Separation and relationship of ten marine scuticociliates (Protozoa, Ciliophora) using RAPD fingerprinting method

On the basis of the random amplified polymorphic DNA-fingerprinting (RAPD) method, 10 morphospecies of scuticociliates from 7 genera, including 15 clones of 13 strains, Uronema marinum,Uronema cf marinum, Parauronema virginianum, P. longum, Metanophrys similis, M. sinensis, Paralembus digitiformis, Mesanophrys carcini, Paranophrys magna and Cohnilembus verminus were analysed using 8 oligonucleotide primers. The genetic similarity among the clones of the same strain measured by a band-sharing index is 0.97￣0.98, while 0.40￣0.52 among strains. This value measured is 0.39￣0.46 among congeners of the same genus, whereas 0.16￣0.47 between different genera. A distance tree was constructed based on 8-primer analysis, in which the scuticociliates investigated were separated into two clusters: one consists of 2 genera, Uronema and Parauronema, and the other was composed of five, Metanophrys, Mesanophrys, Paranophrys, Paralembus and Cohnilembus. It is demonstrated also that the morphospecies Parauronema virginianum may be a species-complex, i. e., it contains different genospecies.

Tolerance of ciliated protozoan Paramecium bursaria (Protozoa, Ciliophora) to ammonia and nitrites

The tolerance to ammonia and nitrites in freshwater ciliate Paramecium bursaria was measured in a conventional open system. The ciliate was exposed to different concentrations of ammonia and nitrites for 2h and 12h in order to determine the lethal concentrations. Linear regression analysis revealed that the 2h-LC50 value for ammonia was 95.94 mg/L and for nitrite 27.35 mg/L using probit scale method (with 95% confidence intervals). There was a linear correlation between the mortality probit scale and logarithmic concentration of ammonia which fit by a regression equation y=7.32x–9.51 (R2=0.98; y, mortality probit scale; x, logarithmic concentration of ammonia), by which 2 h–LC50 value for ammonia was found to be 95.50 mg/L. A linear correla- tion between mortality probit scales and logarithmic concentration of nitrite is also followed the regression equa- tion y=2.86x+0.89 (R2=0.95; y, mortality probit scale; x, logarithmic concentration of nitrite). The regression analysis of toxicity curves showed that the linear correlation between exposed time of ammonia-N LC50 value and ammonia-N LC50 value followed the regression equation y=2 862.85e-0.08x (R2=0.95; y, duration of exposure to LC50 value; x, LC50 value), and that between exposed time of nitrite-N LC50 value and nitrite-N LC50 value followed the regression equation y = 127.15e-0.13x (R2=0.91; y, exposed time of LC50 value; x, LC50 value). The results demonstrate that the tolerance to ammonia in P. bursaria is considerably higher than that of the larvae or juveniles of some metozoa, e.g. cultured prawns and oysters. In addition, ciliates, as bacterial predators, are likely to play a positive role in maintaining and improving water quality in aquatic environments with high-level ammonium, such as sewage treatment systems.

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.

Phylogeny of subclass Scuticociliatia(Protozoa,Ciliophora) using combined data inferred from genetic,morphological,and morphogenetic evidence

Gene sequence-based genealogies of scuticociliates are different from those produced by morphological analyses.For this reason,11 representative scuticociliates and two ambiguously related genera were chosen to test the ability of combined phylogenetic analyses using both gene sequences and morphological/morphogenetic characteristics.Analyses of both the SSrRNA gene sequences and the combined datasets revealed a consistent branching pattern.While the terminal branches and the order level relationships were generally well resolved,the family level relationships remain unresolved.However,two other trees based on ITS1-5.8S-ITS2 region sequences and morphological/morphogenetic characters showed limited information,due to a lack of informative sites in these two datasets.Our data suggest,however,that the combined analysis of morphological/morphogenetic characters and gene sequences did produce some changes to the phylogenetic estimates of this group.

