Discovery, Antitumor Activity, and Fermentation Optimization of Roquefortines from Penicillium sp. OUCMDZ-1435.

Meleagrin and oxaline, which belong to the roquefortine alkaloids with a unique dihydroindole spiroamide framework, have significant bioactivities, especially tumor cell inhibitory activity. In order to discover the requefortine alkaloids, Penicillium sp. OUCMDZ-1435 was fished and identified from marine fungi using molecular probe technology. Meleagrin (1) and oxaline (2) were isolated from it. In addition, we first reported that compounds 1 and 2 could effectively inhibit the proliferation and metastasis of the human HepG2 cell and induce HepG2 cell apoptosis and cell cycle arrest in the G2/M phase. Additionally, the fermentation of Meleagrin (1) was optimized to increase its yield to 335 mg/L. These results provided bioactive inspiration and fungus resources for roquefortine alkaloid development.

Morphology and small-subunit rRNA gene sequences of two novel marine ciliates, Metanophrys orientalis spec. nov. and Uronemella sinensis spec. nov. (Protista, Ciliophora, Scuticociliatia), with an improved diagnosis of the genus Uronemella.

The morphology and infraciliature of two novel marine scuticociliates, Metanophrys orientalis spec. nov. and Uronemella sinensis spec. nov., collected from sandy beaches at Qingdao, China, were investigated using live observation and protargol-staining methods. Metanophrys orientalis spec. nov. is distinguished by the following characteristics: marine habitat and a slender to elongate oval body with pointed anterior end and rounded caudal end, in vivo about 25-50 µm long; buccal field about a quarter to a third of body length; nine or ten somatic kineties with dikinetids approximately in anterior half of body, monokinetids in posterior half; membranelles 1 and 2 almost equal in length and composed of two and three longitudinal rows of kinetids respectively; paroral membrane with zigzag structure extending anteriorly to middle portion of membranelle 2; contractile vacuole pore located at posterior end of somatic kinety 1. The genus Uronemella is redefined as follows: marine form with an elongate-elliptical or inverted pear-shaped body; apical plate conspicuous; buccal field about two-thirds of body length, cytostome subequatorially located; oral apparatus Uronema-like; somatic kineties comprising a mixture of dikinetids and monokinetids. Uronemella sinensis spec. nov. is recognized by having an elongate-elliptical body with truncated apical frontal plate, size in vivo about 25-35 × 15-20 µm, nine or ten somatic kineties, membranelle 1 consisting of two or three basal bodies, contractile vacuole pore at posterior end of somatic kinety 1. This study also compared the small-subunit rRNA gene sequences of these two species with other closely related species to show the sequence divergence, which ranged from 3.53 to 9.60%. Phylogenetic analyses support the contention that the genus Uronemella is monophyletic, while Metanophrys is non-monophyletic.

Morphology and morphogenesis of a soil ciliate, Rigidohymena candens (Kahl, 1932) Berger, 2011 (Ciliophora, Hypotricha, Oxytrichidae), with notes on its molecular phylogeny based on small-subunit rDNA sequence data.

The morphology and morphogenesis of the stylonychine hypotrich Rigidohymena candens (Kahl, 1932) Berger, 2011, isolated from garden soil in Qingdao, China, were investigated using live observation and protargol impregnation methods. The Qingdao isolate possesses all diagnostic morphological characters of R. candens. The main events during binary fission are as follows: (i) the proter retains the parental adoral zone of membranelles entirely, whereas the old undulating membranes dedifferentiate into an anlage that gives rise to the leftmost frontal cirrus and the new undulating membranes of the proter; (ii) five streaks of fronto-ventral-transverse cirral anlagen are segmented in the pattern 3 : 3 : 3 : 4 : 4 from left to right, which form two frontal, four frontoventral, one buccal, five ventral and five transverse cirri, respectively; (iii) dorsal morphogenesis is in the typical Oxytricha pattern; (iv) three caudal cirri are formed, one at the posterior end of each of dorsal kineties 1, 2 and 4; and (v) the postoral ventral cirrus V/3 is not involved in primordia formation. The morphological and morphogenetic observations and phylogenetic analyses based on the small-subunit rDNA sequence data support the validity of Rigidohymena Berger, 2011 and its systematic position in the subfamily Stylonychinae.

