Reevaluation of cortical developmental patterns in Euplotes(s.l.), including a morphogenetic redescription of E.charon (Protozoa,Ciliophora,Euplotida)

We documented the pattern of cell development in Euplotes charon. The ontogenesis of this species was similar to many of its congeners, except for the formation of the caudal cirri. In E. Charon, a caudal cirrus is formed posterior to each of the rightmost two or three dorsal kinety anlage in the proter, and the second rightmost dorsal kinety in the opisthe. In addition, two caudal cirri are formed posterior to the rightmost dorsal kinety in the opisthe. This pattern of development represents a completely new type. Based on our evaluation, and in comparison with previous studies, we also conclude that the pattern of cell development is variable among species in the Euplotes genera. The variation is particularly evident during the formation of frontoventral and caudal cirri. Based on the segmentation pattern of frontal-midventral transverse cirral anlagen, cirri reduction, and migration of frontoventral cirri, we identified five types: the affinis-type, the eurystomus-type, the charon-type, the raikovi-type and orientalis-type. Euplotes (s. l.) can also be divided into three types based on the formation of caudal cirri: focardii-type, vannus-type and charon-type. Indeed, we conclude that the number (one or two) of marginal cirri should be given as much consideration as the genetic separation. Given this, we reassessed the validity of using genetic separation to classify the group. Generally, the morphogenetic data disagreed with the molecular data (SSrRNA gene sequences). Given these discrepancies, it is too early to draw conclusions on the systematic arrangement of this species-rich taxon.

Molecular phylogeny and species separation of five morphologically similar Holosticha-complex ciliates(Protozoa, Ciliophora) using ARDRA riboprinting and multigene sequence data

To separate and redefine the ambiguous Holosticha-complex, a confusing group of hypotrichous ciliates, six strains belonging to five morphospecies of three genera, Holosticha heterofoissneri, Anteholosticha sp. popl, Anteholosticha sp. pop2, A. manca, A. gracilis and Nothoholostichafasciola, were analyzed using 12 restriction enzymes on the basis of amplified ribosomal DNA restriction analysis. Nine of the 12 enzymes could digest the DNA products, four （HinfⅠ, Hind Ⅲ, Msp Ⅰ, Taq Ⅰ） yielded species-specific restriction patterns, and Hind Ⅲ and Taq Ⅰ produced different pattems for two Anteholosticha sp. populations. Distinctly different restriction digestion haplotypes and similarity indices can be used to separate the species. The secondary structures of the five species were predicted based on the ITS2 transcripts and there were several minor differences among species, while two Anteholosticha sp. populations were identical. In addition, phylogenies based on the SSrRNA gene sequences were reconstructed using multiple algorithms, which grouped them generally into four clades, and exhibited that the genus Anteholosticha should be a convergent assemblage. The fact that Holosticha species clustered with the oligotrichs and choreotrichs, though with very low support values, indicated that the topology may be very divergent and unreliable when the number of sequence data used in the analyses is too low.

A General Overview of the Typical 18 Frontal-VentralTransverse Cirri Oxytrichidae s. l. Genera(Ciliophora, Hypotrichia)

Oxytrichidae s. l. ciliates usually have 18 frontal-ventral-transverse cirri which are clustered to six distinct groups usu- ally originating from six longitudinal primordia segregating 1, 3, 3, 3, 4, 4 cirri. During morphogenesis, three dorsal kinetics anlagen are primarily formed. Fragmentation of kinety 3 usually present, while sometimes secondarily lost. Dorsomarginal kineties are formed, while sometimes lost. Oxytrichids tend to have overlapping characters, e.g. cell shape and size, infraciliature, pellicle fea- tures. This makes a great problem for genera separation. In the present work, all typical 18 frontal-ventral-transverse-cirri Oxytrichi- dae s. 1. genera were revised systematically based on their living morphology, ciliature patterns and dorsal morphogenetic features. The outline of the genera, the schematic illustrations, and the key to typical 18 frontal-ventral-transverse-cirri genera of Oxytrichidae s. l. were clarified. Additionally, some morphological and morphogenetic patterns were summarized and compared.