Complementary Notes on a ‘Well-known’ Marine Heterotrichous Ciliate, Folliculinopsis producta (Wright, 1859) Frauré-Fremiet, 1936 (Protozoa, Ciliophora)

The living morphology and infraciliature of a heterotrichous ciliate, Folliculinopsis producta (Wright, 1859) Frauré Fremiet, 1936, which was collected from the north coast of China, were investigated by in vivo observation and protargol impregnation techniques. As a new contribution, a redescription is presented: large Folliculinopsis of green to dark green in color, 800-1500 μm in size; two peristomial lobes of approximately equal size, 300-400 μm in length; adoral zone of membranelles containing about 1000 membranelles, lying along lobe margins and exhibiting two circles within buccal cavity; 50-70 somatic kineties in mid body; macronucleus miniliform, consisting of about 20 beads; lorica smooth, vase shaped, (300-500) μm×(90-130) μm in size, with 5-12 spiral ridges on neck tube; marine habitat.

The Preliminary Report on Rumen Protozoa Grazing Rate on Bacteria with a Fluorescence-Labeled Technique

Studies on the bacterial predation rate by rumen protozoa were carried out under laboratory conditions using a technique of fluorescence-labeled bacteria （FLB）. Four Xuhuai goats were used in this experiment to obtain rumen protozoa and bacteria. Two groups were designed as follows： One group was the whole bacteria which were labeled using fluorescence through removing free bacteria from rumen fluid （WFLB）; the other group was the bacteria which were labeled using fluorescence without removing free bacteria from rumen fluid （FLB）. The result indicated that the bacterial predation rates of rumen protozoa was 398.4 cells/（cell h） for the group WFLB, 230.4 cells/（cell h） for the group FLB, when the corresponding values expressed as bacteria-N, they were 2.15 pg N/（cell h） for the group WFLB, and 1.24 pg N/（cell h） for the group FLB, respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat, there were 103.2 mg N/（d capita） for the group WFLB, and 59.5 mg N/（d capita） for the group FLB, respectively. It was estimated that protein losses due to microbial recycling were 0.645 g pro/（d capita） for the group WFLB and 0.372 g pro/（d capita） for the group FLB, respectively. In addition, the fluorescence-labeled technique would be a potential assay for the determination of bacterial predation rate by rumen protozoa.

Intestinal protozoa and intestinal helminthic infections among schoolchildren in Central Sudan

Objective:To determine the prevalence of intestinal parasitic infections and soil-transmitted helminths(STHs) among primary schoolchildren in El dhayga,Central Sudan.Methods:In this cross-sectional study,three fresh faecal samples were collected from each child,which were examined by direct wet mount,brine flotation,formalin-ether and Kato-Katz techniques.The intensity of each STH infection was expressed as the mean of eggs per gram counts of the three samples.Results:In total,142(90.4%) of 157 children harboured at least one type of intestinal parasite.Ascaris lumbricoides,Hymenolepis nana,Entamoeba histolytica and Giardia lamblia were the most common parasites found,with prevalence rates of 32.5%,30.6%,33.1%and 19.7%,respectively.Out of these 157 children,29(18.5%) harboured more than two intestinal parasitic infections.No cases of Schistosoma mansoni or Enterobius vermicularis were identified. Conclusions:The study demonstrates significant burden of intestinal protozoa and STH infections in this part of Sudan and highlights the need for preventive and intervention measures.

The Importance of Three Protozoa in Corn Straw Decomposition and Nutrient Transformation

Three typical soil protozoa of Bodo edax, Colpoda cucullus and Amoeba proteus were inoculated into the soil amended with corn straw. The soils were then incubated at 25℃ for 60 days. It was found that the protozoa, particularly Bodo edax, significantly reduced soil microbial biomass C. However, the decomposition of corn straw was accelerated by the protozoa. Colpoda cucullus significantly enhanced soil available P content, but Amoeba proteus decreased soil available P content. Colpoda cucullus and Bodo edax did not obviously influence NH4+-N and NO3--N contents. In contrast, Amoeba proteus significantly increased both NH4+-N and NO3--N contents.

The community structure and microbial linkage of rumen protozoa and methanogens in response to the addition of tea seed saponins in the diet of beef cattle

