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.     

Field and laboratory studies of Ophryoglena sp. (Ciliata: Ophryoglenidae) infection in zebra mussels,Dreissena polymorpha (Bivalvia: Dreissenidae)

This study, conducted in the Dnieper-Bug Canal in Belarus, is the first to monitor the seasonal (June-November) dynamics of infection with the parasitic ciliate Ophryoglena sp. in a zebra mussel (Dreissena polymorpha) population. Mean population prevalence and intensity of infection varied, respectively, from 11 to 62% and from 0.9 to 24.1 ciliates/mussel. Mean prevalence was highly correlated with mussel length in mussels <20 mm (R(2)=0.97) and was lower in larger mussels. Mean infection intensity in mussels 1-25 mm long was similarly correlated with their size (R=0.98), reached a maximum in the 20-25 mm size-class, and then sharply decreased, thus providing evidence, albeit limited, that high intensity of infection might be lethal. Transinfection of zebra mussels by Ophryoglena sp. was achieved in the laboratory-a first for a protozoan parasite of D. polymorpha; from an initial complete lack of infection, mean prevalence and intensity rose, respectively, to 86.7% and 8.3 ciliates/mussel.     

Analysis of expressed sequence tags (ESTs) and gene expression changes under different growth conditions for the ciliate Anophryoides haemophila, the causative agent of bumper car disease in the American lobster (Homarus americanus)

The scuticociliate Anophryoides haemophila, causes bumper car disease in American lobster (Homarus americanus) in commercial holding facilities in Atlantic Canada. While the parasite has been recognized since the 1970s and much has been learned about its biology, minimal molecular characterization exists. With genome consortiums turning to model organisms like the ciliates Tetrahymena and Paramecium, the amount of relevant sequence data available has made sequence surveys more attractive for gene discovery in related ciliates. We sequenced 9984 expressed sequence tags (ESTs) from a non-normalized A. haemophila cDNA library to characterize gene expression patterns, functional gene distribution and to discover novel genes related to the parasitic life history. The A. haemophila ESTs were grouped into 843 clusters and singletons with 658 EST clusters having identifiable homologs, while 159 ESTs were unique and had no similarity to any sequences in the public databases. Not unexpectedly, about 67% of the A. haemophila ESTs have similarity to annotated and hypothetical genes from the related oligohymenophorean ciliate, Tetrahymena. Numerous cysteine proteases, hypothetical proteins and novel sequences possess putative secretory signal peptides suggesting that they may contribute to the pathogenesis of bumper car disease in lobster. Real time RT-qPCR analysis of cathepsin L and two homologs of cathepsin B did not show any changes in gene expression under varying in vitro growth conditions or during a modified-in vivo infection which may be suggestive of the opportunistic life history strategy of this ciliate.     

Studies on ciliated protozoa in eutrophic lakes: 2. Field and laboratory studies on the effects of oxygen and other chemical gradients on ciliate distribution

An investigation into the spatial distribution of hypolimnetic ciliates in three small eutrophic lakes during the period of summer stratification was carried out. Peak ciliate densities were found to occur at the oxic/anoxic boundary, ciliate numbers declining with increasing depth within the hypolimnion. The ciliates only occurred in aerobic water where oxygen levels were less than about 0.5 mgl^–1 Laboratory experiments demonstrated that the ciliates swim upwards under anaerobic conditions but swim rapidly downwards under aerobic conditions. Further laboratory experiments showed that although the bulk of the population occured within anaerobic water, the hypolimnetic ciliates are aerobes and cannot survive indefinite anoxia. Despite the demonstrable toxicity of high levels of ammonia and sulphide, it was probably excesive distance from an available source of oxygen that excluded the ciliates from the lowest levels of the hypolimnion. Possible mechanisms which allowed these aerobic ciliates to colonise anaerobic water are considered.     

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.     

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.     

First observations of predation by New Zealand Greenshell mussels (Perna canaliculus) on zooplankton

