Based on the experimental ecological methods ,using hydrostatic system ,early Studing of the ammonia excretion rate to Bugula stoloni f era in northern China .mainly including Bugula stoloni f era in starvation ,Effect of temperature ,interaction of temperature and salinity and temperature and pH to its ammonia excretion rate .Not only the prevention of important significance in marine fouling organ-isms cultured biological hazards ,but also provide a basis for the further exploration and development of effective prevention and treatment methods .Specific test results are : <br> 1 .Temperature has a very significant impact on the creeping stems of Bugula stoloni f era excre-tion rate in the range of 12~27 ℃ ,showed a peak change when its ammonia excretion rate reached the highest value at 24 ℃ . <br> 2 .Interaction of temperature and salinity have a significant <br> impact on its ammonia excretion rate ,at 12~27 ℃ salinity in the range of 16‰ ~40‰ ,showed a peak change in salinity 32‰ ammonia excretion rate reached its highest value . <br> 3 .Interaction of temperature and pH have an extremely significant impact on its ammonia excre-tion rate at 12~27 ℃ conditions ,pH in the range of 5~10 ,showed a peak change in pH 8 of its am-monia excretion rate maximum value .

One complete cDNA of TLR named SiTLR-1 ( GenBank:HQ259110 ) was cloned from sea urchin Strongylocentrotus intermedius through degenerate primer PCR amplification and SmartTM RACE technology. Expres-sion distribution in different tissue after challenge with Vibrio fortis,β-D glucosan,and dsRNA were assessed using quantitative real-time PCR ( qRT-PCR) . The full-length cDNA sequence of SiTLR-1 is 3637 bp,with 16 α-he-lix,33 β-sheet;composed of 1 transmembrane domain (725-750aa),a putative signal peptide (1-32aa), and a leucine-rich repeat ( LRR) . SiTLR-1 was assessed in all tested tissues ( peristomial membrane,gut,coelomic fluid, muscle in Aristotles lantern,tube feet) ,the expression level in coelomic fluid was significantly higher than the others ( P<0 . 05 ); the expression level of SiTLR-1 in coelomic fluid was strongly up-regulated after challenge with V. fortis and β-D glucosan,reach the highest point at 12 h;the expression level of SiTLR-1 showed no significant difference after challenge with dsRNA, just weakly up-regulated at 3 h. The findings showed that, TLR genes par-ticipated in the immune response of sea urchin S. intermedius, SiTLR-1 can specifically identify bacteria andβ-D glucosan.

Flora on eipthelia of diseased juvenile sea cucumber Apostichopus japonicus with body length of 3-5 cm showing body wall lesions was studied by a method of 16S rDNA cloning library. The results showed that the bacte-ria were composed of Alphaproteo bacteria, Betaproteo bacteria and Gammaproteo bacteria, with the predominant flora of Shigella sp. , Acinetobacter sp. and Pseudomonas sp. on the lesions of the diseased sea cucumber as well as sea cucumber without obvious symptom. There was 49% of Shigella sp. , 17% of Acinetobacter sp. and 5% of Pseudomonas sp. in the diseased sea cucumber, while there was 20% of Shigella sp. , 26% of Acinetobacter sp. and 9% of Pseudomonas sp. in those without lesions. The members of Alphaproteo bacteria, Betaproteo bacteria and Gammaproteo bacteria were found in the seawater from the culture ponds as a control group, with predominant phaproteobacteria including Vibrio sp. (75%) and Shigella sp. (12%). Also, a predominant bacterium strain was isolated from the diseased cultured sea cucumber by a traditional method based on bacterial culture, and identified as Vibrio sp. by 16S rDNA cloning library. The findings indicate that the predominant flora is varied with the iden-tifying method including a traditional method and 16S rDNA cloning library.

When eggs from the Chinese tetraploid loach that had 100 chromosomes were fertilized with UV-irradiated sperm, we obtained viable gynogenetic progeny without any additional treatment for the duplication of maternal chromosomes, which survived beyond first feeding towards adult stage of development. Gynogenetic progeny were determined to be diploid since they possessed 50 chromosomes, along with two chromosomes bearing nucleolar organizing regions (NORs), detected by silver nitrate staining (Ag-NORs), chromomycin-A3 (CMA3)-positive sites and fluorescence in situ hybridization (FISH) signals for rDNA loci. In contrast, when gynogens were induced using eggs from diploid loach fertilized by UV-irradiated sperm, but without chromosome doubling, we found that all resultant progeny were non-viable haploid gynogens with 25 chromosomes, along with one NOR-bearing chromosome detected by Ag-NORs, CMA3 and FISH. These observations demonstrate the true genetic tetraploid nature of the Chinese loach possessing 100 chromosomes, and the potential use of this tetraploid as a source of functional diploid gametes for further ploidy manipulation experiments.