Background: This study investigated changes in rumen protozoal and methanogenic communities, along with the correlations among microbial taxa and methane(CH_4) production of six Belmont Red Composite beef steers fed tea seed saponins(TSS). Animals were fed in three consecutive feeding periods, a high-grain basal diet for 14 d(BD period) then a period of progressive addition of TSS to the basal diet up to 30 g/d for 20 d(TSS period), followed by the basal diet for 13 d without TSS(BDP post-control period).Results: The study found that TSS supplementation decreased the amount of the protozoal genus Entodinium and increased Polyplastron and Eudiplodinium genera. During BDP period, the protozoa community of steers did not return to the protozoal profiles observed in BD period, with higher proportions of Metadinium and Eudiplodinium and lower Isotricha. The addition of TSS was found to change the structure of methanogen community at the subgenus level by decreasing the abundance of methanogens in the SGMT clade and increasing the abundance of methanogens in the RO clade. The correlation analysis indicated that the abundance of SGMT clade methanogens were positively correlated with Isotricha, and Isotricha genus and SGMT clade methanogens were positively correlated with CH_4 production. While RO clade were positively correlated with the proportion of Metadinium genus, which was negatively correlated with CH_4 emission.Conclusions: These results suggest that different genera of rumen protozoa ciliates appear to be selectively inhibited by TSS, and the change in methanogen community at the subgenus level may be due to the mutualistic relationships between methanogens and rumen ciliates.

Molecular detection of vector-borne bacteria and protozoa in healthy hunting dogs from Central Italy

Objective:To determine the pi'evalence of vector-bome bacteria and protozoa in hunting dogs living in Central Italy.Methods:Molecular testing was executed on DNA which was extracted from blood specimens collected from 117 asymptomatic dogs to detect Anaplasma phagocytophilum,Babesia canis(B.canis),Bartonella spp..Coxiella burnetii(C.burnetii).Ehrlichia canis.Hepatozoon canis.and Leislnnania infantum.Results:A total of 48 dogs(41.0%) were infested by Ixodes ricinus and Rhipicephalus sanguineus ticks.Tick-borne infections were observed in 64(54.7%) animals.More in detail.38 dogs(32.5%) screened positive for Hepatozoon canis,24(20.5%) for Bartonella rinsonii subsp.berkhoffii.20(17.1%) for Leishmania infantum,6(5.1%) for C.burnetii,5(4.3%) for B.canis(3 B.canis vogeli and 2 B.canis canis),3(2.5%) for Anaplasma phagocytophilum,and 2(1.7%) for Ehrlichia canis.Mixed infection by 2 agents occurred in 17(14.5%) subjects,by 3 agents in 7(6.0%) dogs,and by 4 agents in 1(0.9%) animal.Conclusions:The results demonstrated that several vector-borne pathogens were circulating in this region and dogs infected by these agents were usually asymptomatic.A relevant finding was the presence of DNA of C.burnetii,a severe zoonotic agent,in the 5.1% of tested dogs,which can be source of infection for their owners not only through tick bites,but also directly with urine,feces and birth products.

Evolution of discocephalid ciliates:Molecular,morphological and ontogenetic data support a sister group of discocephalids and pseudoamphisiellids (Protozoa,Ciliophora) with establishment of a new suborder Pseudoamphisiellina subord.n.

Discocephalids and pseudoamphisiellids are possibly two of the most confused groups among hypotrichous/euplotid ciliates regarding their systematic position and phylogenetic relationships.The former were often regarded as related to euplotids while the latter,in the absence of molecular data,were mostly assigned to the urostylid-like hypotrichs.In the present work,the small subunit rRNA genes of several rarely observed discocephalid and pseudoamphisiellid genera were analyzed to obtain insights into the phylogenetic relationships of these highly ambiguous Spirotrichea.Four different tree reconstruction algorithms yielded nearly identical topologies,which indicated both groups belong to the same assemblage.This assemblage is clearly isolated as a deep-branching clade and invariably positioned between Euplotida and Hypotricha.The sister group relationship of the Pseudoamphisiellidae and Discocephalidae supports the previous suggestion that they might represent an ordinal taxon,the Discocephalida.Both morphological and morphogenetic features indicate that the pseudoamphisiellids should be placed in the order Discocephalida but as a sister group to other typical discocephalids.Thus we propose establishing a new suborder,Pseudoamphisiellina subord.n.The new taxon is diagnosed by the following characteristics:(i) two distantly separated midventral rows that are morphogenetically formed with an urostylid mode;(ii) absence of the "frontoterminal row",which is formed from the posterior-most frontoventral-transverse cirral anlage in all other typical urostylids;(iii) numerous caudal cirri that derive from each of the dorsal kinety anlagen;(iv) right marginal row that has a unique de novo origin;and (v) inhabiting periphytic communities.The validity of the suborder Pseudoamphisiellina is firmly supported by molecular data.

