Marine Heterotrophic Amoebae, Flagellates and Heliozoa From Belize (Central America) and Tenerife (Canary Islands), With Descriptions of New Species, Luffisphaera Bulbochaete N. Sp., L. Longihastis N. Sp., L. Turriformis N. Sp. and Paulinella Intermedia N. Sp.

ABSTRACT. Thirty four taxa of heterotrophic protists (amoebae, flagellates and heliozoa) were encountered in cultures established from marine samples from Belize (Central America) and Tenerife (Canary Islands). Most species are flagellates drawn from the choanoflagellates, the cryptophyceans, the euglenids, the kinetoplastids, the bicosoecids, the chromulinids, the pedinellids and a variety of laxa of uncertain affinities (Protista incertae sedis). the identity of the thecate choanoflagellates Salpingoeca ringens Kent, 1880, and S. tuba Kent, 1880, is discussed, and four new species of heterotrophic protists are described: one new species of the amoeba genus Paulinella (Paulinella intermedia n. sp.) and three new species of the incertae sedis genus Luffisphaera Belcher & Swale, 1975 ( Luffisphaera bulbochaete n. sp.; L. longihastis n. sp.; L. turriformis n. sp.).     

Trypanosoma Manulis N. Sp. From the Russian Pallas Cat Felis Manul

ABSTRACT. The morphology of Trypanosoma manulis n. sp. is described from living and stained specimens obtained from the blood of a Pallas cat, Felis manul , from Kazakhstan. the cat was also infected with a Hepatozoon sp. and feline immunodeficiency virus. the morphology of the trypanosome most closely resembles that of Trypanosoma mpapuense Reichenow and Trypanosoma heybergi Rodhain found in bats. Trypanosoma manulis does not grow well in conventional media, but co-culture with African green monkey kidney cells in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum at approximately 27° C resulted in luxuriant growth of trypanosomes. Under these growth conditions, epimastigotes adhered to the surface of the culture flask and to African green monkey kidney cells, as well as forming large rosettes. At 37° C, although growth was poor, transformation of the epimastigotes into the bloodstream forms occurred. This represents the first report of a trypanosome of the subgenus Megatrypanum in a felid.     

Isospora atrata N. Sp. (Apicomplexa, Eimeriidae): a New Coccidium Isolated from Carduelis atrata (Passeriformes, Fringillidae)

ABSTRACT Large numbers of coccidian oocysts belonging to the genus Isospora were obtained from the intestinal contents of 98 Carduelis atrata imported into Italy from South America during the months of August through December 1994. The oocysts are sub-spherical and average 21 × 20.3 μm (19.4–23.5 μm × 18.5–22 μm), have a bilayered wall, and an oval polar granule (rarely two). The sporocysts are elliptical and measure 18.8 μm × 10.3 μm (17.5–18.94 μm × 9.5–11.0 μm). The Stieda body protrudes slightly from the end of the sporocyst. A large sporocyst residuum is present, consisting of many granules that may be in a compact mass or scattered. Since this Isospora sp.does not resemble any other species of Isospora previously described from birds of the genus Carduelis , it has been named Isospora atrata n. sp. after the host. Disseminated asexual stages were found in mononuclear cells derived from formalin-fixed post mortem material, suggesting this coccidian may represent an Atoxoplasma -like parasite. Four coccidia-free Serinus canarius L. cohabitated for a long period (4 mo) with infected C. atrata but oocysts were never found in the stool of these birds.     

Gametogenesis and Sporogony of Hepatozoon Mocassini (Apicomplexa: Adeleina: Hepatozoidae) In an Experimental Mosquito Host, Aedes Aegypti

ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti , an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiatioe were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a panoitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporazoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endopbsmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.     

A Redescription of Entamoeba Histolytica Schaudinn, 1903 (Emended Walker, 1911) Separating It From Entamoeba Dispar Brumpt, 19251

ABSTRACT. Explaining the low incidence of invasive disease (10%) in humans infected with Entamoeba histolytica has occupied the attention of generations of both clinical and nonclinical investigators. One possible explanation would be the existence of two morphologically identical species—one an invasive pathogen, the other noninvasive. This was first proposed by Brumpt in 1925, but his explanation was virtually ignored until 1978 when the first of several publications appeared suggesting that E. histolytica did indeed consist of two species. We have reexamined Brumpt's claim in light of recent biochemical, immunological and genetic studies and conclude that the data derived from these investigations provide unequivocal evidence supporting his hypothesis. With this in mind, we redescribe the invasive parasite retaining the name Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911), and set it apart from the noninvasive parasite described by Brumpt, Entamoeba dispar Brumpt, 1925.     