Population dynamics of marine ciliate Euplotes vannus (Protozoa, Ciliophora) in different artificial seawaters

To study population dynamics of marine ciliates in different artificial seawaters （ASW）, the population growth dynamics of a common marine ciliate Euplotes vannus were investigated using beef extract media and rice media for five types of ASW and natural seawater （NSW）. The results show that： （1） the population growth rate was in the order of NSW〉Flack ASW〉Nakamula ASW〉Schmadz ASW〉Oshima ASW〉Subow ASW and was considerably higher in rice media than in beef extract media （apart from Subow ASW）; （2） the maximum density of E. vannus in stationary phase in each treatment was ranked as Hack ASW〉Nakamula ASW〉Schmadz ASW〉NSW〉Oshima ASW〉Subow ASW, and was again higher in rice media than in beef extract media （except for Subow ASW）; （3） the exponential and stationary phases were longer in rice media than in beef extract media; （4） strains of E. vannus that had been domesticated for 〉1 year in ASW grew significantly slower, with lower maximum density and longer stationary phase than those isolated and maintained in NSW. It was demonstrated that： （1） E. vannus may grow well in Flack, Nakamula and Schmads ASW compared with NSW （mainly in terms of growth rate）; and （2） Oshima ASW is the preferred choice for stock cultures of E. vannus, but the ASWs Flack, Nakamula and Schmadz are preferred for mass culture. These findings suggest that these three ASWs are effective for the cultivation of marine protozoa for experimental studies on ecology, toxicology and molecular biology.

Temporal population dynamics of dinoflagellate Prorocentrum minimum in a semi-enclosed mariculture pond and its relationship to environmental factors and protozoan grazers

The ecological processes and interrelationships between protists,either autotrophic or heterotrophic,and environmental factors in mariculture ponds are largely unknown.This study investigated the temporal dynamics of potentially harmful dinoflagellate,Prorocentrum minimum (Pavillard) Schiller,and its relationship to physico-chemical factors and protozoan grazers over a complete cycle in a semi-enclosed shrimp-farming pond near Qingdao,Northern China.P.minimum occurred frequently in low numbers from June to August,followed by a sharp increase from the middle of August,reaching a single maximum peak value of 2.2×105 cells L-1 in October.Temporal variation in abundance was positively correlated with dissolved nitrogen,but showed a significant inverse relationship to abundance of the dominant ciliates,Tintinnopsis lohmanni and Askenasia stellaris.The results provide statistical evidence that the number of P.minimum increased with increasing nitrogen,and the suppression or shortening of algal bloom may be associated with protozoan grazers,such as Tintinnopsis lohmanni,in mariculture ponds.

Characterization of marine microplankton communities of Qingdao coastal areas using randomly amplified polymorphic DNA(RAPD)

Microplankton communities of three coastal sites of Qingdao,Shandong Province,China were investigated using RAPD（random amplified polymorphic DNA） molecular markers and morphological observations.Eight RAPD-primers were selected to amplify the DNA polymorphy.The genetic distances inferred from the pairwise similarities were calculated for the phylogenetic tree construction.Meantime,the traditional microscopic determination,a way of visualizing the species composition,was performed to detect the major taxa of microplanktons from all samples.Results showed that：（1） the band sharing index values were in the range of 0.504 2-0.763 2 among samples from the same sampling site at different time scales,while 0.406 5-0.685 7 among the samples from different stations at the same time scales,indicating that spatial variations of microplankton communities were more pronounced than temporal ones;（2） samples from the same station basically clustered together,corresponding to the geographic distribution of the sampling sites;（3） diversity derived from genetic and morphological data did not correspond with each other well.