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.

Preliminary Study on Applicability of Microsatellite DNA Primers from Parasite Protozoa Trypanosoma cruzi in Free-living Protozoa

In this paper, we took the lead in studying on specificity of the microsatellite DNA loci and applicability of mi crosatellite DNA primers in protozoa. In order to study characters of microsatellites in free living protozoa, eight microsatellite loci primers developed from Trypanosoma cruzi (MCLE01, SCLE10, MCLE08, SCLE11, MCLF10, MCLG10, MCL03, MCL05) were employed to amplify microsatellite in four free living protozoa, including Bodo designis, Euglena gracilis FACHB848, Paramecium bruzise and Tetrahymena thermophila BF1. In the amplification systems of P. bruzise, four loci (SCLE10, SCLE11, MCLF10, MCL03) were amplified successfully, and four amplification fragments were in proper size. In genome of E. gracilis FACHB848, five of eight primers brought five clear amplification bands. In B. designis, three (No.4, 5 and 7) of eight loci produced clear and sharp products without stutter bands, whereas no bands appeared in T. thermophila BF1. Further, eight 300-500 bp amplification fragments were cloned and sequenced. Nevertheless, all sequenced products did not contain corresponding microsatellite sequence, although Bodo is in the same order and has the nearest phylogenetic relation with Trypanosoma among these four species. Thus, the microsatellite DNA primers can not be applied among order or more far taxa, and the specificity of microsatellite DNA is very high in protozoa. The results of this study will contribute to our understanding of microsatellite DNA in protozoa.

Spatio-temporal variability of periphytic protozoa related to environment in the Niyang River,Tibet,China

The Niyang River, a main tributary of the Yarlung Zangbo River, is an important and typical plateau fiver ecosystem in Tibet, China. At present, few studies have focused on its aquatic living resources and fiver ecology. In this study, the composition, abundance, and diversity of periphytic protozoa were investigated across four seasons from 2008 to 2009 to better understand their spatio-temporal patterns and relationship to the environment. Our investigation shows that periphytic protozoa in the Niyang River contained 15 genera, belonged to Tubulinea, Alveolata, Discosea and Rhizaria, Alveolata possessed most genera, up to nine, with highest share in abundance, exceeding 50%, Difflugia and Glaucoma were dominant genera. Moreover, four diversity indices of periphytic protozoa, including species richness, total abundance, Shannon-Wiener diversity index and Pielou＇s evenness index, displayed a significant descending trend as the seasons continued, in the order of winter, spring, summer and autumn; with a significant difference existing between winter and summer （or autumn） for Shannon-Wiener diversity index and species richness （P〈0.05）. Four of these diversity indices also presented a V-shaped pattern between the upper middle course of the Niyang River and the confluence of the Niyang River and Yarlung Zangbo River, with the lowest value occurred in the middle course of the Niyang River. However, no significant variation was found through the Niyang River （P〉0.05）. In addition, canonical correlation analysis （CCA） shows that the densities of Difflugia, Glaucomais, Enchelydium, Cyphoderia, and Enchelys correlate with water temperature, alkalinity, hardness, pH, and dissolved oxygen, respectively. Lastly, the relationship between periphytic protozoa diversity and the environmental factors of the Niyang River can be predicted using classification and regression trees （CART） annalysis, which suggests that the total abundance and Shannon-Wiener diversity index would be higher when the elevation is above 3 308 m. On the other hand, the Shannon-Wiener diversity index and Pielou＇s evenness index would be lower when pH and ammoniacal nitrogen have lower or higher values. Finally yet importantly, close attention should be paid to periphytic protozoa and its environment to ensure sustainable development of the Niyang River ecosystem.

PRELIMINARY STUDIES ON THE ROLE OF PROTOZOA IN THE FOOD WEB OF DONGHU LAKE

Plankton from Station I of Donghu Lake, an eutrophic lake, was divided into three groups (2-32 μm, 32-112 μm and >112 μm) by filtering the water sample through 2, 32, 112 μm pores. It was supposed that the 2-32 μm Protozoa (group 1) was fed on by the 32-112 μm Protozoa and small rotifers (group 2) and >112 μm large rotifers, cladocerans and copepods (group 3). The feeding rate of zooplankton in situ was estimated by counting the protozoan individuals and also by the 14 C NaHCO 3 method. The P/B ratio of the small Protozoa (group 1) was 2.9 for 6 h and 1.2 for 24 h. The feeding rate of the 32-112 μm zooplankton on small Protozoa (2-32 μm) was about 2/3 the total feeding rate of zooplankton, and that of >112 μm zooplankton on the 2-32 μm Protozoa was 1/3 of the total feeding rate. The lower the density of feeders, the higher were the feeding rates. The daily production of the 2-32 μm protozoans was 1.559 mg/L, which equaled to 25% of the daily food consumption of other zooplankton. Isotope experiments showed result similar to that from the above direct counting method.

