Paramecium calkinsi andP. putrinum (Ciliophora, Protista) harboring alpha-subgroup bacteria in the cytoplasm

Summary New intracellular bacteria were detected in the cytoplasm ofParamecium calkinsi andP. putrinum. Some of the bacteria were not evenly distributed in the cytoplasm of the host but were found in the center of the cell, eventually near the nuclei, but not in the cortex area, whereas another species was found in the cortex area. These peculiarities of intracellular bacteria localization in the host suggest that the conditions in various parts of the cytoplasm favor bacterial maintenance to different extent. Due to the results obtained by transmission electron microscopy and in situ hybridization using appropriate oligonucleotide probes, the bacteria, three or possibly four species, are Gram-negative and belong to the alpha-subgroup of proteobacteria. Bacteria from one stock ofP. calkinsi were found to be infectious for bacteria-free cells ofP. calkinsi andP. nephridiatum.     

Geosiphon pyriforme,a fungus forming endocytobiosis withNostoc (Cyanobacteria), is an ancestral member of the glomales: Evidence by SSU rRNA Analysis

Geosiphon pyriforme inhabiting the surface of humid soils represents the only known example of endocytobiosis between a fungus (Zygomycotina; macrosymbiont) and cyanobacteria (Nostoc; endosymbiont). In order to elucidate the taxonomical and evolutionary relationship ofGeosiphon pyriforme to fungi forming arbuscular mycorrhiza (AM fungi), the small-subunit (SSU) ribosomal RNA genes ofGeosiphon pyriforme andGlomus versiforme (Glomales; a typical AM fungus) were analyzed and aligned with SSU rRNA sequences of several Basidiomycetes, Ascomycetes, Chytridiomycetes, and Zygomycetes, together with all AM-fungal (Glomales) sequences published yet. The distinct group of the order Glomales, which includesGeosiphon, does not form a clade with any other group of Zygomycetes. Within the Glomales, two main lineages exist. One includes the families Gigasporaceae and Acaulosporaceae; the other one is represented by the genusGlomus, the members of which are very divergent.Glomus etunicatum andGeosiphon pyriforme both form independent lineages ancestral to the Glomales. The data provided by the present paper confirm clearly thatGeosiphon represents a fungus belonging to the Glomales. The question remains still open as to whether or notGeosiphon is to be placed within or outside the genusGlomus, since this genus is probably polyphyletic and not well defined yet.Geosiphon shows the ability of aGlomus-like fungus to form a “primitive” symbiosis with a unicellular photcautotrophic organism, in this case a cyanobacterium, leading to the conclusion that a hypothetical association of aGlomus-like fungus with a green alga as a step during the evolution of the land plants appears probable. Correspondence to: H. Gehrig     

Morphological description of three marine ciliates (Ciliophora, Scuticociliatia), with establishment of a new genus and two new species

Three marine scuticociliates, Falcicyclidium fangi nov. gen., nov. spec., Falcicyclidium atractodes nov. spec., and Cristigera media Kahl, 1928 were investigated using live observation and silver impregnation methods. The genus Falcicyclidium is distinguished by the combination of: (i) dorsoventrally flattened body, (ii) hook-like (falciform) paroral membrane, (iii) anterior end of paroral membrane posterior to anterior end of membranelle 1, and (iv) multiple caudal cilia. Falcicyclidium fangi nov. spec., the type of the new genus, can be recognized by the combination of its large size, extremely dorsoventrally flattened (3:1) body, consistently 10 somatic kineties, and the broad, elongate buccal area occupying 60% of the body length. Falcicyclidium atractodes nov. spec. is mainly characterized by a unique spine projecting from both the anterior and posterior end. The uncommon form, Cristigera media is redescribed based on the population from Qingdao, the statistic data and additional features, especially the morphology of the living cells, are documented.     

