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Ultrastructure of infection, development and gametocyst formation of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in its mosquito host, Aedes albopictus (Diptera: Culicidae).

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.

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Laboratory susceptibility of Wyeomyia smithii (Diptera: Culicidae) to Ascogregarina taiwanensis (Apicomplexa: Lecudinidae).

Gregarines in the genus Ascogregarina are not known to develop in sabethine mosquitoes, but we successfully infected larvae of Wyeomyia smithii with Ascogregarina taiwanensis in the laboratory. Ascogregarina taiwanensis is a natural parasite of the exotic Asian tiger mosquito, Aedes albopictus. Only 18% to 70% of the W. smithii larvae had visible trophozoites, with a range of 1-92 per larva. Trophozoites persisted in the midgut for more than 37 d, and one adult female W. smithii had gametocysts in its Malphigian tubules, which indicated that A. taiwanensis might fully develop in W. smithii. After 50 d, gregarines were not found in W. smithii larvae.

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The fine structure of the gamont of Pterospora floridiensis (Apicomplexa: Eugregarinida).

Transmission electron microscopy of the gamont stage of Pterospora floridiensis has revealed a number of features. The gamont's surface varies from smooth to crenulate, with numerous pockets and folds. The pellicle is composed of an outer membrane, a middle lucent region, and an inner dense layer comprised of two tightly appressed membranes. Short ridges on the pellicle are 200-300+ nm long, 75-100 nm wide, and have a height of approximately 50 nm. The thickness of the pellicle is 100 nm when measured from the inner membrane to the top of a ridge. The ridges are formed by the plasma membrane and an underlying structure that is circular in cross-section. The surface folds and the pellicular ridges are distributed over the soma and the cell's unusual branching arms, though both are reduced near the junction between two gamonts in syzygy, and are absent at the central area of the junctional site. The cell has numerous active Golgi complexes associated with vesicles, as well as scattered dense mitochondria, lipid droplets, and paraglycogen granules. The nucleus has a large (13 microm) endosome, eccentrically located, and peripheral chromatin along the inner nuclear membrane.

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Role of gregarine parasite Ascogregarina culicis (Apicomplexa: Lecudinidae) in the maintenance of Chikungunya virus in vector mosquito.

Ascogregarina culicis and Ascogregarina taiwanensis are common gregarine parasites of Aedes aegypti and Aedes albopictus mosquitoes, respectively. These mosquito species are also known to transmit dengue and Chikungunya viruses. The sporozoites of these parasites invade the midgut epithelial cells and develop intracellularly and extracellularly in the gut to complete their life cycles. The midgut is also the primary site for virus replication in the vector mosquitoes. Therefore, studies were carried out with a view to determine the possible role of these gregarines in the vertical transmission of dengue and Chikungunya viruses from larval to adult stage. Experiments were performed by exposing first instar mosquito larvae to suspensions containing parasite oocysts and viruses. Since Ascogregarina sporozoites invade the midgut of first instar larvae, the vertical transmission was determined by feeding the uninfected first instar larvae on the freshly prepared homogenates from mosquitoes, which were dually infected with viruses and the parasite oocysts. Similarly, the role of protozoan parasites in the vertical transmission of viruses was determined by exposing fresh first instar larvae to the dried pellets of homogenates prepared from the mosquitoes dually infected with viruses and the parasite oocysts. Direct vertical transmission and the vertical transmission of CHIK virus through the oocyst of the parasites were observed in the case of Ae. aegypti mosquitoes. It is suggested that As. culicis may have an important role in the maintenance of CHIK virus during the inter-epidemic period.

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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.

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Extrachromosomal DNA in the Apicomplexa.

Malaria and related apicomplexan parasites have two highly conserved organellar genomes: one is of plastid (pl) origin, and the other is mitochondrial (mt). The organization of both organellar DNA molecules from the human malaria parasite Plasmodium falciparum has been determined, and they have been shown to be tightly packed with genes. The 35-kb circular DNA is the smallest known vestigial plastid genome and is presumed to be functional. All but two of its recognized genes are involved with genetic expression: one of the two encodes a member of the clp family of molecular chaperones, and the other encodes a conserved protein of unknown function found both in algal plastids and in eubacterial genomes. The possible evolutionary source and intracellular location of the plDNA are discussed. The 6-kb tandemly repeated mt genome is the smallest known and codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two bizarrely fragmented rRNAs. The organization of the mt genome differs somewhat among genera. The mtDNA sequence provides information not otherwise available about the structure of apicomplexan cytochrome b as well as the unusually fragmented rRNAs. The malarial mtDNA has a phage-like replication mechanism and undergoes extensive recombination like the mtDNA of some other lower eukaryotes.

