Coleps shanghaiensis n. sp. challenges the validity of the genus Levicoleps (Cilophora, Prostomatida).

Coleps is a common genus of pelagic ciliates in freshwater and brackish water habitats. Classification and phylogeny of Coleps species are, however, still full of confusion. In this study, we investigated Coleps shanghaiensis n. sp., collected from a river in Shanghai, China, by living observation, protargol staining, and molecular methods. Coleps shanghaiensis is about 70-90 µm × 35-55 µm in size, has a barrel-shaped body with three posterior spines, and possesses 21-24 ciliary rows, each composed of two perioral dikinetids and 19-22 monokinetids, and six caudal cilia. In SSU rRNA gene phylogenies, C. shanghaiensis fell within the clade of subspecies of Levicoleps biwae, which questions the validity of the genus Levicoleps. Furthermore, the biogeography of the genus Coleps is discussed.

Biogeographical and biodiversity patterns of planktonic microeukaryotes along the tropical western to eastern Pacific Ocean transect revealed by metabarcoding.

Microeukaryotic plankton (0.2-200 µm), which are morphologically and genetically highly diverse, play a crucial role in ocean productivity and carbon consumption. The Pacific Ocean (PO), one of the world's largest oligotrophic regions, remains largely unexplored in terms of the biogeography and biodiversity of microeukaryotes based on large-scale sampling. We investigated the horizontal distribution of microeukaryotes along a 16,000 km transect from the west to the east of the PO. The alpha diversity indices showed a distinct decreasing trend from west to east, which was highly correlated with water temperature. The microeukaryotic community, which was clustered into the western, central, and eastern PO groups, displayed a significant distance-decay relationship. Syndiniales, a lineage of parasitic dinoflagellates, was ubiquitously distributed along the transect and dominated the community in terms of both sequence and zero-radius operational taxonomic unit (ZOTU) proportions. The prevailing dominance of Syndiniales-affiliated ZOTUs and their close associations with dinoflagellates, diatoms, and radiolarians, as revealed by SparCC correlation analysis, suggested that parasitism may be an important trophic strategy in the surface waters of the PO. Geographical distance and temperature were the most important environmental factors that significantly correlated with community structure. Overall, our study sheds more light on the distribution pattern of both alpha and beta diversities of microeukaryotic communities and highlighted the importance of parasitisms by Syndiniales across the tropical PO.IMPORTANCEUnderstanding the biogeographical and biodiversity patterns of microeukaryotic communities is essential to comprehending their roles in biogeochemical cycling. In this study, planktonic microeukaryotes were collected along a west-to-east Pacific Ocean transect (ca. 16,000 km). Our study revealed that the alpha diversity indices were highly correlated with water temperature, and the microeukaryotic communities displayed a distinct geographical distance-driven pattern. The predominance of the parasitic dinoflagellate lineage Syndiniales and their close relationship with other microeukaryotic groups suggest that parasitism may be a crucial survival strategy for microeukaryotes in the surface waters of the Pacific Ocean. Our findings expand our understanding of the biodiversity and biogeographical pattern of microeukaryotes and highlight the significance of parasitic Syndiniales in the surface ocean.

Equine Piroplasmosis in Asymptomatic Horses of Western Iran: Comparison of Microscopic Examination and Multiplex PCR.

PURPOSE: Piroplasmosis is responsible for anemia, fever, loss of physical activity and even death in equines. In epidemiological studies, accurate diagnostic tests are essential for detecting asymptomatic carriers. This study aimed to investigate the prevalence of infection in asymptomatic horses from Lorestan province, western Iran by developing a multiplex PCR. METHODS AND RESULTS: Blood samples were examined by microscopy and multiplex PCR targeting the SSU rRNA gene of Theileria equi and Babesia caballi. Out of the total of 165 horses, 19 (11.51%) and 31 (18.78%) cases were positive for piroplasms by microscopy and PCR, respectively. The detection rates of both genera were significantly higher in multiplex PCR compared to microscopy (p < 0.0001). Compared with multiplex PCR, the sensitivities of microscopy for the detection of Babesia were only 28.5%. The prevalence of T. equi infection was significantly higher in summer (p = 0.035). The prevalence of B. caballi was significantly higher in males (p = 0.038). CONCLUSION: Findings indicate that the multiplex PCR described here is a sensitive technique for the detection of piroplasm DNA in carriers. Furthermore, asymptomatic carriers must be considered as an important source of infection for equids living in this region.

