Morphology and Phylogeny of Two Novel Ciliates, Arcanisutura chongmingensis n. gen., n. sp. and Naxella paralucida n. sp. from Shanghai, China.

The morphologies of two novel ciliates, Arcanisutura chongmingensis n. gen., n. sp. and Naxella paralucida n. sp., collected from Shanghai, China, have been investigated using live observation and silver staining methods. Arcanisutura n. gen. can be easily distinguished from related genera by its inconspicuous, oblique anterior suture. Arcanisutura chongmingensis n. sp. is mainly recognized by its elongated body with a tail-like posterior end, 25-33 somatic kineties, and 4-11 excretory pores. Naxella paralucida n. sp. can be distinguished from its congeners based on its two short nassulid organelles, fusiform trichocysts, 37-49 somatic kineties, and 16 nematodesmal rods. The small-subunit (SSU) rRNA gene sequences of these two species are presented, revealing the phylogenetic positions of Arcanisutura and Naxella. Phylogenetic analyses show that Arcanisutura forms a sister clade to other synhymeniid genera, namely, Chilodontopsis, Orthodonella, and Zosterodasys; Naxella is most closely related to Nassula spp. and is located within the monophyletic clade of the family Nassulidae.

Ultrastructural features of the tomont of Cryptocaryon irritans (Ciliophora: Prostomatea), a parasitic ciliate of marine fishes.

Numerous studies have been conducted on the cellular morphology of Cryptocaryon irritans. However, details regarding the tomont stage of its life cycle remain lacking. In this study, we investigated the morphology of the tomont stage throughout encystment and cell division using light and electron microscopy. Results showed that there was no secretion of encystation-specific secretory vesicles or extrusomes during formation of the cyst wall. Instead, the synthesis and construction of the C. irritans cyst wall materials may involve molecular events at the pellicle. The somatic cilia and the cytostome were present during encystment and covered by the newly formed cyst wall. New somatic cilia were continuously created between old cilia and showed various lengths during cell division, a process that was similar to morphogenesis in many free-living ciliates. During cell division inside the tomont, dividing daughter cells formed temporary cell chains with no oral primordia before separating from each other into dissociative tomite precursors. The process of cell division may not be accompanied by stomatogenesis, and new oral primordia in offspring cells likely formed before the dividing cell chains split into dissociative spherical tomites. Mitochondrial autophagy was observed in encysting C. irritans cells. Numerous endosymbionts and Golgi structures were observed in the tomont cytoplasm. Cellular metabolic activity in the C. irritans tomont was quite high, with large amounts of materials or cellular organelles potentially being synthesized and prepared for the following infective theront stage.

The differentiation of cellular structure during encystment in the soil hypotrichous ciliate Australocirrus cf. australis (Protista, Ciliophora).

Ciliates are able to form resting cysts as a survival strategy in response to stressful environmental factors. Studies on the characteristics of cellular structure during encystment may provide useful information for further understanding of the regulatory mechanism of cellular patterns and supply new clues regarding the phylogeny of ciliates. Scanning and transmission electron microscopies were used to observe the ultrastructure of cells during encystment of the soil ciliate Australocirrus cf. australis. The dedifferentiation of ciliature was revealed for the first time. Ciliary shafts first shortened, and the remaining ciliature, including basal bodies and the fibrillar cirral basket, retracted into the cytoplasm and was surrounded by the autophagic vacuoles and then gradually digested. A large number of autophagic vacuoles were observed in mature resting cysts. Autophagy might not only be necessary for the differentiation of cellular structures during encystment but might also be important to sustain the basic life activities in the resting stage. Australocirrus cf. australis formed a kinetosome-resorbing cyst and contained four layers in the cyst wall: the ectocyst, mesocyst, endocyst and granular layer. The ciliature resorbing state and the number of layers in the cyst wall were consistent with those found in other oxytrichous ciliates. However, the phenomenon wherein the two macronuclear nodules are not fused during encystment is not commonly observed among oxytrichids. Additionally, the octahedral granules in the mesocyst of this species exhibit different morphology from the congeners.