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The development and fine structure of Lankesterella cf. dicroglossi (Apicomplexa: Lankesterellidae) infecting frogs in Niger, West Africa.

One of four Hoplobatrachus occipitalis (Günther, 1859) frogs received from Niger, West Africa was heavily infected with Lankesterella blood and pre-erythrocytic stages. Infected blood and tissues from this frog were force-fed to the remaining three frogs. Two survived to necropsy on days 14 and 27 post-feeding and were found to be infected with gamogonic and oogonic stages, respectively. The source of infection is inconclusive, as a natural origin cannot be excluded. Microgamont, macrogamont, oocyst and sporozoite structure and fine structure are described and found to conform in general, but not in detail, to previous descriptions. Gamonts and oocysts occurred predominantly in the liver and spleen. Walled sporulating oocysts were situated within macrophage centres. Oocysts yielded a progeny of 32 sporozoites. Pre-erythrocytic sporozoites developed within expanded inclusions, within their host cell, from which they massively invaded the liver and spleen, and to a lesser extent the lungs and kidneys. Sporozoites occurred in a parasitophorous vacuole in the erythrocytes. Conspecificity with Lankesterella dicroglossi Paperna et Ogara, 1996 reported from the same host species in Kenya remains uncertain due to several structural and developmental differences.

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Ultrastructure of developmental stages of Hemolivia mariae (Apicomplexa: Haemogregarinidae), natural parasite of the Australian sleepy lizard, in experimentally infected deviant hosts.

Mabuya vitatta (Olivier) (Scincidae) and Agama stellio (L.) (Agamidae) were infected with Hemolivia mariae Smallridge et Paperna, 1997 by ingestion of tick viscera from Amblyomma limbatum Neumann, fed as nymphs on naturally infected Australian sleepy lizards, Tiliqua rugosa Gray. The unnatural infection apparently interfered with the developmental schedule of the parasites. Transmission electron microscopic images of merogonic stages were obtained, as well as images of early developing gametocytes. Tissue and intraerythrocytic meronts were bound by a hardened wall. Intraerythrocytic gametocytes were lodged in a parasitophorous vacuole, which was filled with granular material, and were bound by a two-membrane wall. Small and large osmiophilic bodies were located in a sub-pellicular position. With differentiation, the wall membranes tightened with the parasitophorous vacuole wall, and the osmiophilic bodies disappeared. The outer parasite membrane consolidated into a thick encasing with distinct sutures. Late infection in A. stellio comprised gametocytes only.

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Haemogregarines of the genus Hepatozoon (Apicomplexa: Adeleina) in rodents from northern Europe.

We studied the prevalence and distribution of Hepatozoon infections in small rodents from Finland and other areas in northern Europe. Hepatozoon infections were more common in voles (Arvicolinae) than mice (Murinae) and more prevalent in voles of the genus Clethrionomys than in voles of the genus Microtus. Transmission electron microscopical examination of Hepatozoon erhardovae Krampitz, 1964 from bank voles Clethrionomys glareolus (Schreber) showed that intracellular lung meronts were located in alveolar septa. Meronts consisted of varying numbers of merozoites packed with amylopectin vacuoles inside electron-lucent parasitophorous vacuole. The size of the meronts was approximately 19 x 14 microm. Monozoic or dizoic cysts were frequent findings in the lung alveoles; the size of cysts was approximately 10 x 6 microm. Gametocytes were found inside eosinophilic granulocytes in the capillaries of lung tissue. Ultrastructurally, micronemes, microtubules, mitochondria, nuclei and lipid droplets were visible.

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Intraerythrocytic merogony in Haemogregarina koppiensis (Apicomplexa: Adeleorina: Haemogregarinidae).