Observations made overseas of predation by blue mussels and zebra mussels on mesozooplankton (>200 μm) have raised concern within New Zealand that the Greenshell mussel, Perna canaliculus, which is cultured in large tonnages throughout hundreds of marine farms within the New Zealand coastal zone, could exert ecologically detrimental effects by preying on zooplankton. We conducted experiments at Clova Bay, Pelorus Sound in May 2002 to determine the rates that P. canaliculus ingests prey, up to and including the mesozooplankton size range. Single mussels from farms were incubated with seawater enriched with zooplankton (>60 μm) in gently circulated 15-l pails. Depletion of chlorophyll-a (chl-a), ciliate microzooplankton, and nauplii, copepodites, and adults of copepods was determined over 5 h, relative to controls with no mussels. Two experiments were made over consecutive days. Gut contents of these experimental mussels, and of mussels examined soon after collection from a farm, were described.Gut contents of experimental and of freshly collected mussels (standard shell length ∼90 mm) had numerous copepod parts, whole copepods and larval bivalves present. Experimental mussels cleared chl-a and ciliates from 59- to 137-l individual⁻¹ day⁻¹, respectively, averaged across the two experiments. Faster ciliate than chl-a clearance was probably caused by the high proportion (56%) of phytoplankton below the retention size for P. canaliculus (ca. 5 μm) and by faster ciliate grazing in controls than treatments. The average clearance rates of adult, copepodite, and naupliar copepod stages by mussels were 20, 31, and 49 l individual⁻¹ day⁻¹, respectively. The clearance rates of each copepod stage were not significantly different between the two experiments. Clearance of nauplii was significantly greater than of adults and copepodites, while adult and copepodite clearance rates were nearly significantly different. The mean lengths of the adult, copepodite, and naupliar copepods were 430, 265, and 165 μm, respectively. The decreasing clearance rates with increasing size and development of prey (from ciliates, through naupliar, copepodite to adult copepods), suggested that prey escape ability, related to body size and/or morphology, affected capture rates. Mussel faecal samples indicated complete digestion of the gut contents. Pseudofaecal samples showed very low rejection rates of mesozooplankton by mussels. The results are considered in context of current biophysical modelling studies of impacts of large mussel farms in New Zealand. Designs of future experiments to improve accuracy of estimates of mesozooplankton clearance rates by P. canaliculus are considered.     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.     

Sequence conservation in the internal transcribed spacers and 5.8S ribosomal RNA of parasitic scuticociliates Miamiensis avidus (Ciliophora, Scuticociliatia)

The scuticociliate Miamiensis avidus is a histophagous parasite that causes high mortality in cultured marine fishes, with clinical signs of severe ulcers and hemorrhages in the skeletal muscle. The internal transcribed spacer (ITS) region, which is widely used in taxonomy and molecular phylogeny because of a high degree of variation, was compared for 21 cloned strains of M. avidus (Ciliophora, Scuticociliatia). These strains were isolated from olive flounder Paralichthys olivaceus, ridged-eye flounder Pleuronichthys cornutus and spotted knifejaw Oplegnathus fasciatus in Korea and Japan. The ITS1 (140 bp), ITS2 (236 bp) and 5.8S (119 bp) regions from 21 strains were identical, indicating that these regions are highly conserved in M. avidus. Phylogenic analysis of ITS2 shows that the ciliate should be included in the Philasterida with a close relationship to Pseudocohnilembus hargisi. This study exhibits the first detailed analysis of the ITS1, 5.8 S and ITS2 rRNA regions of M. avidus.     

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2

In eukaryotes, mature rRNA sequences are produced from single large (45S) precursor (pre-rRNA) as the result of successive removal of spacers through a series of rapid and intricate actions of endo- and exonucleases. The excision of internal transcribed spacer (ITS2), a eukaryotic-specific insertion, remains the most elusive processing step. ITS2 is the element mandatory for all eukaryotic pre-rRNAs that contain at least three processing cleavage sites for precise 5.8S and 28S formation. Conserved core sequences (cis-elements) binding to trans-factors provide for precise rRNA processing, whereas rapidly diverging regions between the core sequences preserve internal complementarity, which guarantees the spatial integrity of ITS2. Characteristic differences in the formation of such insertions during evolution should reflect the relationships between taxa. The phylogeny of the reptiles and the relationships between taxa proposed by scientists are controversial. To delineate the structural and functional features preserved among reptilian ITS2s, we cloned and sequenced 58 ITS2s belonging to four reptile orders: Squamata, Crocodilians, Aves, and Testudines. We studied the subsequent alignment and folding of variable regions. The sizes and packing of the loop–stems between conserved consensus segments in reptiles vary considerably between taxa. Our phylogenetic trees constructed on the basis of the reptile ITS2s primary structural alignments revealed a split between Iguania clade and all other taxa. True lizards (suborder Scleroglossa) and snakes (suborder Serpentes) show sister relationships, as well as the two other reptilian orders, Crocodilia + Aves and Testudines. In summary, our phylogenetic trees exhibit a mix of specific features deduced or, to the contrary, rejected earlier by other authors.     