Integrative data of a novel ciliate (Alveolata, Ciliophora) propose the establishment of Heterodeviata nantongensis nov. sp.

BACKGROUND: As unicellular eukaryotes, ciliates are an indispensable component of micro-ecosystems that play the role of intermediate nutrition link between bacteria or algae and meiofauna. Recent faunistic studies have revealed many new taxa of hypotrich ciliates, indicating their diversity is greater than previously thought. Here we document an undescribed form isolated from an artificial brackish water pond in East China. Examination of its morphology, ontogenesis and molecular phylogeny suggests that it represents a new species. RESULTS: The morphology and morphogenesis of the new brackish-water deviatid ciliate, Heterodeviata nantongensis nov. sp., isolated from Nantong, China, were investigated using live observations and protargol staining. The diagnostic traits of the new species include three frontal cirri, one buccal cirrus, one or two parabuccal cirri, an inconspicuous frontoventral cirral row of four to six frontoventral cirri derived from two anlagen, three left and two right marginal rows, two dorsal kineties, dorsal kinety 1 with 9-14 dikinetids and dorsal kinety 2 with only two dikinetids, and one to three caudal cirri at the rear end of dorsal kinety 1. Its main morphogenetic features are: (i) the old oral apparatus is completely inherited by the proter except undulating membranes, which are reorganized in situ; (ii) anlagen for marginal rows and the left dorsal kinety develop intrakinetally in both proter and opisthe; (iii) dorsal kinety 2 is generated dorsomarginally; (iv) five cirral anlagen are formed in both proter and opisthe; (v) in the proter, anlagen I and II very likely originate from the parental undulating membranes and the buccal cirrus, respectively, anlage III from anterior parabuccal cirrus, anlage IV originates from the parental frontoventral cirri and anlage V from the innermost parental right marginal row; and (vi) anlagen I-IV of the opisthe are all generated from oral primordium, anlage V from the innermost parental right marginal row. Phylogenetic analyses based on SSU rRNA gene sequence data were performed to determine the systematic position of the new taxon. CONCLUSIONS: The study on the morphology, and ontogenesis of a new brackish-water taxon increases the overall knowledge about the biodiversity of this ciliate group. It also adds to the genetic data available and further provides a reliable reference for environmental monitoring and resource investigations.

Anaeramoeba pumila sp. nov. and Anaeramoeba sp. OCE22C represent two novel types of symbiosis of Anaeramoebae and prokaryotes.

Anaeramoebae is a recently described phylum of anaerobic, marine amoebae, and amoeboflagellates belonging to the Metamonada supergroup. So far, six species have been described based on light microscopic morphology and sequences of the SSU rRNA gene. Here we present three new strains of Anaeramoeba with a description of their morphology, ultrastructure, and phylogenetic position based on the analysis of SSU rRNA gene sequences. Two of the strains represent a new species, Anaeramoeba pumila sp. nov., that has the smallest cells of all known Anaeramoeba species, and one that represents a species from the newly recognized Anaeramoeba flamelloides complex. Anaeramoebae are known to have a syntrophic relationship with prokaryotes. Our strains display two novel, remarkable types of symbioses, previously unknown from Anaeramoebae.

Morphology, morphogenesis, and molecular characterization of Castula specialis sp. nov. (Ciliophora, Armophorea, Metopida).