Balamuthia Mandrillaris, N. G., N. Sp., Agent of Amebic Meningoencephalitis In Humans and Other Animals

ABSTRACT. We recently reported the isolation of a leptomyxid ameba from the brain of a mandrill baboon that died of meningo-encephalitis. Based on light and electron microscopic studies, animal pathogenicity tests, and immunofluorescence patterns, we conclude that our isolate differs fundamentally from the other two amebas ( Leptomyxa and Gephyramoeba ) included in the Order Leptomyxida. We therefore created a new genus, Balamuthia , to accommodate our isolate and described it as Balamuthia mandrillaris to reflect the origin of the type species. Briefly, B. mandrillaris is a pathogenic ameba that causes amebic encephalitis in humans and animals. It has trophic and cyst stages in its life cycle, and is uninucleate with a large vesicular nucleus and a central nucleolus. Mature cysts have a tripartite wall consisting of an outer loose ectocyst, an inner endocyst and a middle mesocyst. Unlike Acanthamoeba and Naegleria , the other two amebas that cause amebic encephalitis in humans, Balamuthia will not grow on agar plates seeded with enteric bacteria. However, Balamuthia grows on a variety of mammalian cell cultures and kills mice following intranasal or intraperitoneal inoculation. Based on immunofluorescence testing, 35 cases of amebic encephalitis in humans and three in other animals have been identified worldwide as being caused by Balamuthia .     

Isospora Normanlevinei N. Sp. and Isospora Coluzzii N. Sp. (Apicomplexa: Eimeriidae) In Emberiza Cirlus (Cirl Bunting) (Passeriformes-Emberizidae)

ABSTRACT. The morphological characteristics of two new species of Isospora observed in Emberiza cirlus (Cirl Bunting) from Italy are reported. the oocysts of Isospora normanlevinei n. sp. are spherical or sub-spherical, with a smooth double-layered wall, and measure 24.2 times 23.7 (21.0-26.5 times 21.5-25.5) μm; each oocyst contains 2 to 10 polar granules. No micropyle or residuum was observed. the piriform sporocysts measure 19.4 times 11.2 (17.0-21.0 times 10.0-12.5) μm and contain a dispersed residuum. the Stieda body is flat; the substiedal body, with scattered clear and dark granules, may be either symmetrical or asymmetrical. the oocysts of I. coluzzii n. sp. are asymmetrical and rounded shape and measure 28.6 times 24.2 (25.0-31.5 times 21.5-26.0) μm. the oocyst has a double-layered wall and 2 to 3 polar granules. Neither micropyle nor residuum is present. the sub-ellipsoidal sporocyst, measuring 18.2 times 10.0(16.5-20.0 times 9.0-11.0) μm, has a dispersed sporocyst residuum. the Stieda complex is symmetrical.     

Genetic differences between two spider crabs Pisoides bidentatus (A. Milne-Edwards, 1873) and Pugettia quadridens (de Haan, 1839) (Decapoda: Brachyura: Majoidea) from the Sea of Japan

Biochemical genetics was applied to elucidate the taxonomic position of the spider crab Pisoides bidentatus. Two morphologically similar spider crabs, P. bidentatus and Pugettia quadridens, were compared with four sympatric brachyuran species, Eriocheir japonicus, Hemigrapsus sanguineus, Hemigrapsus penicillatus (Varunidae), and Cancer amphioetus (Cancridae), using 20 allozyme loci. High genetic identity-value between P. bidentatus and P. quadridens (I = 0.758 ± 0.092) suggests that they should evidently be placed to one genus. This conclusion is supported by the similar identity-value between two species of the genus Hemigrapsus (I = 0.821 ± 0.078) and the low genetic similarity (I = 0.125 ± 0.05) between different genera, Hemigrapsus and Eriocheir, within the family Varunidae.     

Diversity and genetic structure of three species of Dioon Lindl. (Zamiaceae, Cycadales) from the Pacific seaboard of Mexico