Temporal dynamics of phytoplankton communities in a semi-enclosed mariculture pond and their responses to environmental factors

Variations in physical-chemical factors, species composition, abundance and biomass of nano-and micro-phytoplankton assemblages, as well as their responses to environmental factors, were investigated over a complete cycle (6 months) in a semi-enclosed shrimp-farming pond near Qingdao, northern China. The aim was to establish the temporal patterns of phytoplankton communities and to evaluate protists as suitable bioindicators to water quality in mariculture systems. A total of 34 taxa with nine dominant species were identified, belonging to six taxonomic groups (dinoflagellates, diatoms, cryptophyceans, chlorophyceans, euglenophyceans and chrysophyceans). A single peak of protist abundance occurred in October, mainly due to chlorophyceans, diatoms and chrysophyceans. Two biomass peaks in July and October were primarily due to dinoflagellates and diatoms. Temporal patterns of the phytoplankton communities significantly correlated with the changes in nutrients, temperature and pH, especially phosphate, either alone or in combination with NO3-N and NH3-N. Species diversity, evenness and richness indices were clearly correlated with water temperature and/or salinity, whereas the biomass/abundance ratio showed a significant correlation with NO3-N. The results suggest that phytoplankton are potentially useful bioindicators to water quality in semi-enclosed mariculture systems.

Molecular phylogeny of oligotrich genera Omegastrombidium and Novistrombidium(Protozoa,Ciliophora) for the systematical relationships within Family Strombidiidae

The phylogeny of the oligotrich ciliates is currently a hot debate despite the availability of both morphological and molecular data.In the present paper,further small subunit rRNA(SS rRNA) genes were analyzed from the Genera Omegastrombidium and Novistrombidium,as well as from Strombidium,and combined with three new SS rRNA sequences from Strombidium basimorphum,S.sulcatum population QD-1,and Novistrombidium testaceum population GD.The phylogenetic positions of these organisms were inferred using Bayesian inference,Maximum Likelihood,and Maximum Parsimony methods.The main results are:(1) the SS rRNA gene sequence analyses match the recent findings about the molecular evolution of oligotrichs,indicating that the family Strombidiidae is paraphyletic;(2) the Genus Omegastrombidium is separated from the Genus Strombidium,as shown in recent cladistic analyses;(3) morphospecies in Genus Novistrombidium,based on similarity of somatic ciliature,are separated from each other in all topological trees,indicating that this genus could be a paraphyletic group;(4) the molecular data indicate a possibility of paraphyly for the genus Strombidium;and(5) the similarities of the SS rRNA gene of specimens identified as S.sulcatum and S.inclinatum are 99.8%-100%.However,present knowledge on the oligotrichs sensu stricto is still insufficient and further studies based on both molecular and other technologies are required.

Morphogenetic Processes in Division of Uronemella filificum (Ciliophora, Scuticociliatida)

The morphogenetic processes of Uronemella filificum (Kahl, 1931) Song and Wilbert ①, were studied using protargol method. The stomatogenesis of U. filificum can be summarized as follows: membranelles 1-2, paroral membrane and scutica of the opisthe originate from the parental paroral membrane, while membranelle 3 in the opisthe develops from the parental scutica. The paroral membrane of the proter developes from the parental paroral membrane. The stomatogenetic sequences are similar to the previous descriptions for other related taxa. The phylogenetic position of Uronematidae is discussed in the light of morphogenetic data obtained.

Taxonomic description of a new marine ciliate,Euplotes qingdaoensis n. sp.(Ciliophora:Euplotida)

The characteristics and infraciliature of a new euplotid ciliate, Euplotes qingdaoensis n. sp., discovered from the coastal waters of Jiaozhou Bay, northern China, were studied using live observation and silver impregnation. The new marine species is characterized by a slightly flattened body （90-110 um×70- 80 um, in vivo）, an adoral zone of 26-29 membranelles, 10 strong frontoventral and five transverse cirri, two caudal cirri, a single left marginal cirrus, seven dorsal kineties, a middle kinety with about 15 pairs of basal bodies, a silverline system of the double-eurystomus type, and a C-shaped macronucleus.