During October 2003, a specimen of Amblyrhynchotes honckenii (Bloch, 1795) was captured at low tide, with a hand net, in a rock pool at Koppie Alleen, De Hoop Nature Reserve, South Africa. This fish was heavily parasitized by unidentified gnathiid praniza larvae, caligid copepods identified as Caligus tetrodontis Barnard, 1948, cymothoid isopods identified as Cinusa tetrodontis (Schioedte et Meinert, 1884), and the blood protozoan Haemogregarina koppiensis Smit et Davies, 2001. Giemsa-stained blood smears from this fish revealed new and unusual stages of merogony for H. koppiensis that included small, rounded, likely intraerythrocytic merozoites arranged in circles of eight around the host nucleus. Host cells appeared ghost-like and enlarged compared with normal erythrocytes. Identical merozoites, usually in clusters of up to 16, were also observed free of host cells. The pattern of merogony seen in H. koppiensis is unusual for a fish haemogregarine.

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Morphologic and morphometric analysis of Hepatozoon spp. (Apicomplexa, Hepatozoidae) of snakes.

Hepatozoon species are the most abundant hemoparasites of snakes. Its identification has been based mainly on the morphologic characterization of the gamonts in the peripheral blood of the vertebrate host and also of the cysts found in the internal organs of the vertebrate and invertebrate hosts. Using a computerized image analysis system, we studied five species of Hepatozoon from recently captured snakes in Botucatu, State of S o Paulo, Brazil, to evaluate the importance of the morphology and morphometry of the gamonts for the characterization of Hepatozoon species and to analyze the morphologic changes induced in the erythrocytes by the parasite. The studied species were H. terzii of Boa constrictor amarali, Hepatozoon sp. of Crotalus durissusterrificus, H. philodryasi of Philodryas patagoniensis, and H. migonei and H. cyclagrasi of Hydrodynastes gigas. We observed three different groups, one of them including the species H. terzii, H. philodryasi and Hepatozoon sp. of C. durissus terrificus; and the other two consisting of H. migonei and H. cyclagrasi. Degree of alterations in the erythrocytes was variable and it may be useful for characterization of Hepatozoon species.

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Transmission of Hepatozoon americanum (Apicomplexa: Adeleorina) by ixodids (Acari: Ixodidae).

American canine hepatozoonosis (ACH) caused by Hepatozoon americanum Vincent-Johnson, Macintire, Lindsay, Lenz, Baneth, and Shkap is an emerging, often fatal, tick-borne protozoal disease of dogs in the United States of America. Dogs acquire infection by ingesting ticks that contain oocysts. To understand the invertebrate (definitive) host range of H. americanum, experiments were carried out using four ixodids, Rhipicephalus sanguineus (Latreille), Dermacentor variabilis Say, Amblyomma americanum (L.), and Amblyomma maculatum Koch. Laboratory-reared nymphal ticks were fed on dogs that were either naturally or experimentally infected with H. americanum; when these ticks molted to the adult stage they were either fed to susceptible dogs or were dissected and examined for the presence of oocysts. Mature H. americanum oocysts were found in >90% of A. maculatum (both males and females), whereas oocysts were not found in any of the other three species. These results confirm that A. maculatum is an excellent host and vector for H. americanum and also suggest that this apicomplexan may have a narrow invertebrate host range, at least among ixodid ticks that are likely candidate vectors in the United States.

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Hepatozoon species (Apicomplexa: Hepatozoidae) of the corn snake, Elaphe guttata (Serpentes: Colubridae) and the pigmy rattlesnake, Sistrurus miliarius barbouri (Serpentes: Viperidae) in south Florida.