Genetic characterization of Zostera asiatica on the Pacific Coast of North America

We gathered sequence information from the nuclear 5.8S rDNA gene and associated internal transcribed spacers, ITS-1 and ITS-2 (5.8S rDNA/ITS), and the chloroplast maturase K (matK) gene, from Zostera samples collected from subtidal habitats in Monterey and Santa Barbara (Isla Vista) bays, California, to test the hypothesis that these plants are conspecific with Z. asiatica Miki of Asia. Sequences from approximately 520 base pairs of the nuclear 5.8S rDNA/ITS obtained from the subtidal Monterey and Isla Vista Zostera samples were identical to homologous sequences obtained from Z. marina collected from intertidal habitats in Japan, Alaska, Oregon and California. Similarly, sequences from the matK gene from the subtidal Zostera samples were identical to matK sequences obtained from Z. marina collected from intertidal habitats in Japan, Alaska, Oregon and California, but differed from Z. asiatica sequences accessioned into GenBank. This suggests the subtidal plants are conspecific with Z. marina, not Z. asiatica. However, we found that herbarium samples accessioned into the Kyoto University Herbarium, determined to be Z. asiatica, yielded 5.8S rDNA/ITS sequences consistent with either Z. japonica, in two cases, or Z. marina, in one case. Similar results were observed for the chloroplast matK gene; we found haplotypes that were inconsistent with published matK sequences from Z. asiatica collected from Japan. These results underscore the need for closer examination of the relationship between Z. marina along the Pacific Coast of North America, and Z. asiatica of Asia, for the retention and verification of specimens examined in scientific studies, and for assessment of the usefulness of morphological characters in the determination of taxonomic relationships within Zosteraceae.     

Highly divergent SSU rRNA genes found in the marine ciliates Myrionecta rubra and Mesodinium pulex

Myrionecta rubra and Mesodinium pulex are among the most commonly encountered planktonic ciliates in coastal marine and estuarine regions throughout the world. Despite their widespread distribution, both ciliates have received little attention by taxonomists. In order to better understand the phylogenetic position of these ciliates, we determined the SSU rRNA gene sequence from cultures of M. rubra and M. pulex. Partial sequence data were also generated from isolated cells of M. rubra from Chesapeake Bay. The M. rubra and M. pulex sequences were very divergent from all other ciliates, but shared a branch with 100% bootstrap support. Both species had numerous deletions and substitutions in their SSU rRNA gene, resulting in a long branch for the clade. This made the sequences prone to spurious phylogenetic affiliations when using simple phylogenetic methods. Maximum likelihood analysis placed M. rubra and M. pulex on the basal ciliate branch, following removal of ambiguously aligned regions. Fluorescent in situ hybridization probes were used with confocal laser scanning microscopy to confirm that these divergent sequences were both expressed in the cytoplasm and nucleolus of M. ruisra and M. pulex. We found that our sequence data matched several recently discovered unidentified eukaryotes in Genbank from diverse marine habitats, all of which had apparently been misattributed to highly divergent amoeboid organisms.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.     

Intestinal ciliate composition found in the feces of the Turk rahvan horse Equus caballus, Linnaeus 1758

Species composition and distribution of large intestinal ciliates were investigated in the feces from 15 Turk rahvan horses, living in the vicinity of Izmir, Turkey. Twenty-two ciliate genera consisting of 36 species were identified. This is the first report on intestinal ciliates in Turk rahvan horses and no previously unknown species were observed. The mean number of ciliates was 14.2 ± 13.9 × 10⁴ cells ml⁻¹ of feces and the mean number of ciliate species per host was 9.9 ± 7.1. No ciliates were observed in 2 horses. Bundleia and Blepharocorys were considered to be the major genera since these ciliates were constantly found in high proportions. In contrast, Paraisotricha, Didesmis and Gassovskiella were only observed at low frequencies. The ciliates found in this survey had almost the same characteristics as those described in previous reports, suggesting that there was no significant geographic variation in the intestinal ciliate fauna of equids.     

Diversity and Distributional Patterns of Ciliates in Guaymas Basin Hydrothermal Vent Sediments

Little is known about protists at deep‐sea hydrothermal vents. The vent sites at Guaymas Basin in the Gulf of California are characterized by dense mats of filamentous pigmented or nonpigmented Beggiatoa that serve as markers of subsurface thermochemical gradients. We constructed 18S rRNA libraries to investigate ciliate assemblages in Beggiatoa mats and from bare sediments at the Guaymas vent site. Results indicated a high diversity of ciliates, with 156 operational taxonomic units identified in 548 sequences. Comparison between mat environments demonstrated that ciliate and bacterial assemblages from pigmented mats, nonpigmented mats, and bare sediments were significantly different and highly correlated with bacterial assemblages. Neither bacterial nor ciliate assemblages were correlated with environmental factors. The most abundant ciliates at Guaymas were more likely to be represented in clone libraries from other hydrothermal, deep‐sea, and/or anoxic or microaerophilic environments, supporting the hypothesis that these ciliate species are broadly distributed. The orange mat environment included a higher proportion of ciliate sequences that were more similar to those from other environmental studies than to cultured ciliate species, whereas clone libraries from bare sediments included sequences that were the most highly divergent from all other sequences and may represent species that are endemic to Guaymas.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.     

