Taxonomy and molecular phylogeny of a new freshwater ciliate Frontonia apoacuminata sp. nov. (Protista, Ciliophora, Oligohymenophorea) from Qingdao, PR China.

The morphology and ciliature of a new freshwater ciliate, Frontonia apoacuminata sp. nov., isolated from an artificial pond in Qingdao, PR China, were investigated using live observation and silver staining methods. The main features separating F. apoacuminata sp. nov. from its congeners are as follows: a broad elliptical body that is slightly pointed at the posterior end, four ophryokineties, one or two spherical micronuclei of a 'compact' type, a dorsally positioned contractile vacuole, and peniculi 1-3 each with five kinetosome rows though the left-most two rows in peniculus 3 are extremely shortened (with only two or three kinetosomes each). Additionally, an improved diagnosis of F. acuminata is provided. Phylogenetic analyses based on the small subunit ribosomal RNA (SSU rRNA) gene show that F. apoacuminata sp. nov. clusters with F. atra, F. minuta, F. acuminata and F. terricola. These five species group with Disematostoma, Marituja and Stokesia rather than with other Frontonia species, causing polyphyly of the genus Frontonia.

Molecular characterization and phylogenetic analyses of Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970 based on 18S rRNA gene sequence data.

In the present study, we provided the first 18S rRNA gene sequence data of two Tripartiella species, Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970, which were isolated from Tachysurus fulvidraco (Richardson, 1846) and Hemibarbus maculatus Bleeker, 1871 in Chongqing, China, respectively. Morphologically, both species fall within the morphometry range of the original descriptions and are very similar to the original populations in the overall appearance of the adhesive disc. Tripartiella macrosoma can be easily distinguished from the other Tripartiella species by possessing the denticle with a long strip and conspicuously inclined backward blade and a robust and short ray. Tripartiella obtusa is mainly characterized by a broad blade and a relatively long ray. Phylogenetically, T. macrosoma clustered with Trichodinella myakkae (Mueller, 1937) Raabe, 1950 and further with Trichodinella sp., which was sister to a group that includes four populations of Trichodinella epizootica (Raabe, 1950) Srámek-Husek, 1953; finally, they formed a small clade with T. obtusa. This result suggested that T. macrosoma had a closer relationship with Trichodinella spp. than with T. obtusa and T. obtusa diverged earlier than T. macrosoma and Trichodinella spp. By combining morphological and molecular data, the polyphyletic characteristics of Tripartiella and Trichodinella were further analyzed, and the results revealed that the validity of the genus Tripartiella is doubtful.

First report of Trichodinella and new geographical records of trichodinids in Nile tilapia (Oreochromis niloticus) farmed in Brazil / Primeiro relato de Trichodinella e novos registros geográficos de tricodinídeos em tilapia do Nilo (Oreochromis niloticus) cultivada no Brasil

Abstract Massive occurrence of trichodinids is frequently accompanied by serious disease in fish farms. In this study, trichodinid species from the gills and skin of Nile tilapia (Oreochromis niloticus) farmed in the central-western region of Brazil (state of Goiás) were morphologically characterized. Dried slides were prepared from the parasites and were impregnated with silver nitrate (2%). Morphometric characteristics were determined and schematic drawings of the denticles were made using photomicrographs produced from the slides. Seven species of trichodinid ectoparasites (Protozoa: Ciliophora: Trichodinidae) were found parasitizing the gills: four of the genus Trichodina Ehrenberg, 1838; one of Tripartiella Lom, 1959; one of Paratrichodina Lom, 1963; and one of Trichodinella Ṧrámek-Huṧek, 1953. On the body surface, three specimens of the genus Trichodina were identified. This study presents new geographical records of trichodinids in Brazil, thus confirming that Trichodina centrostrigeata, Trichodina compacta, Trichodina heterodentata, Paratrichodina africana and Tripartiella orthodens are widely distributed worldwide. Additionally, the first record of the genus Trichodinella in Brazil is presented.