Ultrastructure observation on the cells at different life history stages of Cryptocaryon irritans (Ciliophora: Prostomatea), a parasitic ciliate of marine fishes.

Cells of Cryptocaryon irritans at different life history stages were studied using both light and electron microscopy. The characteristics of several organelles were revealed for the first time at the ultrastructural level. It was confirmed that the cytostome of trophonts, protomonts and theronts was surrounded by cilium-palp triplets rather than ciliary triplets. The nematodesmata underlying the circumoral dikinetids were single bundles, whereas these were always paired in Prorodontids. Toxicysts were present in late-stage tomonts and theronts, but were absent in trophonts and protomonts. We posited that toxicysts might play a role in infection and invasion of host-fish tissue by theronts. The adoral brosse was unlike that of any other family of the class Prostomatea based on its location and morphology. Membranous folds were present in trophonts, protomonts and theronts. These folds were longer and more highly developed in C. irritans than in exclusively free-living prostome ciliates suggesting that they might be linked to parasitism in C. irritans. Trophonts, protomonts and theronts had multiple contractile vacuoles. The basic ultrastructure of the contractile vacuole of C. irritans was similar to that of other kinetofragminophoran ciliates. They might play different roles in different stages of the life cycle since their ultrastructure varied among trophonts, protomonts and theronts.

New data on the subgenus Gnathopaederus of the genus Paederus (Coleoptera: Staphylinidae: Paederinae) of mainland China.

Paederus (Gnathopaederus) xuei Peng & Li sp. n. (Yunnan: Dawei Shan) and P. (G.) cheni Peng & Li sp. n. (Hunan: Badagong Shan) are described and illustrated. Additional records of P. (G.) bursavacua Willers, 2001, P. (G.) szechuanus (Chapin, 1927) and P. (G.) yunnanensis Willers, 2001 are reported.

Phylogenetic positions of four hypotrichous ciliates (Protista, Ciliophora) based on SSU rRNA gene, with notes on their morphological characters.

 The morphology and infraciliature of the four hypotrichous ciliates; Rigidohymena inquieta (Stokes, 1887) Berger, 2011, Pattersoniella vitiphila Foissner, 1987, Notohymena australis Foissner & O' Donoghue, 1990, and Cyrtohymena (Cyrtohymenides) australis (Foissner, 1995) Foissner, 2004, collected from east China, were investigated by using live observation and protargol impregnation method. An improved diagnosis for R. inquieta was supplied based on descriptions of present and previous populations. New morphology and morphogenesis information based on Chinese populations of another three hypotrichids were also supplemented. The Small-subunit rRNA (SSU rRNA) gene sequences of the four species were characterized and their phylogenetic positions were revealed by means of Bayesian inference and Maximum-likelihood analysis. The analyses shows that R. inquieta clusters with other members of the subfamily Stylonychinae, which confirms the monophyly of the subfamily and verified R. inquieta as a separated species from R. candens though it differs from others mainly by body size. C. (C.) australis occupying the basal position of the clade which contains cyrtohymenids and some other groups, declines the idea of separating Cyrtohymena into two subgenus. Notohymena australis and China population of Pattersoniella vitiphila respectively clustering with their congeners correspond well with the systematics revealed by morphological similarities.

On the Lathrobium fauna of the Donggong Mountains, eastern China.

Material of the paederine genus Lathrobium Gravenhorst, 1802 from the Donggong Mountains, eastern China, is examined. Fifteen species are identified, seven of them described previously and eight undescribed. Seven species are described and illustrated for the first time, all of them micropterous and locally endemic: L. haoae Peng & Li sp. n., L. songi Peng & Li sp. n., L. yui Peng & Li sp. n., L. fengyangense Peng & Li sp. n., L. liyangense Peng & Li sp. n., L. mu Peng & Li sp. n. and L. yani Peng & Li sp. n. One probably undescribed species remains unnamed. The female sexual characters of L. baishanzuense Peng & Li, 2012 and L. zhui Peng & Li, 2014 are described and illustrated for the first time. Including the new taxa, 196 Lathrobium species are currently known from mainland China.