Genetic diversity and phylogenetic position of the subclass Astomatia (Ciliophora) based on a sampling of six genera from West African oligochaetes (Glossoscolecidae, Megascolecidae), including description of the new genus Paraclausilocola n. gen

To more confidently assess phylogenetic relationships among astome ciliates, we obtained small subunit (SSU) rRNA sequences from nine species distributed in six genera and three families: Almophrya bivacuolata, Eudrilophrya complanata, Metaracoelophrya sp. 1, Metaracoelophrya sp. 2, Metaracoelophrya intermedia, Metaradiophrya sp., Njinella prolifera, Paraclausilocola constricta n. gen., n. sp., and Paraclausilocola elongata n. sp. The two new species in the proposed new clausilocolid genus Paraclausilocola n. gen. are astomes with no attachment apparatus, two files of contractile vacuoles, and an arc-like anterior suture that has differentiations of thigmotactic ciliature on the anterior ends of the left kineties of the upper surface. Phylogenetic analyses were undertaken using neighbor-joining, Bayesian inference, maximum likelihood, and maximum parsimony. The nine species of astomes formed a strongly supported clade, showing the subclass Astomatia to be monophyletic and a weakly supported sister clade to the scuticociliates. There were two strongly supported clades within the astomes. However, genera assigned to the same family were found in different clades, and genera assigned to the same order were found in both clades. Thus, astome taxa appear to be paraphyletic when morphology is used to assign species to genera.     

Reconciling classical and molecular phylogenies in the stichotrichines (Ciliophora, Spirotrichea), including new sequences from some rare species

We performed a comparative morphological and molecular study on oxytrichid and urostylid stichotrichs (=part of the former hypotrichs). Included are new small subunit (18S) ribosomal RNA (rRNA) gene sequences from five rare oxytrichids (Gonostomum namibiense, Cyrtohymena citrina, Hemiurosoma terricola, Onychodromopsis flexilis, Orthoamphisiella breviseries) and published sequences, based on cultures provided by the senior author, of two key stichotrichid genera, viz., Gastrostyla and Engelmanniella. These and other sequences, altogether 27 species representing 23 genera, were used to analyze how 18S rRNA-based phylogenetic trees can be reconciled with the morphological and ontogenetical data. In 18S rRNA trees, the oligotrichine family Halteriidae invariably clusters within the oxytrichid clade, usually near Oxytricha granulifera, type species of the genus. This position is hardly supported by morphological and ecological evidence and, especially, it contradicts the current ontogenetic findings; possibly, it is an artifact caused by taxa undersampling and/or special molecular evolutionary events. In contrast, most morphological and DNA sequence data of the stichotrichs can be harmonized with the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis which suggests that the urostylid midventral pattern evolved from an oxytrichine ancestor, developing a second time within the Oxytrichidae. The systematic position of one of the two key genera could be clarified with the 18S rRNA sequences: Gastrostyla is a stylonychine oxytrichid. Based on the molecular data and a reassessment of ontogenesis, a new genus, Styxophrya nov. gen., is established for Onychodromus quadricornutus Foissner, Schlegel & Prescott, 1987.     

Detection of bacteria originally isolated from Alexandrium spp. in the midgut diverticula of Mytilus edulis after water-borne exposure

Bacteria associated with toxic dinoflagellates have been implicated in the production of paralytic shellfish poisoning (PSP) toxins, but it has not been substantiated that bacteria are truly capable of autonomous PSP toxin synthesis or what role bacteria may play in shellfish toxification. In this study, different putatively PSP toxin producing bacteria originally isolated from toxic Alexandrium spp. were exposed to the blue mussel Mytilus edulis. To document that these bacteria accumulated in the digestive tract of the mussels, hybridization techniques that use rRNA targeted oligonuceotides for in situ identification of these bacteria were applied. The mussel hepatopancreas was dissected and paraffin and frozen sections were made. The dissected glands were hybridized with digoxigenin-labelled 16S rRNA oligonucleotide probes. Results demonstrate that mussels will readily uptake and accumulate these bacteria in the hepatopancreas. However, the mussels were not rendered toxic by the ingestion of the bacteria as determined by HPLC with UV detection for PSP toxins and determination of sodium channel blocking activity using the mouse neuroblastoma assay. Thus, although the role that bacteria play in mussel toxification remains unclear, methods are now available which will aid in further investigation of this relatively unexplored area.     