The morphology, morphogenesis, and molecular phylogeny of a new metopid ciliate, Castula specialis sp. nov., comprising three strains from geographically distant (China, Mexico, Czech Republic) anoxic freshwater habitats, were studied based on microscopic observation of live and protargol-stained specimens as well as SSU rRNA gene sequence data. The new species is characterized as follows: size in vivo 105-220 × 25-70 µm, body oblong to elongated ellipsoidal and asymmetrical; preoral dome distinctly projecting beyond the body; 32-46 adoral membranelles; 31-52 somatic kineties; and 4-7 setae. This study brings the first morphogenetic investigation of a member of the genus Castula. The morphogenesis of the type population (China) of the new species proceeds as in Metopus spp. comprising drastic changes in body shape and a pleurotelokinetal stomatogenesis; however, the main difference is the origin of the opisthe's paroral membrane that derives from all perizonal rows and some adjacent dome kineties. Phylogenetically, the genus Castula is paraphyletic.

Morphogenesis of an anaerobic ciliate Heterometopus palaeformis (Kahl, 1927) Foissner, 2016 (Ciliophora, Armophorea) with notes on its morphological and molecular characterization.

The morphology, morphogenesis, and molecular phylogeny of Heterometopus palaeformis (Kahl, 1927) Foissner, 2016 were studied using microscopical observations on live and protargol-stained specimens as well SSU rRNA gene sequencing. The morphogenetic data for the genus are presented for the first time. Compared to other metopids, the morphogenesis of H. palaeformis is distinct since its (1) perizonal stripe rows 4 and 5 are involved in the formation of the opisthe's adoral polykinetids; (2) perizonal stripe rows 3-5 and two adjacent preoral dome kineties contribute to most of the opisthe's paroral membrane while perizonal stripe rows 1 and 2 contribute very little; (3) four kinety rows are formed to the left of the opisthe's adoral zone of polykinetids. The Chinese population resembles the original and neotype populations well in terms of general morphology - characterized by a life size of 55-120 × 10-20 µm, an elongate ellipsoidal body with a hardly spiralized flat preoral dome, about 18 somatic kineties and 20 adoral polykinetids. The SSU rDNA sequence of the present population exhibits a disparity of 1.33%-2.22% divergence from sequences of other populations. Nevertheless, phylogenetic analysis reveals that populations of H. palaeformis form a separate, stable cluster within the paraphyletic Metopidae clade.

Prevalence and Genetic Diversity of a Microsporidian Parasite in the Black Imported Fire Ant and Its Social Parasitic Ant (Formicidae: Myrmicinae: Solenopsis) in Buenos Aires Province, Argentina.

Microsporidia are natural pathogens of arthropods and have been used as biological control against insect pests. In the United States, efforts to control the invasive Red Imported Fire Ant, Solenopsis invicta, and Black Imported Fire Ant, Solenopsis richteri, have included the use of the microsporidium, Kneallhazia solenopsae. However, there is limited information about the genetic differences among the microsporidian variants found in S. invicta and in S. richteri. In this study, we assessed the prevalence and genetic diversity of K. solenopsae in native populations of S. richteri in Argentina (South America). Additionally, we examined the social parasitic ant, Solenopsis daguerrei, which is found in some S. richteri nests, for the presence of this microsporidium. The survey of 219 S. richteri nests revealed K. solenopsae infections in all five sites analyzed, with 28 colonies (12.8%) positive for the microsporidium. Among the 180 S. daguerrei individuals collected, seven ants (3.9%) from three sites tested positive for K. solenopsae. Phylogenetic analyses of the microsporidian variants present in S. richteri and S. daguerrei based on partial small subunit ribosomal gene sequences (SSU rRNA) showed that both ant species shared the same variant, which is different from the ones found in S. invicta. Further studies are needed to determine the pathogenicity of genetically different K. solenopsae variants among Solenopsis species.

A new contribution to the raptorial ciliate genus Lacrymaria (Protista: Ciliophora): a brief review and comprehensive descriptions of two new species from Changjiang Estuary.