We have estimated levels of genetic diversity and partitioning in the Mexican endemic cycad species Dioon sonorense, Dioon tomasellii, and Dioon holmgrenii, whose populations are exclusively distributed along the Pacific seaboard. For the three species, the patterns of variation at 19 allozyme loci in a total of 11 populations were evaluated. The average number of alleles per locus was in the range 2.05–1.68, corresponding to the northernmost population of D. sonorense (Mazatán), and the southernmost population of Dioon holmgrenii (Loxicha), respectively. In turn, the percentage of polymorphic loci peaked (94.73) in the El Higueral and Altamirano populations of Dioon tomasellii, and was estimated to be lowest (57.89) in the Loxicha population of D. holmgrenii. The mean expected heterozygosis varied markedly between taxa, with relatively high indices for D. sonorense and D. tomasellii (H_E = 0.314 and 0.295, respectively) and substantially lower values for D. holmgrenii (H_E = 0.170). Comparison of the inferred genetic structure based on F‐statistics for the three species also indicated differences along the north‐south Pacific seaboard axis. For D. sonorense and D. tomasellii, local inbreeding (F_IS) was zero but global inbreeding (F_IT) values were positive and significantly different from zero (0.130 and 0.116, respectively). By contrast, values of both F_IT and F_IS were negative and significantly different from zero (?0.116 and ?0.201, respectively) for D. holmgrenii. The genetic differentiation between populations (F_ST) had positive values in all taxa and corresponded with their geographic location along the north‐south axis: according to this statistic, D. sonorense was the most differentiated species (F_ST = 0.151), D. tomasellii had intermediate values (F_ST = 0.145), and D. holmgrenii was the less differentiated taxon (F_ST = 0.069). Finally, a phenogram representing Nei's genetic distances among populations displayed three major groups, each one corresponding to each of the studied species. Within D. tomasellii (of intermediate geographic distribution), a further division into two clusters corresponded precisely to the pair of populations that are geographically divided by the Trans Mexican Neovolcanic Mountains. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94 , 765–776.     

Identification of Naegleria fowleri and other Naegleria spp. (free-living amoebae) using cellulose acetate membrane electrophoresis of glucose phosphate isomerase

Abstract A simple isoenzyme cellulose acetate membrane electrophoresis method with respect to glucose phosphate isomerase (GPI) was developed for the differentiation of the human pathogenic free-living amoeba Naegleria fowleri from other Naegleria spp. A single GPI band was detected in all the species tested, the relative mobility of which could be used to identify N. fowleri . Of the other Naegleria spp., only N. italica and N. jadini shared a common GPI mobility. No intraspecies variation in GPI profile was detected, regardless of whether the strains were cultured in monoxenic or axenic media. The technique is proposed as a useful means of identifying N. fowleri soon after isolation from the environment.     

Genetic Variation and Divergence in the Genus Carcinus (Crustacea,Decapoda)

The extent of genetic differentiation of the decapod crustaceans Carcinus maenas (L.) and C. aestuarii Nardo was studied in nine nearshore locations comprising various European marine and brackish-water habitats, (Norway, Germany, France, Spain, Italy and Yugoslavia), using starch gel electrophoresis. The heterozygosity for all 19 allozyme loci was 0.026–0.016 (C. maenas) and 0.011–0.003 (C. aestuarii); one locus (Pgm) was the most polymorphic. The populations had rather low levels of genetic variability, comparable to those found in other decapods. In general, a light clinal variation in allele frequencies was discovered at the Pgm locus of C. maenas as latitude decreased. Because of similar morphologies and a closed genetic relationship (I = 0.89), the two forms should be considered subspecies. Therefore, the designation of the Atlantic subspecies C. maenas maenas is proposed, and the term of the Mediterranean subspecies C. maenas aestuarii is recommended.     

Description of Opisthonecta Matiensis N. Sp. (Protozoa, Ciliophora), A New Peritrich Ciliate From Wastewater

Opisthonecta matiensis n. sp. was isolated from the inlet water of a wastewater treatment plant near Madrid, Spain, and studied in vivo, with silver methods, and using electronic and indirect immunofluorescence microscopy. This new species shows an amphora-like cell shape and has a size of 45-73 microns (x 58.2) x 25-40 microns (x 31.3). The oral infraciliature is formed by one haplokinety, three polykineties, and a short row of kinetosomes (epistomial membrane). The aboral infraciliature is made up of the trochal band and the scopula. From the trochal band arise three fibrillar systems: oral fibers, aboral fibers, and oblique fibers. The myoneme system is composed of a delicate peristomial ring, longitudinal branched fibers that reach the trochal band and of radial fibers extending from the scopula to the trochal band. The silverline system consists of an average of 147 lines. This new species is separated from other known forms by its smaller size, the presence of one single vacuole, and its higher number of silverlines.     