Assessing mariculture water quality with the structural and functional characteristics of a ciliate community

Ciliated protozoa play important roles in micro-ecosystems, especially in marine biotopes. However, few studies have been carried out on the periphytic, or aufwuch, forms in mariculture waters so far. In this study, we sampled periphytic ciliate communities in two closed mariculture ponds (ponds CP1 and CP2) and a natural seawater reservoir (pond RP) using a glass slide method to evaluate their colonizing processes and general ecological features, as well as their application as water quality indicators. We analyzed species compositions, structural parameters (species number, richness, diversity, evenness, abundance and dBP) and functional parameters (G, Seq and T90%). Pond RP was characterized by higher levels of structural parameters (except for abundance and dBP) and more equal proportion of the major taxonomic groups. The values of Seq were significantly higher in pond RP and similar in both pond CP1 and CP2. It was also demonstrated that environmental factors, including NO2-H, NO3-H, NH3-H, soluble reactive phosphate, temperature and pH, were the first principal factors affecting the communities. Among them, temperature and chemical factors were all significantly and negatively correlated with species number (P<0.01), richness (P<0.01), diversity (P<0.01), and positive correlated with abundance (P<0.01). Opposite correlations between pH and structural parameters were observed. This study showed that there were significant differences in species composition, structural parameters and functional parameters of the periphytic ciliate communities among the ponds, which were in agreement with the water quality. Results of this study confirmed the periphytic ciliate communities to be useful bioindicators of water quality in intensive mariculture waters.

Planktonic protist communities in semi-enclosed mariculture waters: temporal dynamics of functional groups and their responses to environmental conditions

The functional groups of planktonic protist communities and their responses to the changes of environmental conditions were investigated in a semi-enclosed shrimp-farming pond in Qingdao,Shandong Province,China,during a six-month study period （a complete shrimp-culture cycle） from May to October 2002.The results reveal that： （1） the protist communities represented five trophic and functional groups of the species identified,about 60% were photoautotrophs,20% algivores,12% bacterivores,5% raptors and about 3% non-selectives;（2） the photoautotrophs,algivores and bacterivores were the primary contributors to the changes in the protist communities in short temporal scales,the succession of dominance typically being bacterivores→photoautotrophs→algivores,with the raptors dominating the protist communities in a single sample （early June）;（3） the photoautotrophs and non-selectives were the primary contributors to the peak of protist abundance in early October whereas the photoautotrophs,bacterivores,raptors and non-selectives mainly gave rise to two bimodal peaks of biomass in July and October respectively;（4） five functional groups of protist communities represented significant correlations with water nutrients （i.e.,NH 3-N,NO 3-N,and PO 4 ）,either alone or in combination with temperature,of which algivores and raptors were strongly correlated with phosphate and the concentration of Chl a,while bacterivores were strongly related to nitrogen and the concentration of bacteria.These findings confirm that planktonic protists are potentially useful bioindicators of water quality in the semi-enclosed mariculture system.

Diversity of oligotrich ciliates(Ciliophora, Spirotrichea) in the northern coast of South China Sea as revealed in LSU rDNA sequences

Although the taxonomy of oligotrich ciliates has been widely investigated,yet the species diversity remains poorly known.We newly designed a pair of oligotrich-specific LSU rDNA primers covering the 600-bp D1/D2 region,and it was effective for detecting oligotrich species.Using the primers,we constructed the cloning libraries to investigate the species diversity of oligotrichs in the northern coastal waters of the South China Sea.In total,165 oligotrich sequences were obtained from five widely separated sampling sites.Sixty operational taxonomic units(OTUs)were obtained at 99%similarity threshold,and low-abundance OTUs with no more than two sequences contributed most of these(about 78%).Our findings are consistent with previous morphological studies,Strombidium was found the most abundant and widely distributed genus in this area.In addition,the BLAST search in the NCBI database resulted in 95%OTUs matching with named oligotrich species in similarity below 99%.Therefore,oligotrich morphospecies diversity has been underestimated as low-abundance species,and the LSU rDNA oligotrich sequence database needs to be better promoted.