Ciliates serve as excellent indicators for water quality monitoring. However, their utilization is hindered by various taxonomic confusions. The ciliate genus Lacrymaria Bory de Saint-Vincent, 1824 is commonly found in different aquatic habitats, but its taxonomy has been sparsely investigated using state-of-the-art methods. This study investigated two new Lacrymaria species from Nanhui Wetland, Shanghai, China, using living observation, protargol staining, and molecular phylogeny methods. Lacrymaria songi sp. nov. is 180-340 × 20-25 µm in size and possesses 12-16 somatic kineties, 1 terminal contractile vacuole, 2 macronuclear nodules, and 2 types of rod-shaped extrusomes. Lacrymaria dragescoi sp. nov. is distinguished from its congeners by its cell size of 210-400 × 25-35 µm, 14-17 somatic kineties, 1 terminal contractile vacuole, 1 macronucleus, and 2 types of rod-shaped extrusomes. Phylogenetic analyses based on SSU rRNA gene sequences indicate that Lacrymariidae is monophyletic but Lacrymaria is not. Additionally, a brief review of the genus Lacrymaria is provided in this study. We suggest that L. bulbosa Alekperov, 1984, L. lanceolata Kahl, 1930, and L. ovata Burkovsky, 1970 be removed from the genus and propose Phialina lanceolata nov. comb. and Phialina ovata nov. comb. for the latter two. ZooBank registration: Present work: urn:lsid:zoobank.org:pub:CDFB1EBD-80BD-4533-B391-CEE89F62EDC4 Lacrymaria songi sp. nov.: urn:lsid:zoobank.org:act:417E7C2D-DAEC-4711-90BB-64AB3CD2F7D5 Lacrymaria dragescoi sp. nov.: urn:lsid:zoobank.org:act:8778D6B0-1F2E-473C-BE19-3F685391A40D.

Prevalence and molecular characterization of Enterocytozoon bieneusi and a new Enterocytozoon sp. in pet hairless guinea pigs (Cavia porcellus) from China.

Enterocytozoon bieneusi, the most common microsporidian species, has been detected in humans and a variety of animals worldwide. However, limited information is available on the prevalence and molecular characterization of this parasite in guinea pigs. In this study, we conducted the first investigation of E. bieneusi infection in hairless guinea pigs recently introduced into China as new exotic pets. A total of 324 fecal samples were collected from hairless guinea pigs from a pet market and four breeding facilities in China. Sequence alignment of the internal transcribed spacer (ITS) revealed an infection rate of 14.2% (46/324) and two known ITS genotypes, S7 and PGP. Genotype S7 was the dominant genotype in these animals (42/46, 91.3%). Due to significant ITS sequence divergence, four and two PGP isolates from hairless and regular guinea pigs, respectively were further identified by PCR and phylogenetic analysis based on the small subunit (SSU) rRNA gene, as well as phylogenetic analysis of the ITS locus using E. hepatopenaei and two related genera Enterospora and Nucleospora as the outgroup. Three out of the six PGP isolates were successfully sequenced and generated the same sequences. Phylogenetic analysis of SSU rRNA and ITS loci revealed that PGP isolates formed a separate clade that was distinct and far away from E. bieneusi, suggesting that they represent a new species of Enterocytozoon. These findings indicate the dominance of zoonotic E. bieneusi genotype S7 in hairless guinea pigs and the existence of a cryptic Enterocytozoon species in guinea pigs.