Coccidia of Brazilian Mammals: Eimeria Marajoensis N. Sp. (Apicomplexa: Eimeriidae) From the Anteater, Tamandua Tetradactyla (Xenarthra: Myrmecophagidae)

ABSTRACT Feces from a juvenile specimen of the anteater Tamandua tetradactyla from Ponta de Pedras, Marajó, Pará, northern Brazil, contained three different coccidial oocysts: Eimeria tamanduae Lainson, 1968; E. corticulata Lainson & Shaw, 1990; and a third species previously unrecorded and described here as Eimeria marajoensis n. sp. Oocysts of the latter parasite are spherical to subspherical, 13.9 ± 1.5 times 13.4 ± 1.4 (11.1-16.5 times 11.1-16.5) μm, shape index (length/width) 1.0 (1.0-1.2). the oocyst wall is a single, Colorless layer about 0.6-1.0 μm thick with no striations or micropyle. There is no oocyst residuum, but a single, round, oval or irregularly shaped polar granule of about 0.75-2.5 μm is consistently present. the sporocysts are broadly ellipsoidal, 7.1 ± 0.7 ± 5.3 ± 0.6 (6.0-8.8 times 4.0-5.7) μm, shape index 1.3 (1.2-1.5), with a delicate wall bearing a minute stieda body. No sub-stieda body was visible. the sporocyst residuum consists of some 10-20 rounded granules, lying between the two slightly curved sporozoites which measure approximately 6.5 times 2.0 μm. Sporocyst refractile bodies were not discernablc.     

Revision of the Family Spironemidae Doflein (Protista, Hemimastigophora), With Description of Two New Species, Spironema Terricola N. Sp. and Stereonema Geiseri N, G., N. Sp.

ABSTRACT. Two new hemimastigophoran flagellates are described using light and electron microscopy, and the family Spironemidae is revised. Spironema terricola n. sp. occurs in soil from the Grand Canyon (southwest USA). It moves in a conspicuously euglenoid manner and differs from S. multiciliatum Klebs by its vermiform shape and shorter kinetics. Spironema terricola is similar to Goodey's Spironema multiciliatum from soil in England. However, Goodey's vermiform species has a very elongate nucleus and is thus neither identical with S. terricola , which has a roundish nucleus, nor with Klebs' lanceolate S. multiciliatum ; we consider it a new species, Spironema goodeyi n. sp, Stereonema geiseri n. g., n. sp. was discovered in the Aufwuchs (periphyton) of a river in Bavaria, Germany. the new genus differs from Spironema by its a contractility, and from Hemimastix by its shorter kinetics and less plicate cortex. the fine structure of Spironema and Stereonema is very similar to that of Hemimastix Foissner et al., viz., the cortex is composed of two plates having diagonal symmetry and the flagellated basal bodies have associated a short and a long microtubular ribbon. All species have unique extrusomes of the same type. the main differences between the three genera and five species recognized are contractility, length of kinetics, body size, shape of cell and nucleus, and particulars of the cortex and extrusomes. the phylogenetic relationships of the Hemimastigophora are still uncertain. However, the diagonal symmetry of the cortical plates and the pronounced euglenoid movement of Spironema spp. suggest a common ancestor with euglenids.     

Sarcocystis Idahoensis Sp. N. In Deer Mice Peromyscus Maniculatus (Wagner) and Geopher Snakes Pituophis Melanoleucus (Daudin)*,†

SYNOPSIS Deer mice Peromyscus maniculatus (Wagner) were trapped near Hammett, Idaho, as a possible source of Besnoitia jellisoni Frenkel and species of Sarcocystis to be used for life cycle studies. Forty-nine deer mice were necropsied; 20 (40.8%) were positive for sarcocysts structurally identical with those of Sarcocystis idahoensis sp. N. the source of S. idahoensis used for life cycle studies was a Great Basin gopher snake Pituophis melanoleucus deserticola Stejneger killed near Hammett, Idaho; 20 sporulated sporocysts measured 11.1 × 13.4 (11-12 × 13-14) μm. Structurally identical sporocysts were found in 7 of 14 Pacific gopher snakes P. m. catenifer (Blainville), and in 6 of 10 San Diego gopher snakes, P. m. annectens Baird & Girard. Totals of 148 deer mice and 17 gopher snakes were necropsied in the course of life cycle studies. Development of the first generation meronts took place within the hepatocytes of deer mice 2-10 days post-inoculation (PI) with sporulated sporocysts. Rosette-shaped meronts (6-8 days PI) contained tachyzoites attached by their posterior poles to a residual body. After release from the residual body, tachyzoites were initially retained in a meront wall and later released from the hot cells Within muscle cells a single tachyzoite-shaped structure was found 11 days PI and PAS-negative metrocyte-containing sarcocysts (2nd generation meronts) 13-34 days PI. PAS-positive material was first seen in sarcocysts 34 days II at which time bradyzoite formation became apparent. At 160 days PI, 10 sarcocysts measured 0.4 × 5.8 (0.2-0.9 × 1.8-9.9) μ and appeared to be mature and structurally identical with those from naturally infected deer mice. After ingestion of S. idahoensis-infected deer mice by gopher snakes, bradyzoites developed directly into microgamonts and macrogametes. These stages were first seen 5 days PI. Microgamonts were generally located above and macrogametes below the epithelial host cell nucleus. Seven to 11 days PI microgamonts were seen with mature microgametes, and oocysts which had not yet begun sporogony were found with oocyst walls. Clinical signs of illness were generally not observed in infected gopher snakes; however, one snake developed anorexia and cachexia, and became moribund after repeated ingestion of heavily infected deer mice. Acute hepatitis associated with developing meronts often was noted in deer mice given over 15,000 sporocysts each. Five to 6 days PI anorexia, weakness, ataxia, and dyspnea were observed: these clinical signs increased in severity until 6-8 days PI, when mice became recumbent and died, or were killed while moribund. Hepatosplenomegaly, petechial hemorrhage o the serosal and cut surfaces of the liver, and icterus were common. Diffuse coagulative necrosis with cellular infiltration (primarily neutrophils) was noted on microscopic examination.     

