A new species of Nassarius (Gastropoda: Nassariidae) from the western Pacific Ocean

This paper describes a new species of Nassarius from the South China Sea,which was recognized when re-sorting the collection of Nassariidae in the Marine Biological Museum,Chinese Academy of Sciences,Qingdao,China.The shells were collected during several investigations,including the National Comprehensive Oceanic Survey in 1958-1959,and the China-Vietnam Co-Investigation on Marine Resource of the Beibu Gulf during 1959-1962.The morphology of the shell and the radula places the new species of Nassarius within the subgenus Zeuxis.It is named Nassarius(Zeuxis) nanhaiensis sp.nov.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ （COⅡ） gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A＋T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

A new species of Lataxiena Jousseaume, 1883 (Gastropoda: Muricidae) from the East and South China Seas

A new muricid gastropod species,L ataxiena lutescena sp. nov.,is described and illustrated. The new species was recognized during reidentification of the Muricidae collection in the Marine Biological Museum,Chinese Academy of Sciences,Qingdao. The specimens of the new species were collected from the East and South China Seas off Fujian,Guangdong,and Hainan Provinces. Lataxiena lutescena sp. nov. is similar to Lataxiena blosvillei(Deshayes,1832) in general shape,but can be distinguished from the latter by the shell sculpture and radular characteristics. Lataxiena lutescena sp. nov. also resembles Lataxiena bombayana(Melvill,1893),but differs from that species in the shell shape and anal notches and in lacking short spines on the shell.     

Construction of cDNA subtractive library from pearl oyster (Pinctada fucata Gould) with red color shell by SSH

The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COΙ. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COΙ so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.     

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.     

The Relationships Between Toxicity,Species and Populations in Nassarius Based on Toxin Detection and Multiple Gene Barcoding

Nassarius causes many poisonings in China and other Asian countries. The toxicity of Nassarius is related to species and populations. However, the molecular assignment of different toxic Nassarius samples is still unclear, which deteriorates the poisoning. Our study combined toxicity, toxin detection and multiple genes DNA barcoding to assign the various toxic Nassarius species and populations from China coast. The toxicity test indicated that different Nassarius species and different populations within one Nassarius species had different toxicity. Based on High Performance Liquid Chromatography, the toxin was confirmed to be TTX. The toxin content varied in different samples. The COI, 16 S and ITS sequences revealed different toxic Nassarius species as monophyletic clades. All the samples tested have been assigned to species level. However, the different toxic populations within N. succinctus, N. variciferus and N. semiplicata did not form separated clades, and the toxic and nontoxic species did not form two clades separately. Our study constructed the relationship between toxicity and taxonomy assignment of different toxic Nassarius samples based on toxicity detection and multiple gene DNA barcoding, which provides the basis for controlling poisoning and studying the toxin-resistance difference in Mollusca. It is also indicated that the toxicity of Nassarius is really related with species at the genetic level, but not absolutely related with populations.     

Characterization and in-vivo evaluation of potential probiotics of the bacterial flora within the water column of a healthy shrimp larviculture system

A thorough understanding of the normal bacterial flora associated with shrimp larviculture systems contributes to probiotic screening and disease control.The bacterial community of the water column over a commercial Litopenaeus vannamei larval rearing run was characterized with both culture-dependent and culture-independent methods.A total of 27 phylotypes at the species level were isolated and identified based on 16 S rDNA sequence analysis.Denaturing gradient gel electrophoresis(DGGE) analysis of the V3–V5 region of 16 S rRNA genes showed a dynamic bacterial community with major changes occurred from stages zoea to mysis during the rearing run.The sequences retrieved were affiliated to four phyla,Proteobacteria,Actinobacteria,Bacteroidetes,and Firmicutes,with the family Rhodobacteraceae being the most frequently recovered one.Subsequently,13 representative strains conferred higher larval survival than the control when evaluated in the in-vivo experiments;in particular,three candidates,assigned to Phaeobacter sp.,Arthrobacter sp.,and Microbacterium sp.,significantly improved larval survival( P 0.05).Therefore,the healthy shrimp larviculture system harbored a diverse and favorable bacterial flora,which contribute to larval development and are of great importance in exploiting novel probiotics.     

Phylogenetic relationships within the genus Aspidisca (Protozoa, Ciliophora, Euplotida) revealed by ITS1-5.8S-ITS2 region sequences

The internal transcribed spacer regions (ITS1 and ITS2) and 5.8S rRNA genes were sequenced in six populations of four Aspidisca species, namely A. leptaspis, A. orthopogon, A. magna and A. aculeata. Phylogenetic trees were constructed by means of Bayesian inference (BI), Maximum Parsimony (MP), Neighbor-Joining (NJ), and Maximum Likelihood (ML) to assess the inter- and intra-species relationships within the genus Aspidisca. All trees show similar topologies with stable supports and indicate that: (1) four well known groups, i.e., Oligotrichia, Stichotrichia, Choreotrichia and Hypotrichia, are distinctly outlined within the class Spirotrichea, and all are monophyletic other than Hypotrichia; (2) members of Aspidisca can be distinguished well, based on the ITS1-5.8S-ITS2 region sequences, and A. leptaspis and A. magna shared a closer relationship than other species; (3) Aspidisca and Euplotes branch early in the subclass Hypotrichia. To compare the phylogenetic relationships based on different genes, SSU rRNA trees were also constructed with nearly the same species inclusion, which revealed different topologies of inter-species, inter-genera and inter-subclasses.     