Confirmation through genetic analysis of the existence of many local phyloclades of the genus Simocephalus (Crustacea, Cladocera) in China.

Previously, a series of Simocephalus taxa (Cladocera: Daphniidae) from China were described. Most were proposed to be junior synonyms in the last revision of the genus. Using original material from China and data from GenBank, we investigate the biodiversity and phylogeny of Simocephalus using sequences of the cytochrome c oxidase subunit I (COI) and the nuclear 18S genes. In both cases, neighbor-joining, maximum likelihood and Bayesian inference analyses led to highly congruent tree topologies. The grouping of the deeper clades agrees with the inter-generic classification of Orlova-Bienkowskaja (2001). Only the populations of S. serrulatus from Eurasia and North America seem to be closely related, and there are no other shared species between the two continents. Our study unambiguously confirms the existence of many lineages from the subgenera of Simocephalus (Echinocaudus) and Simocephalus s.str. in China, but their morphology needs to be reexamined by taking a wider range of characters (e.g., of female thoracic limbs and adult males) into consideration.

Morphological and molecular characterization of Parafurgasonia zhangi spec. nov. and Chilodonella acuta Kahl, 1931 (Protozoa, Ciliophora), from a soil habitat of Saudi Arabia.

The morphology and infraciliature of two soil ciliates, Parafurgasonia zhangi spec. nov. and Chilodonella acuta Kahl, 1931, collected from Saudi Arabia, were investigated by observations of both living cells and specimens after standard staining methods. P. zhangi differs from its congeners by the combination of the following features: excretory pore quite near posterior end of paroral membrane, 16 or 17 somatic kineties with about 11 kinetids in each one on dorsal side, paroral membrane gently curved and composed of about 15 dikinetids, and hypostomial organelle composed of four or five files of kinetids with four monokinetids each. The diagnosis of Chilodonella acuta was renewed to include characteristics revealed by the silver impregnation method: cells in vivo measuring 33-45) × 18-26) µm, dorsal hump and tail-like podite present, two contractile vacuoles, seven left and five right kineties, 9-11 nematodesmal rods, and dorsal brush containing about 11 basal bodies. Phylogenetic analyses based on small-subunit rRNA gene sequences showed that P. zhangi was closer to species of the Colpodidiidae rather than the Furgasoniidae represented by Furgasonia blochmanni, and Chilodonella acuta clustered with its congener Chilodonella uncinata but was a well-outlined species of the genus.

Phylogeny of the poorly known ciliates, microthoracida, a systematically confused taxon (ciliophora), with morphological reports of three species.

Three species of Microthoracids, Lopezoterenia paratorpens n. sp., Trochiliopsis australis Foissner et al., 1988 and Discotricha papillifera Tuffrau, 1954, collected from Chinese coastal waters, were investigated using live observation and protargol staining methods. Lopezoterenia paratorpens n. sp. was characterized by its squarely shaped cortical papillae and by dorsal kineties which contained loosely distributed basal bodies. Trochiliopsis australis was revealed to have two oral membranelles, which was not recorded in the original report. Phylogenetic analyses were carried out based on SSU rRNA gene sequence data from each of the three species, and on other available data for microthoracids. The results showed that the order Microthoracida is not monophyletic because the family Discotrichidae, which contains L. paratorpens and D. papillifera, forms a clade separated from the "core" Microthoracids clade. The topologies of the maximum likelihood and Bayesian inference trees, along with the distinct morphological characteristics found previously, suggest that the family Discotrichidae should not be assigned to the order Microthoracida. We propose to designate a new order, Discotrichida n. ord. which diagnosed as: flattened ciliates with conspicuous cortical papillae on both dorsal and ventral faces, rod-shaped mucocysts, and an asymmetric cytopharyngeal basket. Also, the fact that Leptopharynx clusters with the assemblage including T. australis, and Pseudomicrothorax is located distantly from Leptopharynx indicates that the classification of Pseudomicrothoracidae and Microthoracidae by Foissner (1985) is justified.