Hepatozoon guttata n. sp. is described from the corn snake (Elaphe guttata guttata) from south Florida. Gamonts average 14.6 x 4.6 (13-17 x 3.5-6) and are not recurved, with nucleus situated in the second quarter of the gamont. Erythrocyte cytoplasm rarely appears partially dehemoglobinized. The infected cells are usually distorted and are longer than the uninfected erythrocytes but do not differ in width; nuclei are smaller in length and width than those of uninfected cells. Sporogony in Aedes aegypti occurs within the head and the thorax but rarely in the abdomen. Oocysts are spherical to usually ovoid, 87.2 x 75.9 (45-155 x 40-152.5) and contain on average 7.1 (2-32) sporocysts. Sporocysts are spherical to ovoid, 34.8 x 31.0 (20-55 x 17.5-47.5), with 45.7 (14-89) sporozoites contained within. The pigmy rattlesnake (Sistrurus miliarius barbouri) in south Florida is parasitized by H. sistruri n. sp. Gamonts average 14.1 x 5.6 (12.6-15.8 x 4.7-6.3) in breadth and are not recurved, with the nucleus always situated in the second quarter of the gamont commonly at the midbody. Erythrocyte cytoplasm is not dehemoglobinized. The infected cells are always distorted and similar in length to the uninfected cells but with greater width and enlarged nuclei. Sporogony in A. aegypti occurs within head, thorax, and abdomen. Oocysts are spherical to usually ovoid, 163.6 x 154.7 (92-245 x 82-240) and contain 27.4 (12-42) sporocysts. Sporocysts are round to usually ovoid, 39.7 x 33.5 (25-50 x 20-50), with 45.7 (19-70) sporozoites.

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Efficacy of oral sulfadimethoxine against two gregarine parasites, Protomagalhaensia granulosae and Gregarina cubensis (Apicomplexa: Eugregarinida), infecting the Death's Head cockroach, Blaberus discoidalis.

Gregarines are common parasites of insects in culture, but no effective chemotherapeutic or prophylactic control protocol has been demonstrated. Sulfadimethoxine was administered in 5- and 7-day treatments to Death's Head cockroaches (Blaberus discoidalis) infected with Gregarina cubensis and Protomagalhaensia granulosae to test the efficacy of this sulfonamide against gregarine infection. Sulfadimethoxine significantly reduced the mean intensity of both G. cubensis and P. granulosae. Sulfadimethoxine treatment reduced gregarine intensity by 80% to 85% but had no significant effect on gametocyst production, suggesting that sulfonamide toxicity is directed primarily at sporozoites, trophozoites, and perhaps young gamonts. The possible use of sulfadimethoxine to produce gregarine-free insect cultures and the potential utility of gregarines as target organisms for screening pharmacologically active compounds for use against other intestinal apicomplexans are discussed.

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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.

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Long-term occurrence of Hemolivia cf. mauritanica (Apicomplexa: Adeleina: Haemogregarinidae) in captive Testudo marginata (Reptilia: Testudinidae): evidence for cyclic merogony?

Blood smears from wild-caught, long-term captive tortoises, Testudo marginata, revealed the presence of gametocytes of a Hemolivia mauritanica-like hemogregarine in the erythrocytes of 72% tortoises examined. Significant parasitemia was also found in animals living several years in captivity. Experimentally infected tortoises showed no evidence of a decrease in parasitemia level more than 15 mo after infection. Morphologically, stages found in tortoises' erythrocytes were indistinguishable from those referred to by previous workers as H. mauritanica from Testudo graeca. Moreover, successful experimental transmission to Hyalomma aegyptium confirms the conspecificity with H. mauritanica. The occurrence of H. mauritanica gametocytes in tortoise living up to 8 yr in captivity is suggested to result from continuous, long-lasting cyclic merogony in tortoises' parenchymatous organs, which is an unknown phenomenon in the life cycle of Hemolivia spp.

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Revision of the genus Stictospora and description of Stictospora villani, n. sp. (Apicomplexa: Eugregarinida: Actinocephalidae) from larvae of the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae), in Michigan.

Stictospora villani n. sp. is described from larvae of the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) from southern Michigan. Stictospora villani is distinguished from known species of the genus by differences in gamont size and by structural details of the epimerite. In general, S. villani is smaller than other known species of the genus and possesses an epimerite with a margin of 20-24 sympetalous, pendulate, narrowly to very narrowly elliptoid lamina. Previously described species are characterized by epimerites with notably fewer marginal lamina. A heretofore unrecognized protistan is diagnosed from the coelomic fat bodies and tentatively placed within the Neogregarinorida. We conclude that previous workers have mistaken this neogregarine for the gametocyst and oocyst of Stictospora, which are described in this study for the first time. The generic diagnosis of Stictospora is revised to encompass the epimeritic variation of its constituent species and to correctly reflect the morphology of the oocyst.

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