The Complete Sequence of Mitochondrial COII Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit II (COII) gene of Penaeinae shrimp Fenneropenaeus chinen- sis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COI and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COII gene has a faster evolutionary rate than that of the COI gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ (COⅡ) gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A+T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Morphological changes in development of pharyngeal teeth inMylopharyngodon piceus

Adult pharyngeal teeth inMylopharyngodon piceus are molariform. Based on SEM observations of the developing teeth, this paper describes the morphological diversification of pharyngeal teeth inM. piceus. The larval and juvenile teeth are changed from conical to adult molariform teeth through seven stages. Comparisons are made between each stage and corresponding types in some species of different subfamilies in Cyprinidae. It is considered that the ontogenetic resemblances of the pharyngeal teeth bear relationship to the phylogeny of cyprinids. This project was funded by the International Cooperation of Japan—China and the National Natural Science Foundation of China     

16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.     

Pseudosmittia aizaiensis,a new species of orthocladiinae (Diptera,Chironomidae) from human province of China

Male and female adult midges ofPseudosmittia aizaiensis sp.n. are described and illustrated with figures of morphological characters. The inferior volsella of gonocoxite with large lobes, the long but equal width gonostylus, and the small projection in basal part of outer margin are the characters that may separate this species from others. One larva of this genus is purely aquatic in habitat and is associated with Cricotopus bicinctus,Cardiocladius capucinus andPolypedilum sp. in the same algae debris (Spirogyra sp.) of the Dunghe River. Further, the larva's very short SI and SII bifids, its 5 pairs of lateral teeth on mentum and its absence of premandible brush are the characters that may also separate this species from any known species of this genus.     

Tritonia iocasica sp.nov.,a new tritoniid species from a seamount in the tropical Western Pacific(Heterobranchia:Nudibranchia)

During an expedition to a seamount at Caroline Plate in the tropical Western Pacific,a new species of Tritonia was sampled from the upper bathyal zone at depth of 970-1 262 m.This new species,Tritonia iocasica sp.nov.,represents the first tritoniid nudibranch known to feed on octocoral of the family Melithaeidae.The species is up to 120 mm in length,pinkish in color;with the rhinophoral sheath divided into several petaliform extensions;veil with,about 18 elongate digitiform processes;notal margin with17-19 pairs of secondary gills;anus located below the 5~(th) and 6~(th) secondary gills,and the genitalia below the 3~(rd) secondary gill on the right side of the body.Based on these external features,T.iocasica sp.nov.can be dearly distinguished from all previously described members of the genus.Phylogenetic analyses of two mitochondrial(COI,16 S rRNA) and a nuclear(H3) genes using Bayesian inference,maximum likelihood,and species delimitation analysis also support the separation of T.iocasica sp.nov.from its congeners.     

Seasonal variation of plankton communities influenced by environmental factors in an artificial lake

We evaluated the seasonal variation in plankton community composition in an artificial lake. We conducted microscopic analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNAand 18S rRNAgenes to characterize the plankton community. The clustering of unweighted pair group method with arithmetic mean (UPGMA) was then used to investigate the similarity of these plankton communities. DGGE fingerprinting revealed that samples collected at the different sites within a season shared high similarity and were generally grouped together. In contrast, we did not observe any seasonal variation based on microscopic analysis. Redundancy analysis (RDA) of the plankton operational taxonomic units (OTUs) in relation to environmental factors revealed that transparency was negatively correlated with the first axis (R=-0.931), and temperature and total phosphorus (TP) were positively correlated with the first axis (R=0.736 and R=0.660, respectively). In conclusion, plankton communities in the artificial lake exhibited significant seasonal variation. Transparency, phosphorus and temperature appear to be the major factors driving the differences in plankton composition.     

Preliminary identification of three new isolates in genus Alexandrium (Dinophyceae) from China sea area

The 5.8S ribosomal DNA sequences (5.8S rDNA) and their flanking regions, internal transcribed spacer 1 and spacer 2 (ITS1 and ITS2) of three new isolates in genus Alexandrium (Alexandrium sp. qd1, Alexandrium sp. qd2, Alexandrium sp. gz) from China were amplified, sequenced, and subjected to phylogenetic analysis. Alexandrium sp. gz and Alexandrium sp. qd1 were grouped with high bootstrap values with four strains/species, i.e., A. catenella South Korea strain, A. catenella Japan strain, and two from China, Alexandrium sp. AC03 and Alexandrium sp. AN01 being proposed to be A. catenalla in a previous study. Then Alexandrium sp. gz and Alexandrium sp. qd1 were identified as Alexandrium catenella. As A. catenella was isolated from Qingdao and Guangzhou sea areas, it supposedly distributed at least in these two areas and was genetically different. Alexandrium sp. qd2 differed greatly from species in Alexandrium. It clustered with Symbiodinium californium, Symbiodinium sp. G15 and Gymnodinium sp. Zhao 01 with 100% bootstrap value; so Alexandrium sp. qd2 affiliates to genus Symbiodinium, and is probably a free-living Symbiodinium species.     