Resumo O parasitismo intenso por tricodinídeos está frequentemente relacionado à doença grave em fazendas de peixes. Neste estudo, espécies de tricodinídeos das brânquias e da pele de tilápias do Nilo (Oreochromis niloticus) cultivadas na região centro-oeste do Brasil (estado de Goiás) foram caracterizadas morfologicamente. As lâminas secas foram preparadas a partir dos parasitas e impregnadas com nitrato de prata (2%). As características morfométricas foram determinadas e desenhos esquemáticos dos dentículos foram confeccionados com fotomicrografias produzidas a partir das lâminas. Sete espécies de ectoparasitos tricodinídeos (Protozoa: Ciliophora: Trichodinidae) foram encontradas parasitando as brânquias: quatro do gênero Trichodina Ehrenberg, 1838; um de Tripartiella Lom, 1959; um de Paratrichodina Lom, 1963; e um de Trichodinella Ṧrámek-Huṧek, 1953. Na superfície do corpo, três espécimes do gênero Trichodina foram identificados. Este estudo apresenta novos registros geográficos de tricodinídeos no Brasil, confirmando que Trichodina centrostrigeata, Trichodina compacta, Trichodina heterodentata, Paratrichodina africana e Tripartiella orthodens estão amplamente distribuídas mundialmente. Adicionalmente, é apresentado o primeiro registro do gênero Trichodinella no Brasil.

Detection and Quantification of Two Parasitic Ciliates Boveria labialis and Boveria subcylindrica (Ciliophora: Scuticociliatia) by Fluorescence in situ Hybridization.

The thigmotrich scuticociliates Boveria labialis and Boveria subcylindrica are obligate parasites that may cause high mortality in cultured sea cucumbers and bivalves. Morphological methods can identify these organisms in active state, but are unable to discern them in resting stages. In aquaculture practice, these parasitic ciliates are hard to eradicate when massive infection occurs in sea cucumbers. Thus, early detection and precaution are crucial for the control of these pathogens. Under such circumstances, fluorescence in situ hybridization (FISH) will serve as a fast way to detect and monitor the occurrence of these parasites. We designed two SSU-rDNA targeted oligonucleotide probes labeled with fluorochromes, and optimized the FISH protocols for the detection of B. labialis and B. subcylindrica from the host sea cucumber Apostichopus japonicus and the bivalve Atrina pectinata, respectively. The assays resulted in a clear differentiation of the two similar species by strong fluorescence signals from the oligonucleotide probes. Moreover, we successfully used the FISH protocol to detect the cysts of B. labialis and variation in abundance of active parasites to evaluate the efficacy of chemical treatments. This is the first report and detection of the cysts of B. labialis from the host sea cucumber A. japonicus.

Zoothamnium duplicatum infestation of cultured horseshoe crabs (Limulus polyphemus).

An outbreak of the sessile peritrich Zoothamnium duplicatum in a pilot, commercial-scale Limulus polyphemus hatchery resulted in the loss of ∼96% (40,000) second/third instar larvae over a 61day period. peritrich growth was heavy, leading to mechanical obstruction of the gills and physical damage. The peritrichs were controlled without resultant loss of juvenile crabs by administering 10ppm chlorine in freshwater for 1h and the addition of aquarium grade sand; a medium into which the crabs could burrow and facilitate cleaning of the carapace. Peritrich identity was confirmed from a partial SSU rDNA contiguous sequence of 1343bp (99.7% similarity to Z. duplicatum).

First record of an epibiont protozoan Epistylis sp. (Ciliophora, Peritrichia) attached to Ergasilus chelangulatus (Ergasilidae) in Brazil / Primeiro registro do protozoário epibionte Epistylis sp. (Ciliophora, Peritrichia) aderido à Ergasilus chelangulatus (Ergasilidae) no Brasil

In the present paper, we described the first record of an epibiont protozoan Epistylis sp. Ehrenberg, 1830 (Ciliophora, Peritrichia) attached to Ergasilus chelangulatus Thatcher and Brasil-Sato, 2008, parasite of Pimelodus maculatus Lacépède, 1803 in Brazil, with electron microscope observations. Fish were collected in Veados River, state of São Paulo and the crustacean Ergasilus chelangulatus being registered for the first time in this river, expanding its geographical distribution in Brazil.