Influence of enumeration time periods on analyzing colonization features and taxonomic relatedness of periphytic ciliate communities using an artificial substratum for marine bioassessment.

Colonization features and taxonomic relatedness measures of ciliate communities have been used as useful indicators for marine bioassessment. The influence of enumeration time periods on analyzing colonization features measures of periphytic ciliate communities was studied in coastal waters of the Yellow Sea, northern China, during the period of May-June 2010. Ciliated protozoan samples were collected at depths of 1 m using an artificial substratum and were analyzed with different enumeration schemes. The ciliate species were identified using living observation and silver impregnation. Data analyses were conducted using a range of multivariate statistical routines. Enumeration time periods represented a strong influence on analyzing both colonization and taxonomic relatedness features of periphytic ciliate communities, although no significant changes occurred in colonization patterns between two enumeration schemes (within 24 and 24-48 h after sampling). The delayed enumeration (within 24-48 h) may result in the species richness, individual abundance, colonization rate decreasing to standard errors of >10 % in samples with almost all colonization ages, and in the similarities of the communities being reduced to 11-38 %. However, the species biodiversity (e.g., species diversity and evenness, except species richness) and taxonomic relatedness (taxonomic diversity, taxonomic distinctness and average taxonomic distinctness, except variation in taxonomic distinctness) measures of periphytic ciliate communities were weakly sensitive to disturbance from the delayed enumeration, achieving standard errors of <10 and <5 % during the colonization periods, respectively. These results suggest that the enumeration should be completed as soon as possible within 24 h after sampling to analyze colonization and taxonomic relatedness features of periphytic ciliate communities, and that the species diversity and taxonomic distinctness measures can be used on a robust bioindicator with weak dependence on enumeration time limits for monitoring programs and ecological investigations in marine ecosystems.

Can nonloricate ciliate assemblages be a surrogate to analyze taxonomic relatedness pattern of ciliated protozoan communities for marine bioassessment? A case study in Jiaozhou Bay, Northern China.

Congruency using nonloricate ciliates as a surrogate to analyze taxonomic distinctness pattern of ciliated protozoan communities for assessing water quality was studied. The results showed that (1) with significant consistency with the total ciliate communities, the spatial pattern of nonloricate ciliates was significantly correlated with the changes of environmental status; (2) four taxonomic relatedness measures were significantly correlated with those of the total ciliate communities; (3) spatial variations in four taxonomic diversity indices of nonloricate ciliates were significantly related with the changes of environmental variables, especially nutrients; and (4) the paired taxonomic biodiversity indices of nonloricate ciliates showed a clear decreasing trend of departure from the expected taxonomic breadth in response to water quality. These results suggest that nonloricate ciliate assemblages can be used as a potential surrogate of ciliate communities to assess marine water quality using taxonomic distinctness measures, especially the paired indices based on presence or absence data.

Can body-size patterns of ciliated zooplankton be used for assessing marine water quality? A case study on bioassessment in Jiaozhou Bay, northern Yellow Sea.

INTRODUCTION: In order to reveal the potential relationships between body-size patterns of microzooplankton and environmental status, the spatial patterns in body-size spectra of ciliated zooplanktons were studied based on an annual dataset in a bay of the Yellow Sea, northern China. MATERIALS AND METHODS: A total of 120 samples were collected at a depth of 1 m from each of five sampling sites with a spatial gradient of environmental stress from June 2007 to May 2008. A range of physico-chemical variables were measured synchronously for comparison with biotic parameters. RESULTS: The spatial body-size patterns of ciliated zooplankton represented significant differences among the five sites, and were significantly correlated with the changes of physico-chemical parameters, especially salinity, dissolved oxygen and nutrients. Two paired indices, the average body-size distinctness (AvBSD) and the variation in body-size distinctness (VarBSD), were proposed based on the trait resemblances among ciliate species in body-size pattern. The paired measures showed a clear decreasing trend of departure from the expected body-size spectra in response to water quality status. CONCLUSION: These results suggest that the body-size pattern of ciliated zooplankton might be used as a potential indicator of marine water quality.