Two new species of Sellaphora (Bacillariophyta) from a deep oligotrophic plateau lake, Lake Fuxian in subtropical China

We describe two new species of Sellaphora from Lake Fuxian, Yunnan Plateau, China. Based on both light and scanning electron microscopy, these species are described as S. yunnanensis sp. nov. and S. sinensis sp. nov. The primary features of S. yunnanensis are: elliptical to linear-elliptical valves with broadly rounded ends, straight filiform raphe, almost straight central endings and small, slightly expanding central pores, small central area, symmetrical or slightly asymmetrical central nodule. The primary features of S. sinensis are: elliptical valves, obtusely rounded ends, similar raphe and axial area, transapically less expanded central area, larger, elliptical central nodule. We compare these species to those of a similar shape and morphology.     

Structure analysis of the complete mitochondrial genome in cultivation variety ��Rongfu��

In this report, complete mitochondrial genome sequences of Laminaria cultivation variety ‘Rongfu’ were obtained. The results showed the length of circular molecule of mtDNA was 37 638 bp (64.7% A+T), encoding three rRNAs (23S, 16S and 5S), 25 tRNAs, 35 known mitochondrial proteins and 3 ORFs. Sequence alignment indicated its mtDNA genome was very similar to that of Laminaria japonica. Phylogenetic trees inferred from concatenated 30 mitochondrial genes showed that ‘Rongfu’, Laminaria japonica, Laminaria longipedalis, Laminaria diabolica, Laminaria religiosa and Laminaria ochotensis clustered together. In addition, compared with mitochondrial genome of L. japonica, ‘Rongfu’ mtDNA lacked a non-coding region of 19 nucleotides, which was located between rRNA small subunit gene 3 (rps3) and rRNA small subunit gene 9 (rps9). Seven cultivation varieties of China were divided into two groups based on this non-coding region which was absent in ‘Rongfu’, ‘Fujian’ and ‘Sanhai’ while present in ‘Ailunwan’, ‘Dongfang No.2’, ‘Dongfang No.3’ and ‘Zaohoucheng’. So this variation can be used in germplasm identification of cultivation variety.     

Purification and characterization of novel κ-carrageenase from marine Tamlana sp. HC4

We isolated a bacterial strain (HC4) that is able to degrade κ-carrageenan from a live specimen of the red alga Hyalosiphonia caespitosa. With 16S rRNA gene sequencing, we identified the strain as Tamlana sp., and then purified an extracellular κ-carrageenase from a culture of Tamlana sp. HC4 by ammonium sulfate precipitation, Sephadex G-200 gel filtration chromatography, and DE-cellulose 52 anion-exchange chromatography. The purified enzyme yields a single band on SDS-PAGE with a molecular mass of 66.4 kDa. The optimal pH and temperature for κ-carrageenase activity are at 8.0 and 30°C, respectively. The enzyme is stable over the range of pH 7.2–8.6 below 45°C. The enzyme activity is strongly inhibited by Zn²⁺ and Cu²⁺ at 1 mmol/L. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics with the Michaelis constant (K _m ) at 7.63 mg/ml. Analysis of the degradation products of the κ-carrageenase by ESI-MS and ¹³C-NMR spectroscopy indicates that the enzyme degrades κ-carrageenan down to the level of κ-neocarrabiose sulfate.     

DNA barcoding discriminates Pampus minor (Liu et al., 1998) from Pampus species

Although Pampus minor has been classified as a new species, it still remains controversial. Was used a DNA barcoding technique based on homologous sequence analysis of the16S and COI genes to clarify the confusion over the identification of this species. Among 12 individuals whose genetic distance was 0.002, two haplotypes were found. According to the 16S sequences, the genetic distances ranged from 0.121 to 0.133 between P. minor and other Pampus species. Although the same the genetic distance between the two P. minor haplotypes was generated using COI sequences, the haplotype of Pm22-23, Pm28, and Pm32-33 was the same as that of Pci EF607462 and EF607466, while the haplotype of Pm24-27 and Pm29-31 was the same as that of Pci EF607461 and EF607463-65. In addition, the genetic distance ranged only from 0.002 to 0.005 between P. minor and Pa EF607460 and EF607458. Apart from this, the interspecies genetic distances varied from 0.135 to 0.143 between P. minor and other Pampus species according to the COI sequences. Phylogenetic trees, using combined 16S and COI data, strongly support the viewpoint that all the P. minor individuals form one clade that is in a sister position to Pampus sp. individuals (EU357803, FJ434342-FJ434343, and FJ652423-FJ652427).