No presente trabalho nós descrevemos o primeiro registro de um protozoário epibionte Epistylis sp. Ehrenberg, 1830 (Ciliophora, Peritrichia) aderido à Ergasilus chelangulatus Thatcher e Brasil-Sato, 2008, parasito de Pimelodus maculatus Lacépède, 1803 no Brasil, com observações de microscopia eletrônica. Os peixes foram coletados no Rio dos Veados, estado de São Paulo e o crustáceo Ergasilus chelangulatus está sendo registrado pela primeira vez neste rio, ampliando sua distribuição geográfica no Brasil.

Spatial genetic variation, phylogeography and barcoding of the peritrichous ciliate Carchesium polypinum.

Most recent studies of geographic distribution of microbial eukaryotes have focused on marine rather than freshwater protists. Here, we used the freshwater peritrich ciliate Carchesium polypinum to quantify the degree of genetic diversity of four closely related and previously described lineages and to determine whether patterns of genetic differentiation showed geographic partitioning. Using an expanded dataset of 100 isolates and employing the mitochondrial marker cytochrome oxidase c subunit I (cox-1), we enriched the 6 previously identified clades of Carchesium polypinum. We found a large degree of geographic overlap among the different clades (e.g. to the level of range of sampling), but also a spatially restricted clade (e.g. to the level of one river basin). Furthermore, we present evidence of a clear geographic separation in one of the lineages with Canadian and North Carolinian isolates grouping in two distinct clusters.

Trichodina nobilis Chen, 1963 and Trichodina reticulata Hirschmann et Partsch, 1955 from ornamental freshwater fishes in Brazil / Trichodina nobilis Chen, 1963 e Trichodina reticulata Hirschmann et Partsch, 1955 de peixes ornamentais de água doce no Brasil

In the present work Trichodina reticulata and T. nobilis (Ciliophora: Trichodinidae) are morphologically characterised from ornamental freshwater fish culture in the State of Santa Catarina, Brazil. The prevalence of infection and a list of comparative measurements are discussed. We examined "southern platyfish" Xiphophorus maculatus (n = 35), "goldfish" Carassius auratus (n = 31), "guppy" Poecilia reticulata (n = 20), "sailfin molly" Poecilia latipinna (n = 6), "beta" Betta splendens (n = 2) and "spotted headstander" Chilodus punctatus (n = 1). After being anesthetised in a benzocaine solution, fishes were examined for parasitological evaluation. A total of 51.57% fishes were parasitised by Trichodina spp. Carassius auratus was the most parasitised species, followed by X. maculatus and P. reticulata. Beta splendens, C. punctatus and P. latipinna were not parasitised by any trichodinid species. Two species of Trichodina were collected from the skin of fish: T. nobilis was found in C. auratus, P. reticulata and X. maculatus and T. reticulata was only observed in C. auratus. The importance of adequate handling in ornamental fish culture are also discussed.

No presente trabalho Trichodina reticulata e T. nobilis (Ciliophora: Trichodinidae) de peixes ornamentais de água doce cultivados no estado de Santa Catarina, Brasil são caracterizadas morfologicamente. A prevalência de infecção e uma lista comparative de medidas são discutidas. Foram examinados "platis" Xiphophorus maculatus (n = 35), "kinguios" Carassius auratus (n = 31), "guppys" Poecilia reticulata (n = 20), "molinésias" Poecilia latipinna (n = 6), "betas" Betta splendens (n = 2) e "espada" Chilodus punctatus (n = 1). Após a anestesia com solução de benzocaína, os peixes foram submetidos à avaliação parasitológca. Um total de 51,57% peixes estavam parasitados por Trichodina spp. Carassius auratus foi a espécie mais parasitada, seguida por X. maculatus e P. reticulata. Beta splendens, C. punctatus e P. latipinna não estavam parasitados por tricodinídeos. Duas species de Trichodina foram coletadas da superfície corporal dos peixes: T. nobilis foi encontrada em C. auratus, P. reticulata e X. maculatus e T. reticulata foi observada apenas em C. auratus. A importância do manejo adequado em cultivos de peixes ornamentais também foi discutida.

First isolation of Pseudocohnilembus persalinus (Ciliophora: Scuticociliatida) from freshwater-reared rainbow trout, Oncorhynchus mykiss.