Title: Prévalence et caractérisation moléculaire d'Enterocytozoon bieneusi et d'une nouvelle espèce d'Enterocytozoon chez des cobayes sans poils (Cavia porcellus) en Chine. Abstract: Enterocytozoon bieneusi, l'espèce de microsporidies la plus commune, a été détectée chez les humains et chez divers animaux à travers le monde. Cependant, peu d'informations sont disponibles sur la prévalence et la caractérisation moléculaire de ce parasite chez le cobaye. Dans cette étude, nous avons mené la première enquête sur l'infection par E. bieneusi chez des cobayes sans poils récemment introduits en Chine en tant que nouveaux animaux de compagnie exotiques. Trois cent vingt-quatre échantillons fécaux ont été collectés sur des cobayes sans poils provenant d'un marché d'animaux de compagnie et de quatre établissements d'élevage en Chine. L'alignement des séquences de l'espaceur transcrit interne (ITS) a révélé le taux d'infection (14,2 %, 46 / 324) et deux génotypes ITS connus (S7 et PGP). Le génotype S7 était le génotype dominant chez ces animaux (42 / 46, 91,3 %). En raison d'une divergence significative des séquences ITS, quatre et deux isolats de PGP provenant respectivement de cobayes sans poils et ordinaires ont été identifiés par PCR, analyse phylogénétique basée sur le gène de l'ARNr de la petite sous-unité (SSU), ainsi que par analyse phylogénétique du locus ITS en utilisant E. hepatopenaei et deux genres apparentés Enterospora et Nucleospora comme groupe externe. Trois des six isolats de PGP ont été séquencés avec succès et ont généré les mêmes séquences. L'analyse phylogénétique de l'ARNr SSU et des loci ITS a révélé que les isolats de PGP formaient un clade distinct et éloigné de E. bieneusi, ce qui suggère qu'ils représentent une nouvelle espèce d'Enterocytozoon. Ces résultats indiquent la domination du génotype zoonotique E. bieneusi S7 chez les cobayes sans poils et l'existence d'une espèce cryptique d'Enterocytozoon chez les cobayes.

Further insights into the phylogeny of facultative parasitic ciliates associated with tetrahymenosis (Ciliophora, Oligohymenophorea) based on multigene data.

Tetrahymenosis, caused by about 10 Tetrahymena species, is an emerging problem inflicting a significant economic loss on the aquaculture industry worldwide. However, in the order Tetrahymenida, there are many unresolved evolutionary relationships among taxa. Here we report 21 new sequences, including SSU-rRNA, ITS1-5.8S-ITS2 rRNA and LSU-rRNA, genes of 10 facultative parasitic Tetrahymena associated with tetrahymenosis, and conduct phylogenetic analyses based on each individual gene and a three-gene concatenated dataset. The main findings are: (1) All the parasitic and facultative parasitic species cluster in borealis group. (2) With the addition of new sequences, Tetrahymena is still divided into three groups, namely the "borealis group", the "australis group," and the "paravorax group." (3) the cluster pattern of all the newly sequenced facultative parasitic Tetrahymena species shows that members of the "borealis" group may be more susceptible to parasitism. (4) phylogeny based on concatenated genes show that T. pyriformis, T. setosa, and T. leucophrys have close relationship.

First redescription and molecular phylogeny of Trimyema claviforme Kahl, 1933 with the description of a Chinese population of Plagiopyla nasuta Stein, 1860 (Ciliophora, Plagiopylea).

Ciliates belonging to the class Plagiopylea are obligate anaerobes that are often neglected due to their cryptic lifestyles, difficulty of observation, and overall under-sampling. Here, we investigate two species, namely Trimyema claviforme Kahl, 1933 and Plagiopyla nasuta Stein, 1860, collected in China from marine and freshwater anaerobic sediments, respectively. A complete morphological dataset, together with SSU rRNA gene sequence data were obtained and used to diagnose the species. No molecular sequencing had ever been performed on Trimyema claviforme, with its ciliature also previously unknown. Based on these novel data presented here, the ciliate is characterized by a claviform cell shape, with a size of 35-45 × 10-20 µm in vivo, 28-39 longitudinal somatic ciliary rows forming five ciliary girdles (four complete girdles and a shorter one), two dikinetids left to anterior end of oral kinety 1, and an epaulet. A Chinese population of the well-known ciliate P. nasuta was investigated, and morphological comparisons revealed phenotypic stability of the species. The phylogenetic analyses supported previous findings about the monophyly of the families Trimyemidae and Plagiopylidae, with Trimyema claviforme branching off early in the genus Trimyema. The Chinese population of P. nasuta clusters together with two other populations with full support corroborating their conspecificity.

Bradyrhizobium commune sp. nov., isolated from nodules of a wide range of native legumes across the Australian continent.