Molecular identification of Taenia spp. in wolves (Canis lupus), brown bears (Ursus arctos) and cervids from North Europe and Alaska

Taenia tapeworms of Finnish and Swedish wolves (Canis lupus) and Finnish brown bears (Ursus arctos), and muscle cysticerci of Svalbard reindeer (Rangifer tarandus platyrhynchus), Alaskan Grant's caribou (Rangifer tarandus granti) and Alaskan moose (Alces americanus) were identified on the basis of the nucleotide sequence of a 396 bp region of the mitochondrial cytochrome c oxidase subunit 1 gene. Two species were found from wolves: Taenia hydatigena and Taenia krabbei. The cysticerci of reindeer, caribou and one moose also represented T. krabbei. Most of the cysticercal specimens from Alaskan moose, however, belonged to an unknown T. krabbei-like species, which had been reported previously from Eurasian elks (Alces alces) from Finland. Strobilate stages from two bears belonged to this species as well. The present results suggest that this novel Taenia sp. has a Holarctic distribution and uses Alces spp. as intermediate and ursids as final hosts.     

Vavraia lutzomyiae n. sp. (Phylum Microspora) infecting the sandfly Lutzomyia longipalpis (Psychodidae, Phlebotominae), a vector of human visceral leishmaniasis

Vavraia lutzomyiae (Microsporida; Pleistophoridae) is a new species parasitic in the tropical phlebotomine sandfly, Lutzomyia longipalpis (Diptera, Psychodidae, Phlebotominae), a major vector of Leishmania chagasi in Latin America where human visceral leishmaniasis is endemic. Infected larvae and pupae were parasitized in the abdomen, and some adults were parasitized in Malpighian tubules and midgut. The sporogonial plasmodium divided by multiple divisions into up to 64 uninucleate sporoblasts. These stages were surrounded outside the plasmalemma by a thick, amorphous dense coat and transformed into a merontogenetic sporophorous vesicle within which the sporonts developed into sporoblasts. The mature microsporidian spores were broadly ellipsoidal and measured 6.1+/-0.43 x 3.1+/-0.15 microm. The spore wall consisted of a transparent endospore (approximately 100 nm) and a thin electron dense exospore (approximately 30 nm) with the outer limit slightly undulated. Spores contained a polar filament arranged peripherally in a single layer of eight to nine wide anterior coils (approximately 125 nm diameter), and three to four narrow posterior coils (approximately 70 nm diameter). Transverse sections revealed a concentric layer organization with the internal layer surrounded by numerous (up to 25) longitudinal microfibrils. The angle of tilt of the polar filament was about 65-68 degrees.     

Genetic heterogeneity in internal transcribed spacer genes of Balantidium coli (Litostomatea, Ciliophora)

The species Balantidium coli is the only ciliate that parasitizes humans. It has been described in other primates, and it has been proposed that the species B. suis from pigs and B. struthionis from ostriches are synonyms of B. coli. Previous genetic analysis of pig and ostrich Balantidium isolates found a genetic polymorphism in the ITS region but its taxonomic relevance was not established. We have extended the genetic analysis to Balantidium isolates of pig, gorilla, human and ostrich origin. We have PCR-amplified and sequenced the ITS region of individual Balantidium cells. The predicted ITS secondary structures of the sequences obtained were transferred by homology modelling to the sequences of other Trichostomatia ciliates (Isotricha, Troglodytella, Lacrymaria and Spathidium) and compared to determine the importance of the differences in the primary sequences. The results show that the ITS2 secondary structure of the species considered follows the general pattern of other ciliates, although with some deviations. There are at least two main types of ITS sequence variants in B. coli which could be present in the same cell and they are common to the mammal and avian hosts studied. These data do not support B. suis and B. struthionis as distinct species.     

Distribution of alpha7 and alpha4 nicotinic acetylcholine receptor subunits in several tissues of Triturus carnifex (Amphibia, Urodela)

The distribution of neuronal and non-neuronal mRNAs for alpha7 and alpha4 nicotinic acetylcholine receptor subunits was investigated in Triturus carnifex tissues using the in situ hybridization approach. The findings reveal a composite pattern of expression only partially overlapping for the two subunits; subunit alpha7 seems to be expressed widely throughout nervous, gastrointestinal and skin tissues, while alpha4 is present in a restricted number of cells of nervous and gastrointestinal tissue. We also found a specific pattern for each subunit; alpha7 and alpha4 associated exclusively to the epidermal glands and hypophysis, respectively; this is probably due to alternative roles that nicotinic acetylcholine receptors play in regulating physiological functions of non-neuronal amphibian tissues, rather than as mere neurotransmitters in the nervous system.     