Ciliated protists were isolated from the ovarian fluid of apparently healthy adult rainbow trout (Oncorhynchus mykiss) maintained in freshwater. The organism was identified as Pseudocohnilembus persalinus based on morphometric and morphological analysis of silver-stained specimens obtained from culture and on analysis of ribosomal RNA gene sequences. The cytochrome c oxidase subunit 1 gene sequence of this organism also was characterized. This ciliate has been reported previously as free living only in saline environments and as an endosymbiont in a marine teleost, the olive flounder (Paralichthys olivaceus). A cyst-like stage may have facilitated the novel occurrence of this organism as an endosymbiont in rainbow trout.

Description of Paratetrahymena parawassi n. sp. using morphological and molecular evidence and a phylogenetic analysis of Paratetrahymena and other taxonomically ambiguous genera in the order Loxocephalida (Ciliophora, Oligohymenophorea).

The marine scuticociliate Paratetrahymena parawassi n. sp. is described on the basis of morphology, especially infraciliature, and the sequence of its small subunit (SSU) rRNA gene to become the second known member of its genus. Paratetrahymena and other ciliates in the order Loxocephalida possess a mixture of morphological and morphogenetic features characteristic of the subclasses Hymenostomatia and Scuticociliatia. Accordingly, we used SSU rRNA sequences to analyze the phylogeny of Paratetrahymena and three other loxocephalid genera. Paratetrahymena and Cardiostomatella vermiformis formed a moderately well-supported clade that diverged at a deep level from all other scuticociliates, supporting separation of loxocephalids from other scuticociliates as a suprafamilial taxon. Sathrophilus holtae was a sister taxon to Paratetrahymena and Cardiostomatella in a poorly supported, unresolved relationship; nevertheless, association of all three genera into a single clade was supported by an approximately unbiased (AU) test. Any association of these genera singly or as a group with the Hymenostomatia was rejected decisively by AU tests and by a complete absence in the loxocephalids of the unique nucleotide identities that distinguish hymenostomes. Therefore, the morphological and morphogenetic similarities of loxocephalids to hymenostomes may be plesiomorphies, and the conflicting mix of scuticociliate and hymenostome characteristics seen in loxocephalids may result from differing rates of character evolution. Dexiotrichides pangi and Urocentrum, which is currently classified as a peniculid, formed a small clade that associated with hymenostomes and peritrichs. Monophyly of the Loxocephalida with Dexiotrichides and/or Urocentrum included was not rejected by AU; however, inclusion of Urocentrum in the Peniculia was rejected by AU tests. A hypothesis is offered to explain the lack of resolution of loxocephalid ciliates and Urocentrum in phylogenetic trees, namely that their phylogenetic positions are influenced by a combination of heterogeneous data and long-branch attraction caused by poor representation of taxa in analyses. The well-known genus Cyclidium, a member of the order Pleuronematida, was revealed to be polyphyletic as a byproduct of our analyses of loxocephalids. In particular, Cyclidium porcatum appears to fall outside the clade containing typical members of the subclass Scuticociliatia and thus invites investigation as a possible member of the order Loxocephalida.

First record of Trichodina heterodentata (Ciliophora: Trichodinidae) from channel catfish, Ictalurus punctatus cultivated in Brazil / Primeiro registro de Trichodina heterodentata (Ciliophora: Trichodinidae) em bagre-do-canal, Ictalurus punctatus cultivado no Brasil

This study characterises morphologically Trichodina heterodentata Duncan, 1977 from channel catfish, Ictalurus punctatus (Rafinesque, 1818) in the State of Santa Catarina, Brazil. Body and gill smears were air-dried at room temperature, impregnated with silver nitrate and/or stained with Giemsa. Ten characteristics were selected to compare the present material with other morphological characterisations of T. heterodentata. Prevalence rate was 100 percent, mean intensity 89,333.70 (3,125 to 299,100 parasites per host). Trichodina heterodentata was considered medium-sized trichodinid with mean body diameter 59.4 ± 8.5 μm, denticulate ring 38.5 ± 4.5 μm, adhesive disc 60.2 ± 6.7 μm diameter and 24.4 ± 1.6 denticles. In relation to previous reports of T. heterodentata this material resembles in 90 percent of the analysed characters. This work confirms the biometrical variation that exists in the different populations of T. heterodentata. A list of hosts and comparative measurements of T. heterodentata are presented and the channel catfish is considered a new host.