Bradyrhizobia are particularly abundant in Australia, where they nodulate native legumes growing in the acidic and seasonally dry soils that predominate in these environments. They are essential to Australian ecosystems by helping legumes to compensate for nutrient deficiencies and the low fertility of Australian soils. During a survey of Australian native rhizobial communities in 1994-1995, several Bradyrhizobium genospecies were identified, among which genospecies B appeared to be present in various edaphic and climatic conditions and associate with a large range of leguminous hosts across the whole continent. We took advantage of the recent sequencing of the genome of strain BDV5040T, representative of Bradyrhizobium genospecies B, to re-evaluate the taxonomic status of this lineage. We further characterized strain BDV5040T based on morpho-physiological traits and determined its phylogenetic relationships with the type strains of all currently described Bradyrhizobium species using both small subunit (SSU) rRNA gene and complete genome sequences. The digital DNA-DNA hybridization relatedness with any type strain was less than 35 % and both SSU rRNA gene and genome phylogenies confirmed the initial observation that this strain does not belong to any formerly described species within the genus Bradyrhizobium. All data thus support the description of the novel species Bradyrhizobium commune sp. nov. for which the type strain is BDV5040T (=CFBP 9110T=LMG 32898T), isolated from a nodule of Bossiaea ensata in Ben Boyd National Park in New South Wales, Australia.

New Parabasalia symbionts Snyderella spp. and Daimonympha gen. nov. from South American Rugitermes termites and the parallel evolution of a cell with a rotating "head".

Most Parabasalia are symbionts in the hindgut of "lower" (non-Termitidae) termites, where they widely vary in morphology and degree of morphological complexity. Large and complex cells in the class Cristamonadea evolved by replicating a fundamental unit, the karyomastigont, in various ways. We describe here four new species of Calonymphidae (Cristamonadea) from Rugitermes hosts, assigned to the genus Snyderella based on diagnostic features (including the karyomastigont pattern) and molecular phylogeny. We also report a new genus of Calonymphidae, Daimonympha, from Rugitermes laticollis. Daimonympha's morphology does not match that of any known Parabasalia, and its SSU rRNA gene sequence corroborates this distinction. Daimonympha does however share a puzzling feature with a few previously described, but distantly related, Cristamonadea: a rapid, smooth, and continuous rotation of the anterior end of the cell, including the many karyomastigont nuclei. The function of this rotatory movement, the cellular mechanisms enabling it, and the way the cell deals with the consequent cell membrane shear, are all unknown. "Rotating wheel" structures are famously rare in biology, with prokaryotic flagella being the main exception; these mysterious spinning cells found only among Parabasalia are another, far less understood, example.

Morphological and genetic analysis of Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea.

We describe Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea. C. saurida was found in the gallbladders of 3/13 specimens of its type host, Saurida elongata Temminck and Schlegel 1846 (Aulopiformes). Myxospore characters were consistent with the original description to which we have added small subunit (SSU) rRNA gene data. C. mai sp. nov. was found in gallbladders of 3/13 specimens of S. elongata and 5/13 specimens of Neobythites sivicola Jordan and Snyder 1901 (Ophidiiformes). Mature myxospores of C. mai sp. nov. were crescentic in sutural view, with a deeply concave posterior angle 142.2±8.2° (125.8‒158.2°) and an arched anterior side. Shell valves were smooth and equal, 20.9±1.9 (17.3‒24.7) µm thick and 9.2±0.5 (8.1‒9.9) µm long, and joined at a straight, thin sutural plane passing between two nematocysts (polar capsules). The nematocysts were equal-sized, pyriform, 2.6±0.2 (2.4‒2.9) µm long and 2.7±0.2 (2.4‒3.3) µm wide, with their tapered ends pointed toward each other, located in the anterior third of the spore. Sequences of the SSU rRNA gene and internal transcribed spacer 1 showed that the isolates of C. mai sp. nov. obtained from S. elongata and N. sivicola were identical. The SSU rRNA gene sequence of C. mai sp. nov. was distinct from all known myxosporeans and clustered with C. saurida, and then with Ceratomyxa filamentosi Kalatzis, Kokkari and Katharios 2013, both of which also infect Aulopiformes fishes.