The symbiont Capsaspora owczarzaki,nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea

While investigating the resistance of some strains of Biomphalaria glabrata to infection with Schistosoma mansoni, a unicellular eukaryotic symbiont was noted in the snail haemolymph. It was similar in appearance to Nuclearia sp. reported from B. glabrata. Sequences comprising the 18S, ITS1, 5.8S, ITS2 and the beginning of the 28S rDNA gene regions were obtained from symbionts isolated from three strains of B. glabrata, and compared with the same sequences obtained from a culture of Nuclearia sp. 18S rDNA sequences were identical for all four isolates. 18S rDNA sequences were used in a phylogenetic analysis to produce minimum evolution, maximum parsimony, maximum likelihood and Bayesian trees. All four analyses indicated that the B. glabrata symbiont is not closely related to Nuclearia but instead to the Mesomycetozoea, a recently recognised clade of symbiotic eukaryotes. Based on phylogenetic analysis, life history and morphological differences, the symbiont is described as a new genus and species, Capsaspora owczarzaki. Distinguishing characters are the presence of life cycle stage(s) that occur within snail haemolymph; ability to kill and ingest digenetic trematode larvae; ability to undergo asexual fission to produce daughter cells; absence of flagella, a mucous sheath and membranes containing chitin, elastin, or collagen; and presence of long unbranching pseudopodia and a penetration process. Using both polymerase chain reaction (PCR) and culturing techniques, the S. mansoni-resistant Salvador and 13-16-R1 strains were found to be significantly more likely to harbour the symbiont than the susceptible M line strain. Small but consistent sequence differences were noted among symbiont isolates from different snail strains, raising the possibility that the symbiont has diverged in different snail lineages. This suggests further that the symbiont is not restricted to albino lab-reared snails. A role, if any, of the symbiont in resistance awaits further study.     

Experimental infections of Orchitophrya stellarum (Scuticociliata) in American blue crabs (Callinectes sapidus) and fiddler crabs (Uca minax)

Outbreaks of an unidentified ciliate have occurred on several occasions in blue crabs from Chesapeake Bay held during winter months in flow-through systems. The parasite was initially thought to be Mesanophrys chesapeakensis, but molecular analysis identified it as Orchitophyra stellarum, a facultative parasite of sea stars (Asteroidea). We investigated the host-parasite association of O. stellarum in the blue crab host. Crabs were inoculated with the ciliate, or they were held in bath exposures after experimentally induced autotomy of limbs in order to determine potential mechanisms for infection. Crabs inoculated with the ciliate, or exposed to it after experimental autotomy, rapidly developed fatal infections. Crabs that were not experimentally injured, but were exposed to the ciliate, rarely developed infections; thus, indicating that the parasite requires a wound or break in the cuticle as a portal of entry. For comparative purposes, fiddler crabs, Uca minax, were inoculated with the ciliate in a dose-titration experiment. Low doses of the ciliate (10 per crab) were sometimes able to establish infections, but high intensity infections developed quickly at doses over 500 ciliates per crab. Chemotaxis studies were initiated to determine if the ciliate preferentially selected blue crab serum (BCS) over other nutrient sources. Cultures grown on medium with BCS or fetal bovine serum showed some conditioning in their selection for different media, but the outcome in choice experiments indicated that the ciliate was attracted to BCS and not seawater. Our findings indicate that O. stellarum is a facultative parasite of blue crabs. It can cause infections in exposed crabs at 10–15 °C, but it requires a portal of entry for successful host invasion, and it may find injured hosts using chemotaxis.     

Conservation of sequence in the internal transcribed spacers and 5.8S ribosomal RNA among geographically separated isolates of parasitic scuticociliates (Ciliophora, Orchitophryidae)

Nucleotide sequence from the internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene from the ribosomal RNA gene cluster of isolates of the scuticociliate Orchitophrya stellarum from 4 asteroid hosts were compared. Surprisingly, these data (495 bp) were identical for O. stellarum isolated from the testes of Asterias amurensis from Japan; Pisaster ochraceus from British Columbia, Canada; Asterias rubens from The Netherlands; and Asterias vulgaris from Prince Edward Island, Canada. These sequence data were compared to those from 3 scuticociliates which parasitise crustaceans: Mesanophrys pugettensis, M. chesapeakensis and Anophryoides haemophila. No difference was found in this region between the nucleotide sequence of M. pugettensis and M. chesapeakensis. The sequence of Mesanophrys spp. differed by 9.2% in the ITS1 and 4.7% in the ITS2 from that of O. stellarum. The sequence from the ITS1 (135 bp) and ITS2 (233 bp) of A. haemophila differed by 42.6 and 20.5% respectively from those of O. stellarum. Therefore, nucleotide sequence of the ITS regions in these scuticociliates is highly conserved.