Morphological reports on two species of Dexiotricha (Ciliophora, Scuticociliatia), with a note on the phylogenetic position of the genus.

Two Dexiotricha species (Dexiotricha elliptica nov. comb. and Dexiotricha cf. granulosa), respectively isolated from soil north-west of Riyadh, Saudi Arabia, and freshwater in Shanghai, eastern China, were investigated using standard methods. The species Loxocephalus ellipticus Kahl, 1931 is reclassified here in the genus Dexiotricha and was characterized mainly by constantly showing 16 somatic kineties, three post-oral kineties with the middle one shortened, a contractile vacuole located subcaudally with an excretory pore near the posterior end of somatic kinety 2 and single caudal cilia. A Dexiotricha granulosa-like organism having a subcaudally located contractile vacuole and fewer somatic kineties was designated D. cf. granulosa. The small-subunit rRNA gene (SSU rDNA) sequences of these two species were characterized and their phylogenetic positions based on SSU rDNA sequences were revealed by means of Bayesian inference and maximum-likelihood analysis. Phylogenetic analyses confirmed Dexiotricha as a monophyletic genus and supported its assignment to the order Loxocephalida. However, its family assignment remains unsupported.

Phylogeny of the Poorly Known Ciliates, Microthoracida, a Systematically Confused Taxon (Ciliophora), with Morphological Reports of Three Species.

Three species of Microthoracids, Lopezoterenia paratorpens n. sp., Trochiliopsis australis Foissner et al., 1988 and Discotricha papillifera Tuffrau, 1954, collected from Chinese coastal waters, were investigated using live observation and protargol staining methods. Lopezoterenia paratorpens n. sp. was characterized by its squarely shaped cortical papillae and by dorsal kineties which contained loosely distributed basal bodies. Trochiliopsis australis was revealed to have two oral membranelles, which was not recorded in the original report. Phylogenetic analyses were carried out based on SSU rRNA gene sequence data from each of the three species, and on other available data for microthoracids. The results showed that the order Microthoracida is not monophyletic because the family Discotrichidae, which contains L. paratorpens and D. papillifera, forms a clade separated from the "core" Microthoracids clade. The topologies of the ML and BI trees, along with the distinct morphological characteristics found previously, suggest that the family Discotrichidae should not be assigned to the order Microthoracida. We propose to designate a new order, Discotrichida n. ord. which diagnosed as: flattened ciliates with conspicuous cortical papillae on both dorsal and ventral faces, rod-shaped mucocysts, and an asymmetric cytopharyngeal basket. Also, the fact that Leptopharynx clusters with the assemblage including T. australis, and Pseudomicrothorax is located distantly from Leptopharynx indicates that the classification of Pseudomicrothoracidae and Microthoracidae by Foissner (1985) is justified. This article is protected by copyright. All rights reserved.

The ultrastructure of the extrusomes in Pseudourostyla cristata, a hypotrichous ciliated protozoan.