Occurrence of two myxosporean parasites in the gall bladder of white seabream Diplodus sargus (L.) (Teleostei, Sparidae), with the morphological and molecular description of Ceratomyxa sargus n. sp.

Myxosporeans are widespread cnidarian parasites that usually parasitize fish as part of their complex life cycle, thus constituting a potential threat for the aquaculture industry. White seabream Diplodus sargus (L.) is a commercially valuable sparid fish reared in Southern European aquacultures. Nonetheless, knowledge on myxosporean infections potentially harming the sustainable production of this fish is extremely limited. In this study, a myxosporean survey was conducted on D. sargus specimens reared in two Southern Portuguese fish farms. Two coelozoic myxosporeans were detected infecting the gall bladder, and are herein reported based on microscopic and molecular procedures: Ceratomyxa sargus n. sp. and Zschokkella auratis Rocha et al., 2013, previously described from reared stocks of gilthead seabream Sparus aurata in the same geographic locality. Ceratomyxa sargus n. sp. is the 12th species of the genus to be reported from Southern European sparids, reinforcing a substantial radiation of Ceratomyxa within this fish family and geographic region. SSU rRNA-based Bayesian inference and maximum likelihood analyses revealed C. sargus n. sp. positioned separately from other sparid-infecting Ceratomyxa spp. reported from Southern European countries, demonstrating that this species does not share a more immediate common ancestor with its closest relatives based on host affinity and geography. The recognition of a novel sparid-infecting lineage within the Ceratomyxa clade strengthens the contention that this genus entered sparid fish multiple times, namely in the Southern European region. The identification of Zschokkella auratis infections in D. sargus demonstrates that host shift has occurred among sparids reared in the Southern Portuguese coast. This agrees with the broad host specificity that is usually attributed to this genus, and that may be suggested to be the outcome of the capacity of the Zschokkella morphotype to undergo host shift/switch based on our findings and the limited molecular data available for this genus. Thus, a better understanding of Zschokkella host-associated diversification and dispersal mechanisms requires the increasing availability of molecular data from infections of the same species occurring in multiple hosts and geographical locations.

Morphology and molecular phylogeny of Keronopsis koreana n. sp. (Ciliophora, Hypotricha), a new soil species from South Korea.

The morphology and molecular phylogeny of a new soil ciliate, Keronopsis koreana n. sp., which was discovered in soil from Jindo-gun, South Korea, were investigated. The new species is characterized by the following morphological features: cell more or less ellipsoid or ovoid, both ends broadly rounded; cortical granules irregularly distributed, colorless, 1 µm across; 35-54 adoral membranelles; 12-23 cirri in frontal corona; two frontal and two frontoventral cirral rows; two or three buccal cirri; 23-38 left and 22-37 right marginal cirri; three bipolar dorsal kineties; and two macronuclear nodules and three to five micronuclei. Phylogenetic analyses show that Keronopsis koreana n. sp. is placed within a clade containing K. helluo (type species of Keronopsis) and species belonging to Paraholosticha, Uroleptoides, Parabistichella, Orthoamphisiella, Lamtostyla, and Bistichella in non-dorsomarginalian hypotrichs. Also, the sequences belonging to the Keronopsidae did not cluster together. In addition, a new combination is provided: Parabistichella tasmaniensis (Blatterer and Foissner, 1988) n. comb. (original combination: Keronopsis tasmaniensis).

Molecular evidence of Monocercomonas and Acanthamoeba in the feces of captive reptiles.

Reptiles are frequently kept as pet animals. They are considered as important reservoirs of protozoa with veterinary-medical significance. At a reptile farm in Ireland, fecal samples were collected from 98 captive reptiles, representing 43 species of three orders (Squamata, Testudines, and Crocodylia). After DNA extraction, all samples were screened by conventional PCRs, targeting the ribosomal small subunit (SSU) RNA and alpha-tubulin genes of trichomonads and SSU RNA gene of Acanthamoeba spp. One leopard gecko (Eublepharis macularius) was positive for a not yet reported species/genotype of the genus Monocercomonas, different from M. colubrorum. Various Acanthamoeba genotypes were detected in six reptilian species, i.e., Acanthamoeba genotype T11 in Eunectes notaeus and Heloderma suspectum/horridum; genotype T4 in Varanus exanthematicus, Chlamydosaurus kingii, and Macrochelys temminckii; and the genotype T13 in Iguana iguana. Some of these amoeba species might have clinicopathological significance in both humans and animals. Our findings highlight the importance to monitor pathogenic protozoa in pet as well as wildlife reptiles, as a source of possible infection for animals and humans living nearby.