新建迭部蛤(新名) (Tewomorpha)替代晚出名)(Tewoella Ding and Li, 1987 (非)(Tewoella Sun, 1979)

1993年, 于菁珊和董国义为辽宁上三叠统的非海相双壳类化石建立了一个新属辽宁蚌Liaoningia Yu and Dong, 1993, 以Liaoningia opima Yu and Dong, 1993作为模式种。然而, 早在1979年, 邢裕盛和刘桂芝就已为辽宁晚前寒武纪南关岭组所产的化石标本创建了新属名辽宁水母属Liaoningia Xing and Liu, 1979, 模式种是Liaoningia fuxianensis Xing and Liu, 1979。然而, 多数学者反对将它视为水母类化石, 有人怀疑辽宁水母属是假化石, 迄今学术界并无统一意见, 故本文暂将它归为疑问化石(Problematica)。根据“国际动物命名法规”, 我们提出一个新的属名Liaoningoconcha nom. nov., 用以替代Liaoningia Xing and Liu, 1979的次同义名Liaoningia Yu and Dong, 1993, 仍使用于菁珊和董国义在1993年指定的模式种, 中文译名不变。     

Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics

Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the speciesS. officinarum andS. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n+n transmission of the parental chromosomes instead of the peculiar 2n+n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n=107–115) about 10% were identified as originating fromS. spontaneum and about 10% were identified as recombinant chromosomes between the two speciesS. officinarum andS. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.     

Occurrence of Conchophthirus acuminatus (Protista: Ciliophora) in Dreissena polymorpha (Mollusca: Bivalvia) along the River Shannon, Ireland

Samples of Dreissena polymorpha were collected at several sites along the River Shannon navigation in Ireland, to determine the occurrence and distribution of their obligate host-specific commensalistic ciliate, Conchophthirus acuminatus, in this newly invaded region. Mussels collected by various methods were fixed immediately in 75% ethanol, in which they were later dissected under a stereoscopic microscope, beginning with thorough flushing of the mantle cavity and removal of the gills. One ml of sediment flushed from the mantle cavity and dissection residue of each mussel was examined under a compound light microscope using brightfield, phase-contrast, and differential-interference-contrast optics. Of 180 mussels examined, 125 (69.44%) harbored C. acuminatus. The ciliates were invariably well fixed and easily identifiable in all preparations. Mean sampling intensity for infected mussels was 8.47 ciliates per ml of sediment. Both prevalence and sampling intensity varied between sites, but no pattern was discernible. The present results are consistent with other reports of C. acuminatus being the most widespread and abundant symbiont of D. polymorpha throughout Europe, often occurring where no other symbionts occur. Its occurrence in Ireland indicates introduction of the mussels as adults, since planktonic veliger larvae are not known to harbor ciliates. Following similar reasoning, it is possible that the earlier North American invasion by D. polymorpha included only veligers, since C. acuminatus has not been found on that continent. Using these simple and quick methods, the ciliates could be easily identified and counted to give general comparative data among sites regarding intensity and prevalence. Thus, this method has promise for future efforts to obtain basic information rapidly in newly invaded systems.     

Temperature and host-plant effects on development and population growth of Mecinus janthinus (Coleoptera: Curculionidae), a biological control agent for invasive Linaria spp.

Mecinus janthinus Germar is a European stem-mining weevil that has been established in North America as a biological control agent against the invasive European weeds Linaria vulgaris P. Mill. and Linaria dalmatica (L.) P. Mill. (Scrophulariaceae). Establishment success and impact of the weevil have varied widely among sites. We investigated the hypothesis that some of this variation may be due to a lack of sufficient time for M. janthinus to develop to the adult (overwintering) stage in less favorable climates. Development time of M. janthinus was measured in L. vulgaris and L. dalmatica at four constant temperatures, and logistic regression was used to derive a model for the effect of temperature on development. Development rates were simulated using historic climate data for a site in central Alberta (where establishment was marginal on L. vulgaris) and one in southern British Columbia (where outbreaks occurred, resulting in heavy damage to L. dalmatica). The model showed that, on average, the British Columbia site had 50 more days available for the weevil to lay eggs that could reach the adult stage in time for overwintering than did the Alberta site. This may explain the more rapid population buildup at the British Columbia site. This model could be used to predict the climatic suitability of other areas for establishment of M. janthinus. An unexplained result was the very low survival rate of eggs laid in L. dalmatica under the same experimental conditions.     