Temporal species distributions of planktonic protist communities in semi-enclosed mariculture waters and responses to environmental stress

In order to evaluate the relationships between temporal species succession of planktonic protists and physical-chemical parameters in semi-enclosed mariculture waters, species distributions in response to environmental stress were investigated in a shrimp-farming pond near Qingdao, China during a complete shrimp-culture cycle （May to October 2002）. A clear temporal succession in species distribution was found over the complete farming cycle. For example, before the introduction of the shrimp larvae there was low-variability of species distribution in the protist communities whereas during the stages immediately after, there was higher variability in species composition. Multiple linear/logistic regression analyses demonstrate that 12 protist taxa （e.g., Gyrodinium spirale, Teleaulax acuta, Prorocentrum spp. and Mesodinium pupula） were related to the nutrients, in particular ammonia and phosphates, alone or in combination with water temperature. These results suggest that temporal variations in species distribution of planktonic protist communities might be used in assessing water quality of semi-enclosed mariculture waters.

Morphological Studies on the Infraciliature of a Planktonic Ciliate,Tintinnopsis brasiliensis(Ciliophora:Tintinina)

A poorly-described marine planktonic ciliate, Tintinnopsis brasiliensis Kofoid ＆ Campbell, 1929, collected from the Taiping Cape of Qingdao, China, was morphologically investigated based on permanent preparation after protargol impregnation and was compared with other related congeners. According to the infraciliature, three ciliary groups can be recognized, which reveals a very stable structure among specimens and denotes that the pattern of infraciliature is, apart from the features of the lorica, a highly reliable criterion for species identification.

Acute Toxicities of Potassium Permanganate, Formalin, and Lugol's Iodine Solution to a Marine Ciliate, Pleuronema coronatum (Ciliophora, Scuticociliatida)

Acute toxicities of potassium permanganate, formalin, and Lugol’s iodine solution to a commonly occurred marine ciliate Pleuronema coronatum (Ciliophora, Scuticociliatida) were measured. Linear regression analysis of the results highlighted the close relationships between doses of the medicines and mortalities of the organisms, thus providing a capability to predict toxicity effects from the dose. Toxic effects of the medicines on the ciliates were described in the present paper, and the median lethal concentrations (LC 50 values) were given. Results of measurements indicated that 2 h-LC 50 and 12 h-LC 50 values of formalin on P. coronatum were 59.00×10 -6 and 43.57×10 -6, while those of Lugol’s solutions were 90.13 and 67.84×10 -6 respectively. The tolerance of P. coronatum to formalin is apparently lower than that to Lugol’s iodine solution and potassium permanganate is a suitable medicine to kill ciliates in short time.

Meso-scale spatial distribution of large tintinnids in early summer in southern Yellow Sea

The spatial distribution of some large tintinnid species (nominally>76 μm) was investigated according to samples collected by vertical towing in cruises to the southern Yellow Sea in summer 2000-2002 and 2004. Eight species were identified: Codonellopsis mobilis, Leprotintinnus netritus, Tintinnopsis karajacensis, T. japonica, T. kiaochowensis, T. butschlii, T. radix, and Parafavella sp. With maximum abundance of 158.2 ind/L in June 2004, C. mobilis was the dominant species, lasting from May to July 2004. Tintinnid communities were patchy and distributed mainly in shallow waters along the shore.