By using scanning and transmission electron microscopy, the present study demonstrates a great number of trichocyst-like extrusomes distributed in the cortical cytoplasm of the protozoan Pseudourostyla cristata, a hypotrichous ciliate. Of these, the mature organelles are rod-shaped with a cap consisting of tubular structures, a tip located at the apex of the cap, a body consisting of strateform structures of uneven electron density and an elongated shaft located along the longitudinal central axis of the body. The electron microscopic observations suggest that the extrusive organelles in P. cristata might undergo a morphogenetic process including the following sequential events: the occurrence of the vesicles in the cytoplasm, the condensation of the fibrous substances within the vesicles, the appearance of the electron-dense shaft, and the formation of the cap. In contrast with a large quantity of extrusomes in trophozoit P. cristata, there are no such extrusive organelles in the encysted cells of the ciliate. The phenomena that P. cristata ciliates can readily enter physiological reorganization or encysting phases and discharge a great number of their extrusomes when prepared for SEM and TEM observation suggest that the extrusive process of the extrusomes in P. cristata might have an important influence on the life activity of the ciliate and could be one of the causes leading to the physiological reorganization and the encysting of the ciliate. These reactions of P. cristata might be a protective or defensive response to the environmental changes.

Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis.

In order to get further insight into the organization of the pestiviral replication machinery, characterization of NS3, NS5A and NS5B of classical swine fever virus (CSFV) through mutation and complementation analysis was performed. Mutation analysis in genomic replicons and subgenomic replicons indicated importance of the GDD motif in NS5B, the DEYH motif in NS3 and the conserved sequence C2717-C2740-C2742-C2767 in the NS5A for CSFV recover and viral RNA synthesis. Complementation experiments were performed between subgenomic replicons, between RNA replicons or between RNA replicon and expressed nonstructural protein. Rescue of virus and recover of viral RNA synthesis were examined in these complementation experiments. Results showed that mutations within NS5A, neither NS5B nor NS3, can be trans-complemented, strongly suggesting that NS5B and NS3 function in cis mode for regulation of replication. We assumed that the necessary membrane association of CSFV NS5B and NS3 could occur only when they are being translated and originated from an identical translation template, with the exception of NS5A whose membrane association might occur post-translationally.

The C-terminus of PRK2/PKNgamma is required for optimal activation by RhoA in a GTP-dependent manner.

PRK2/PKNgamma is a Rho effector and a member of the protein kinase C superfamily of serine/threonine kinases. Here, we explore the structure-function relationship between various motifs in the C-terminal half of PRK2 and its kinase activity and regulation. We report that two threonine residues at conserved phosphoacceptor position in the activation loop and the turn motif are essential for the catalytic activity of PRK2, but the phosphomimetic Asp-978 at hydrophobic motif is dispensable for kinase catalytic competence. Moreover, the PRK2-Delta958 mutant with the turn motif truncated still interacts with 3-phosphoinositide-dependent kinase-1 (PDK-1). Thus, both the intact hydrophobic motif and the turn motif in PRK2 are dispensable for the binding of PDK-1. We also found that while the last seven amino acid residues at the C-terminus of PRK2 are not required for the activation of the kinase by RhoA in vitro, however, the extreme C-terminal segment is critical for the full activation of PRK2 by RhoA in cells in a GTP-dependent manner. Our data suggest that the extreme C-terminus of PRK2 may represent a potential drug target for effector-specific pharmacological intervention of Rho-medicated biological processes.

Characterization of interaction of classical swine fever virus NS3 helicase with 3' untranslated region.

The classical swine fever virus (CSFV) full-length NS3 protein (NS3F) and the truncated NS3 protein (NS3H) with postulated helicase domain were expressed and demonstrated to have helicase activity. Further, the electrophoretic mobility shift assays containing NS3H and the viral 3' terminal sequences showed that NS3H specifically bound to the plus- and minus-strand 3'UTR. The minus-strand 3'UTR had higher binding activity. The 21-nt fragments at the 3'-most terminal sequences of both 3'UTRs were essential to NS3H binding. A 12-nt insertion, CUUUUUUCUUUU, present in the 3'UTR of a CSFV live attenuated vaccine strain, was also found to be deleterious to helicase binding. Intact secondary structure of 3' terminal sequence of 3'UTR might be important in helicase binding. Our results show that interaction between the helicase and the viral 3'UTR is similar to that between the replicase and the 3'UTR, suggesting that NS3 helicase is important for CSFV genomic replication.