Insights into the phylogeny of the ciliate of class Colpodea based on multigene data.

In the class Colpodea, there are many unresolved evolutionary relationships among taxa. Here, we report 30 new sequences including SSU-rRNA, ITS1-5.8S- ITS2 rRNA, and the mitochondrial small subunit ribosomal RNA (mtSSU-rRNA) genes of five colpodeans, and conduct phylogenetic analyses based on each individual gene and a two-gene concatenated dataset. For the first time, multi-genes were used to analyze phylogenetic relationships in the class Colpodea. The main findings are: (1) SSU-rRNA, ITS1-5.8S- ITS2 rRNA, and mtSSU-rRNA gene sequences of C. reniformis and C. grandis are provided for the first time, and these two species group into the clade including C. inflata, C. lucida, C. cucullus, and C. henneguyi; (2) clustering pattern and morphological similarity indicate that Bresslauides discoideus has a close relation with Colpodidae spp.; (3) Emarginatophrya genus diagnosis is improved to be 'Hausmanniellidae with sharply shortened and isometric leftmost 1-4 ciliary rows' and Colpoda elliotti is transferred to Emarginatophrya; (4) the genus Colpoda is still non-monophyletic with the addition of 10 populations from five Colpoda species sequences, but there are only two Colpoda groups left based on the present work: Group I comprises C. inflata, C. lucida, C. cucullus, C. henneguyi, C. reniformis, and C. grandis, Group II comprises C. maupasi and C. ecaudata, and the presence of diagonal grooves and the way the vestibular opens might be the two key features that differentiates Colpoda species groups; (5) a close molecular relationship, and highly similar merotelokinetal mode, somatic ciliary pattern, and basic organization of the oral apparatus with P. steinii suggests Bromeliothrix metopoides should be temporarily assigned to Colpodidae.

Development and evaluation of a real-time PCR for genotyping of Cryptosporidium spp. from water monitoring slides.

Cryptosporidium is an important cause of gastroenteritis globally and the main agent of waterborne outbreaks caused by protozoan parasites. Water monitoring for Cryptosporidium oocysts is by detection and enumeration using stained slide microscopy. Species identification (known as genotyping) may be undertaken post hoc and remains a specialist test, only undertaken in some laboratories. The benchmark method is nested PCR-sequencing of part of the SSU rRNA gene, but not all slides are typable and the workflow is cumbersome. We report the development, in-house validation and application of a real-time PCR-sequencing assay based on that gene, using a hydrolysis probe, for the detection and genotyping of all Cryptosporidium spp. The assay was investigated in two formats; a high volume DNA template for analysing all the DNA extracted from Cryptosporidium-positive water monitoring slides with <5 oocysts seen, and a lower volume DNA template permitting several technical replicates from slides with ≥5 oocysts seen where multiple species are more likely to be present. Each format conformed to the MIQE guidelines for amplification dynamics and was specific for Cryptosporidium spp. With high sensitivity, being capable of detecting and genotyping single oocysts by sequencing of a 435 bp amplicon. When 65 water monitoring slides with <5 oocysts seen were tested, slide typeability varied by sending laboratory (n = 9), and ranged from 22 to 60%. Typeability was 75% for slides with ≥5 oocysts seen that were submitted by a single laboratory. The laboratory workflow was improved by using real-time PCR, and decreased the time to result compared with nested PCR-sequencing. In practical application, there was no loss of typeability when the ≥5 oocysts assay was applied to all slides, irrespective of the number of oocysts present.