Ecological functions of ciliated protozoa in marine ecosystem： effects of ammonium on the population growth of Euplotes vannus

The effects of ammonium on the population growth of the marine ciliate, Euplotes vannus, were examined using ecotoxicological method, h is showed that ammonium exerts inhibitory effects on the growth of the ciliate populations in a concentration-dependent way. Statistical analvsis reveals that the population growth dynamics exposed to ammoniunt-N concentration over 100mg/L are significantly different from that in the control at P 〈 0.05 level. Linear regression determined that the 24h, 36h, 48h, 60h, 72h and 84h IC50 values of ammonium-N are 19.68, 201.51, 167.49, 47.86, 50.43 and 43.11 concentration over 100mg/L, respectively （ P 〈 0.05; pH 8.2; salinity 28 ppt; temperature, 25℃ ）. The results indicate that the tolerance to ammonium in E. vannus is considerably higher than that of the larvae or juveniles of some metozoa, such as cuhured prawns and oysters. Therefore, it is believed that the high tolerance to ammonium is necessary for eiliated protozoa to play positive roles in maintaining and improving water quality in marine ecosystems, especially in the intensive aquacuhure waters with high-level ammonium. In addition, the correlation between /C50 values and exposure time was discussed.

Molecular Phylogenetic Lineage of Plagiopogon and Askenasia(Protozoa, Ciliophora) Revealed by Their Gene Sequences

Prostomates and haptorians are two basal groups of ciliates with limited morphological characteristics available for tax- onomy. Morphologically, the structures used to identify prostomates and haptorians are similar or even identical, which generate heavy taxonomic and phylogenetic confusion. In present work, phylogenetic positions lineage of two rare genera, Plagiopogon and Askenasia, were investigated. Three genes including small subunit ribosomal RNA gene （hereafter SSU rDNA）, internal transcribed spacer region （ITS region）, and large subunit ribosomal RNA gene （LSU rDNA） were analyzed, 10 new sequences five species each. Our findings included 1） class Prostomatea and order Haptorida are multiphyletic; 2） it may not be appropriate to place order Cyclotriehiida in subclass Haptoria, and the systematic lineage of order Cyclotrichiida needs to be verified further; 3） genus Plagio- pogon branches consistently within a clade covering most prostomes and is basal of clade Colepidae, implying its close lineage to Prostomatea; and 4） Askenasia is phylogenetically distant from the subclass Haptoria but close to classes Prostomatea, Plagiopylea and Oligohymenophorea. We supposed that the toxicyst of Askenasia may be close to taxa of prostomes instead of haptorians, and the dorsal brush is a more typical morphological characteristics of haptorians than toxicysts.

Insights into the phylogeny of sporadotrichid ciliates (Protozoa, Ciliophora: Hypotricha) based on genealogical analyses of multiple molecular markers

The sporadotrichid ciliates are an especially diverse group. A number of investigators have studied the morphological, morphogenetic, and molecular relationships among members of this group. Despite this, a consistent classification is still lacking and several important questions about the phylogenetic relationships within this group remain unsolved. To improve our understanding of these relationships, we constructed phylogenetic trees using the nucleotide sequences of the small-subunit rRNA (SSrRNA) gene and amino acid sequences of actin I and α-tubulin. Analyses of SSrRNA gene sequences indicated that: 1) the Sporadotrichida sensu Lynn (2008) and the Oxytrichidae are polyphyletic; 2) the Uroleptus species, which are classified to urostylids, formed a sister group with the oxytrichids; 3) Halteria grandinella, which is grouped morphologically with oligotrich species, clustered within the oxytrichids. These results are congruent with previous studies based on SSrRNA gene sequences. However, the amino acid sequences of actin I and α-tubulin yielded different topologies. The main results are: 1) in all phylogenetic trees, the genus Oxytricha was paraphyletic; 2) Uroleptus was sister to a subset of Urostyla and Holosticha, albeit with low supporting values; 3) Halteria grandinella was separated distantly from the Oxytrichidae in trees inferred from actin I amino acid sequences but clustered with oligotrichids in the α-tubulin analysis. The inconsistency among the trees inferred from these different molecular markers may be caused by rapidly accumulated genetic characterizations of ciliates. Further studies with additional molecular markers and sampling of more taxa are expected to better address the relationships among sporadotrichids.