Mallomonas transsylvanica,spec. nova (Chrysophyceae), light and electron microscopical studies

The new speciesMallomonas transsylvanica is described in detail. Its silica armour has been examined by light and electron microscopy. Differences and possible relationships with other species are discussed.     

Cchobo, a hobo-related sequence in Ceratitis capitata

A hobo-related sequence, Cchobo, with high similarity to the Drosophila melanogaster HFL1 and hobo₁₀₈ elements was isolated from the medfly. Thirteen PCR-derived clones, which share 97.9–100% DNA identity, were sequenced, seven of which do not show frame-shift or stop codon mutations in their conceptual translations. The consensus sequence has 99.7% DNA identity with the D. melanogaster hobo element HFL1. In a phylogenetic analysis with other hobo-related elements, Cchobo clusters with the HFL1 and hobo₁₀₈ elements from D. melanogaster and hobo-related elements from D. simulans, D. mauritiana and Mamestra brassicae. These elements may have undergone horizontal transfer in the recent past. The genomic distribution of Cchobo was studied by FISH to mitotic and polytene chromosomes, which revealed that Cchobo is distributed within both the heterochromatin and euchromatin. Intra- and interstrain polymorphisms were detected both at euchromatic and heterochromatic sites. These findings suggest that active copies of the element may be present in the medfly genome.     

Poly(A)+ RNA and cytoskeleton during cyst formation in the cap ray of Acetabularia peniculus

Summary. The configuration and distribution of polyadenylated RNA (poly(A)⁺ RNA) during cyst formation in the cap rays of Acetabularia peniculus were demonstrated by fluorescence in situ hybridization using oligo(dT) as a probe, and the spatial and functional relationships between poly(A)⁺ RNA and microtubules or actin filaments were examined by immunofluorescence microscopy and cytoskeletal inhibitor treatment. Poly(A)⁺ RNA striations were present in the cytoplasm of early cap rays and associated with longitudinal actin bundles. Cytochalasin D destroyed the actin filaments and caused a dispersal of the striations. Poly(A)⁺ RNA striations occurred in the cytoplasm of the cap rays up to the stage when secondary nuclei migrated into the cap rays, but they disappeared after the secondary nuclei were settled in their positions. At that time, a mass of poly(A)⁺ RNA was present around each of the secondary nuclei and accumulated rRNA. This mass colocalized with microtubules radiating from the surface of each secondary nucleus and disappeared when the microtubules were depolymerized by butamifos, which did not affect the configuration of actin filaments. These masses of poly(A)⁺ RNA continued to exist even after the cap ray cytoplasm divided into cyst domains. Thus two distinct forms of poly(A)⁺ RNA population, striations and masses, appear in turn at consecutive stages of cyst formation and are associated with distinct cytoskeletal elements, actin filaments and microtubules, respectively. Correspondence and reprints: Graduate School of Kuroshio Science, Kochi University, 2-5-1 Akebono-cho, Kochi 780-8520, Japan.     

The ultrastructure and reproduction of Amphiamblys capitellides (Microspora, Metchnikovellidae), a parasite of the gregarine Ancora sagittata (Apicomplexa, Lecudinidae), with redescription of the species and comments on the taxonomy

The ultrastructural cytology and reproduction of the hyperparasitic microsporidium Amphiamblys capitellides (Caullery and Mesnil, 1897) is described. Merogonial reproduction was not observed. The sporogony comprises two sequences: a sac-bound sporogony in close contact with the cytoplasm of the host and a free sporogony in parasitophorous vacuoles. The free sporogony, which probably precedes the sac-bound, yields a small number of rounded spores. The sac-bound sporogony is polysporoblastic, generating two rows of elongated spores. All stages have isolated nuclei. Both spore types have an extrusion apparatus of the metchnikovellidean type, with a polar sac devoid of anchoring disc, a polar filament with one manubroid and one bulbous part, and a posterior semicircular membrane fold enclosing rounded or tubular structures. Hosts are gregarines of the species Ancora sagittata living in the intestine of polychaetes of the genus Capitella, probably the species Capitella giardi. The cytology, life cycle and classification are discussed. The species is redescribed and the diagnosis of the genus Amphiamblys Caullery and Mesnil, 1914 is emended.