Influence of sampling sufficiency on biodiversity analysis of microperiphyton communities for marine bioassessment.

INTRODUCTION: With quick responses to environmental changes, easy sampling, relative immobility, increasing availability of easily used taxonomic references, and allowing standardization for temporal and spatial comparisons, the biodiversity measures of microperiphyton communities have widely been accepted as useful indicators to evaluate environmental stress and anthropogenic impact. MATERIALS AND METHODS: The influence of sampling sufficiency for biodiversity analysis of microperiphyton communities was studied using a range of statistical methods in coastal waters of the Yellow Sea, northern China, from May to June 2010. Samples were collected from two depths using an artificial substrate. RESULTS: Sampling sizes represented a significant influence on biodiversity analysis of microperiphyton communities, e.g., 20 slide replicates (350 cm(2)) were sufficient for the microperiphyton communities at both depths, while 10 slide replicates (175 cm(2)) could meet the sampling strategy only for the samples with colonization times of 10 days or more at a depth of 1 m for recovering 90% species during the study period. Otherwise, more slide replicates were required with the increase of water depths and shortening colonization times for recovering microperiphyton species, e.g., for recovering 90% species of a "mature" microperiphyton community (>10 days), ∼10 slide replicates (∼175 cm(2)) were sufficient at a depth of 1 m, while for the "young" samples (>10 days) much more (15-30) slide replicates were required at both depths in this study. Furthermore, to achieve <10% standard errors, six (105 cm(2)) and nine (∼160 cm(2)) slide replicates were required for biodiversity analysis of the microperiphyton communities with various colonization times at depths of 1 and 3 m during the summer season, respectively. CONCLUSION: These results suggest that sampling sizes represented a significant influence on biodiversity analysis of microperiphyton communities for monitoring programs and ecological conservation researches in marine ecosystems.

An approach to determining potential surrogates for analyzing ecological patterns of planktonic ciliate communities in marine ecosystems.

INTRODUCTION: In order to identify a potential surrogate of planktonic ciliate communities for marine bioassessments and evaluating biological conservations, the different taxonomic/numerical resolutions and taxa as surrogates were studied in Jiaozhou Bay, northern China during a 1-year cycle (June 2007-May 2008). MATERIALS AND METHODS: Samples were collected biweekly from a depth of 1 m at each of five sites. A range of physicochemical parameters were also measured in order to determine water quality. RESULTS: The genus- and family-level resolutions maintained sufficient information to evaluate the ecological patterns of the ciliate communities in response to environmental status. The non-loricate oligotrichous ciliate assemblages in both abundance and occurrence may be used as a surrogate of planktonic ciliate communities. Heavy data transformations were an optimal strategy for the species level of planktonic ciliates, while mild data transformations were for the higher. The ordination patterns based on species biomass, occurrence, and biomass/abundance ratio matrices were significantly consistent with that of species abundance data. CONCLUSION: The results suggest that the use of simplifications at both taxonomic and numerical resolutions are time-efficient and would allow improving sampling strategies of large spatial/temporal scale monitoring programs and biological conservation researches in the marine ecosystem with a relative paucity of scientists.

An approach to determining the sampling effort for analyzing biofilm-dwelling ciliate colonization using an artificial substratum in coastal waters.