Este estudo caracteriza morfologicamente Trichodina heterodentata Duncan, 1977 em bagre-do-canal, Ictalurus punctatus (Rafinesque, 1818) no Estado de Santa Catarina, Brasil. Esfregaços do corpo e brânquias foram secados à temperatura ambiente, impregnados com nitrato de Prata e/ou corados com Giemsa. Dez características foram selecionadas para comparar o presente material com as diferentes caracterizações morfológicas de T. heterodentata. A taxa de prevalência foi de 100 por cento, a intensidade média foi de 89.333.75 (3.125 a 299.100 parasitos por hospedeiro). Trichodina heterodentata foi considerado um tricodinídeo de tamanho médio com a média do diâmetro do corpo de 59,4 ± 8.5 μm, anel denticulado 38,5 ± 4,5 μm, disco adesivo 60,2 ± 6,7 μm de diâmetro e 24,4 ± 1,6 dentículos. Em relação a registros prévios de T. heterodentata, 90 por cento das características foram semelhantes. Este trabalho confirma a variação biométrica que existe em diferentes populações de T. heterodentata. Uma lista de hospedeiros e medidas comparativas de T. heterodentata são apresentadas e o bagre-do-canal considerado um novo hospedeiro.

In vitro culture and cryopreservation of Uronema marinum isolated from farmed New Zealand groper (Polyprion oxygeneios).

An in vitro culture method was developed for the ciliated protozoa Uronema marinum isolated from New Zealand aquacultured groper (Polyprion oxygeneios). Both formulated media and sterile seawater supplemented with homogenised fish tissue as a food source supported growth of U. marinum achieving cell densities of up to 1 x 10(5)cells/mL in culture. A cryopreservation method based on a cryomix formula of 20% glycerol, 10% fetal bovine serum and 70% cultured U. marinum, incorporating a slow freeze method to -80 degrees C, then liquid nitrogen storage, allowed cryogenic storage of cells and successful re-culture up to 12 months in storage.

High-level genetic diversity but no population structure inferred from nuclear and mitochondrial markers of the peritrichous ciliate Carchesium polypinum in the Grand River basin (North America).

Studies that assess intraspecific genetic variation in ciliates are few and quite recent. Consequently, knowledge of the subject and understanding of the processes that underlie it are limited. We sought to assess the degree of intraspecific genetic variation in Carchesium polypinum (Ciliophora: Peritrichia), a cosmopolitan, freshwater ciliate. We isolated colonies of C. polypinum from locations in the Grand River basin in Southwestern Ontario, Canada. We then used the nuclear markers--ITS1, ITS2, and the hypervariable regions of the large subunit rRNA--and an 819-bp fragment of the mitochondrial cytochrome c oxidase I gene (cox-1) to investigate the intraspecific genetic variation of C. polypinum and the degree of resolution of the above-mentioned markers at the population level. We also sought to determine whether the organism demonstrated any population structure that mapped onto the geography of the region. Our study shows that there is a high degree of genetic diversity at the isolate level, revealed by the mitochondrial markers but not the nuclear markers. Furthermore, our results indicate that C. polypinum is likely not a single morphospecies as previously thought.

Intraspecific phylogeography of Carchesium polypinum (Peritrichia, Ciliophora) from China, inferred from 18S-ITS1-5.8S ribosomal DNA.

Based on the variation of site 34, 46, 241, 305 and 322 in the 18S-ITS1 rDNA sequence, 19 Carchesium polypinum populations collected from eight provinces of China were separated into northern and southern population along the delineation between the Yangtze River and the Pearl River. This geographic distribution pattern of Carchesium polypinum maybe results from two factors: the vicariance resulting from the formation of the delineation between the Pearl River and the Yangtze River accompanied with the uplift of Qinghai-Xizang Plateau, and the different dispersal paths of C. polypinum affected by the climate.