization, life cycle and practical importance

Advances in sarcosporidian research within the latest decade are critically reviewed and analysed in respect to some recent cytological findings of the author and her colleagues on the subject. Three rather than two morpho-functional cell types (metrocytes and merozoites) are distinguished within the sarcocyst, the third one being the intermediate cell. Division by endodyogeny in the cyst of Sarcocystis is very likely confined to the latter cell type. The pattern of nuclear chromatin and the constancy in DNA value per nucleus in the cystic merozoites, revealed by flow cytometry, is rather indicative of their incapability of dividing within the cyst, i.e. in the intermediate host. This enabled us to consider these merozoites as homologs of coccidian gamonts. The obvious differences between cysts and cystic stages in Sarcocystis and Toxoplasma may account in part for the rare, if any, reported cases of congenital sarcocystosis in the intermediate host.     

Purification and properties of a secondary alcohol dehydrogenase from the parasitic protozoan Tritrichomonas foetus

A novel secondary alcohol dehydrogenase has been isolated from Tritrichomonas foetus, the protozoan parasite which is responsible for bovine trichomonal abortion. The enzyme has been obtained in apparently homogeneous form after a 120-fold purification from cell homogenates, thus indicating that this activity constitutes an unusually high 1% of the total cytosolic protein. The native Mr = 115,000, determined by polyacrylamide gel electrophoresis. Mobility on sodium dodecyl sulfate gels suggests that the enzyme is composed of 6-8 subunits, identical as to molecular size (Mr = 17,000). The enzyme catalyzes the reversible oxidation of 2-propanol to acetone, using NADP+ (and not NAD+) as the redox-active co-substrate. Other small secondary alcohols, such as 2-butanol, 2- and 3-pentanol, cyclobutanol, and cyclopentanol are substrates, as are the corresponding ketones of these alcohols. Primary alcohols, such as ethanol and 1-propanol, are oxidized at rates less than 5% of that observed for 2-propanol. Product inhibition studies demonstrate an ordered kinetic mechanism, wherein the co-substrate (NADP+/NADPH) binds to the enzyme prior to binding of the substrate (alcohol/ketone).