A new approach to determining sampling effort for analyzing biofilm-dwelling ciliate colonization was studied in the coastal waters of the Yellow Sea, northern China, from May to June 2010. The optimal sample size for evaluating biofilm-dwelling ciliate colonization increased with shortening exposure time, and can be determined according to the probability of recovering those species with a specified cumulative contribution to communities. More slide-replicates were required at a depth of 3 m than at 1 m to recover equivalent proportions of the ciliate communities. For routine colonization dynamics analyses, 10 slide-replicates (175 cm(2)) were sufficient to achieve a 95% probability of recovering those species with a cumulative contribution of >90% to the ciliate communities at a depth of 1 m. These results suggest that 10 slide-replicates immersed at a depth of 1 m may be an optimal sampling strategy for analyzing the colonization dynamics of biofilm-dwelling ciliate communities in marine habitats.

An approach to analyzing spatial patterns of planktonic ciliate communities for monitoring water quality in Jiaozhou Bay, northern China.

Spatial patterns of planktonic ciliate communities for assessment of marine environmental status were studied from June 2007 to May 2008 in Jiaozhou Bay, Qingdao, northern China. Ciliate communities were sampled biweekly at five sampling sites with a spatial gradient of environmental stress. Multivariate/univariate analyses demonstrated that: (1) the planktonic ciliate community structures represented significant differences among the five sites; (2) spatial patterns of the ciliate communities were significantly correlated with environmental variables, especially the nutrients nitrate nitrogen (NO3-N) and soluble reactive phosphates (SRP); (3) five dominant species (e.g., Rimostrombidium veniliae, Strombidium capitatum, Mesodinium pupula and Strombidinopsis acutum) were significantly correlated with nitrogen and/or SRP; and (4) both species richness and species diversity indices were correlated with NO3-N and salinity. These results suggest that planktonic ciliated protozoa might be used as a robust bioindicator of marine water quality.

Cell division and morphology of the marine ciliate, Condylostoma spatiosum Ozaki and Yagiu (Ciliophora, Heterotrichida) based on a Chinese population.

The morphology and morphogenesis of the large marine heterotrichous ciliate, Condylostoma spatiosum Ozaki & Yagiu in Yagiu, 1944, isolated from Jiaozhou Bay off Qingdao (Tsingtao), China, were investigated using live observation and protargol impregnation. A detailed redescription is given since no infraciliature data about this "well-studied" species are available. It is characterized by: size in vivo 400-700 x 90-140 microm; buccal field about 1/3 of body length; adoral zone composed of 130 membranelles on average; invariably two prominent membrane-like frontal cirri, which are parallel to each other; about 58 somatic kineties; macronucleus composed of 13-22 fragments. Morphogenesis proceeds basically as described in a previous report and can be summarized as follows: (1) the parental adoral zone of membranelles is partly dedifferentiated and then renewed in the posterior portion; (2) in the proter, both the frontal cirri and the paroral membrane are newly formed by anlagen derived from the disaggregated old structures; (3) in the opisthe the anlagen of the paroral membrane and the frontal cirri develop from the right margin of the oral primordium; (4) two frontal cirri are formed one after the other by the frontal cirral anlage; (5) during morphogenesis, no recognizable duplication of basal bodies takes place in somatic kineties; and (6) the macronucleus divides after prior fusion. One reorganizer was observed showing that the basic process of reorganization is obviously similar to that in division.

[Monoclonal antibodies prepared and used for detection of acute virus necrobiotic disease virus in scallop Chlamys farreri by indirect ELISA].

For developing monoclonal antibodies against acute virus necrobiotic disease (AVND) virus, mice of Balb/c strain were immunized with AVND virions which were isolated from the infected scallop Chlamys farreri. The spleen cells from immunized mice were then fused with NS-1 myeloma cells and the hybridomas were screened by means of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). Finally, 4 stable MAbs of IgG isotype were obtained. Moreover, the combined position of these 4 MAbs to this virus was examined by immunogold electron microscopy (IEM). The results demonstrate that all 4 MAbs recognized epitopes on the envelope of the virions. Subsequently, a MAb-based ELISA was developed and used for detection of the infection rate and densities of the scallops which were sampled during different seasons from mid-April to mid-October, 2003. The result exhibited that both of the infection rate and infection densities sharply rose in mid-July and reached to the spikes, which right corresponds with their mortality during this period.