SPECIES COMPOSITION AND SEASONAL DENSITY DYNAMICS OF CILIATED PROTOZOA IN BAOAN LAKE WITH PARTICULAR REFERENCE TO THE SUBMERGED MACROPHYTES

From April 1996 to October 1997, regular samplings were carried out monthly at 8 stations in a macrophytic basin of Baoan Lake. From the water samples, 47 genera, 96 species of ciliata were identified. Stations covered with macrophytes had greater number of ciliate species and higher percentage of sessile species. The mixotroph Strombidium viride bearing algal endosymbionts dominated numerically the whole ciliate communities; most of the other dominants were bactivores. Total ciliate density in Lake Baoan was 6170-34310 ind./L. The seasonal density fluctuations of the dominant species populations were also investigated. Maximum abundances were observed in spring and winter during the decay of macrophytes and minimum densities were observed during the summer months of luxuriant macrophytes growth.

Protozoa of Lough Neagh,Northern Ireland,UK

This paper made the first records of taxa composition of protozoa in Lough Neagh,the largest freshwater lake in British Isles. One hundred and eight species(genus)were identified from the examinations of approximately 150 PFU artificial substrates and various types of natural substrate from May to December, 19g2. Of 108 species or genus,30 core species were found on PFU substrate and 17 on natural ones.The ecological characteristics of protozoa were studied by showing the variations of species richness and individual abundance as a function of season or water temperature.Protozoa on PFU were compared at two sampling stations as well.

A Porous Simulation Experiment of Protozoa Movement with Glass Bead

Protozoa can move due to their flagella, cilia or pseudopodia, and play an important role in the transformation and cycling of matter and energy in ecosystems. In this study, the glass beads with the diameters of 1. 0, 0.5, 0. 25 and 0.1 mm were used to simulate soil pores of 0. 292 - 0. 258, 0.155 - 0.116, 0.056 - 0.040 and 0.028 - 0.020 mm respectively. Three typical soil protozoa of Bodo edax, Colpoda cucullus and Amoeba proteus were examined for the relation of their movement to pores. It was found that the smallest protozoan of Bodo edax could be leached through all the pores. However, no more than 26% of the added protozoan was leached out of the column. The leached protozoa were decreased with the pore sizes and leaching duration. The bigger one of Colpoda cucullus was moved out from the column filled with the glass beads of > 0. 5 mm in diameter. Less than about 15% of the added protozoa were leached out of the column. The biggest one of Amoeba proteus could not leached out from any of the pores. It is evident that most of the protozoa remained in the column due to their occupation of the pores.

An improved glucose-DAB-nickel immunohistochemistry method and its application on protozoan Stylonychia mytilus

Using a modification of the very sensitive glucose-DAB-nickel(GDN)immunohistochemistry method,FOS(the expression protein of the oncogene c-fos)-,neuropeptide Y(NPY)-,somatostatin(SOM)-,leu-enkephalin(L-Enk),cholecystokinin(CCK)-,neurotensin(NT)-,and tyrosine hydroxylase(TH)-immunoreactivities were firstdemonstrated in the protozoan,Stylonychia mytilus.The GDN method and expression ofFOS,neuropeptides and TH in the Stylonychia mytilus were discussed.

Research Progress in NTPase of Toxoplasma gondii and Other Protozoa

Nucleoside triphosphate hydrolase （NTPase） is a multifunctional enzymatic family widely existing in vivo. They can hydrolyze NTP to NMP or dNTP to dNMP to produce energy. In this article, the structure of Toxoplasma gondii NTPase is analyzed. The research progress in NT- Pase of Toxoplasrna gondii, Trypanosoma cruzi, Sarcocystis neurona and Neospora caninum was briefly reviewed.