Description of the Infraciliature and Morphogenesis in the Ciliate Urotricha ondina N. Sp. (Prorodontida, Urotrichidae)

Recent works on prostomatid ciliates show that some genera of this group have a differentiated oral infraciliature and that their stomatogenesis during division involves the proliferation of only a few somatic kineties. These findings have significant implications regarding the iaxonomic status of these genera and also on the terminology used for the oral structures. In Urotricha ondina , the oral infraciliature consists of (1) a paroral kinety formed of paired kinetosomes that encircle the cytostome at the anterior pole of the cell and (2) 3 adoral organelles, each formed of 2 rows of kinetosomes, ventral in position and obliquely disposed, lying above 3 short somatic kineties that do not reach the anterior pole of the cell. This oral ciliature —formerly known as the corona and brosse, respectively—originate during stomatogenesis from the proliferation of 4 somatic kineties that lie posterior to the adoral organelles of the parental cell.     

Microfilum Lutjani N. G. N. Sp. (Protozoa Microsporida), A Gill Parasite of the Golden African Snapper Lutjanus Fulgens (Valenciennes, 1830) (Teleost Lutjanidae): Developmental Cycle and Infrastructure

ABSTRACT Microfilum lutjani n. g., n. sp. (Microsporida) was found on the gill filaments of Lutjanus fulgens (Teleost) inhabiting the coasts of Senegal. This microsporidium forms xenomas distinguished by the microvilli covering the plasma membrane. At all stages of development individuals have isolated nuclei and are in direct contact with the host cytoplasm. Merogony is binary and sporogony is tetrasporoblastic. the spore (4.75 times 2.60 μm)) is characterized by a manubrium inserted on a laterally offset anchoring disc and extending into a very short, noncoiled polar filament (no longer than 500 nm) in the form of a hook. This type of polar filament has not been described previously in the Microsporida.     

Revision of the Genus Askenasia Blochmann, 1895, with Proposal of Two New Species, and Description of Rhabdoaskenasia minima N. G., N. Sp. (Ciliophora, Cyclotrichida)

ABSTRACT. The planktonic ciliate genus Askenasia Blochmann, 1895 is reviewed and the new genus Rhabdoaskenasia n. gen. is established. Askenasia is characterized by three circumferential kinety belts and a circumoral wreath of paired argyrophilic granules without recognizable cilia and nematodesmata. A "brush" is absent. Askenasia apparently lacks the key characters of the Haptorida and is thus transferred to the Cyclotrichida, family Mesodiniidae. Rhabdoaskenasia differs from Askenasia in having single files of basal bodies in all kinety belts and club-shaped extrusomes. It possesses a circumoral kinety composed of dikinetids from which nematodesmata originate, forming a distinct rhabdos. Although very similar to Askenasia in its general appearance, R. minima n. sp. could belong to another order. Based on an extensive review of the literature and on silver impregnated specimens the following Askenasia species are recognized and described in detail: A. volvox (Eichwald, 1852) Kahl, 1930, A. stellaris (Leegaard, 1920) Kahl, 1930, A. acrostomia n. sp., and A. chlorelligera n. sp. Askenasia faurei Kahl, 1930 and A. humilis Gajewskaja, 1928 are transferred to the genus Cyclotrichium: C. faurei (Kahl, 1930) n. comb., C. humilis (Gajewskaja, 1928) n. comb. The systematic position of the genus Askenasia is discussed and keys to the genera of the Mesodiniidae and to the species of Askenasia are provided.