In the loach, or Oriental weatherfish Misgurnus anguillicaudatu s (Teleostei: Cobitidae), diploid (2n = 50) and tetraploid individuals (4n = 100) are often sympatric in central China. The evolutionary mechanism of this tetraploidization was analyzed with the observation of meiotic behavior of chromosomes in both the germinal vesicles of mature oocytes and the primary spermatocytes in diploid and tetraploid loaches. Whereas diploid specimens usually showed 25 bivalents in meiotic cells, tetraploid loaches exhibited 0–6 quadrivalents and 38–50 bivalents in both sexes, with the modal number of quadrivalents as three in females and four in males. In the diploid specimens, the two largest metacentric chromosomes bearing nucleolar organizing regions (NORs) identified by chromomycin A 3 staining and fluorescence in situ hybridization with a 5.8S + 28S rDNA probe formed one bivalent with terminal association. In the tetraploids, four NOR-bearing chromosomes never formed a quadrivalent, but were organized into two terminally-associated bivalents. These findings suggest an autotetraploid origin of the natural tetraploid loach and subsequent rediploidization of whole genome. The latter process, however, seems still in progress as inferred from the concurrence of up-to several quadrivalents and the majority of bivalents.

The chromosomes of the diploid and tetraploid loach Misgurnus anguillicaudatus were analyzed by staining with Ag, chromomycin A 3 (CMA 3 )/distamycin A (DA), and DA/4′,6-diamidino-2-phenylindole (DAPI), and using fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Nucleolus organizer regions (NORs) were mapped to the telomeric region of the short arms of the largest (first) metacentric chromosome pair in the diploid loach with 2 n = 50 and the homologous quartet in the tetraploid loach with 4 n = 100. The NORs were positive at the same region of the first metacentric chromosome for Ag and CMA 3 /DA stainings, but negative for DA/DAPI staining. Four signals at the homologs within the same quartet suggest the duplication of the entire genome from diploid to tetraploid status. However, a size difference was detected between the rDNA signals by FISH and CMA 3 banding.

Eleven morphometrical items were measured to evaluate the morphological variations in Amur weatherfish Misgurnus mohoity,northern weatherfish Misgurnus bipartitus and oriental weatherfish Misgurnus anguillicaudatus by three methods of multivariate analysis.Cluster analysis showed that northern weatherfish was close to oriental weatherfish,and distantly related to Amur weatherfish in genetic relationship.The principal component analysis revealed that the contributory ratio was 50.430% for the principal component 1,20.880% for the principal component 2 and 10.381% for the principal component 3,with the cumulative contributory ratio of 81.691%.Discriminant analysis showed that three discrimination functions and the discrimination accuracy were 71.4%-100% for P1and 71.4%-100% for P2,the integrative discrimination accuracy being 80.8%.

Intraspecific genetic diversity and intraspecific relationship were analyzed in loach species,Misgurnus mohoity,Misgurnus bipartitus and Misgurnus anguillicaudatus.Five loci were polymorphic in these species and a total 27 alleles were found.The number of alleles per locus ranged from 3 to 7.The loci was highly polymorphic and PIC was 0.526-0.722.Thus,these loach species had high genetic variability.In M.mohoity,M.bipartitus and M.anguillicaudatus,the observed heterozygosity(H0) was 0.978,0.889 and 0.742,and the expected heterozygosity(He) was 0.683,0.781 and 0.721,respectively.The genetic distance of three species of loach was 0.189-0.559.The minimum genetic distance(0.189) was between M.anguillicaudatus and M.bipartitus,which showed two species had a close relationship.The genetic distance between M.anguillicaudatus and M.mohoity was maximum(0.559),which showed they had a distant relationship. Ho and He heterozygosity values showed that 5 microsatellite loci examined didn't deviate from H-W equilibrium.The cluster analysis showed that M.mohoity,M.bipartitus and M.anguillicaudatus were clusteredinto two clades.M.anguillicaudatus and M.bipartitus were clusted,then grouped with M.mohoity.

Nature tetraploid male loaches collected from Hubei Province were crossed with diploid female from Dalian,and all-triploid loach seeds were produced.The results showed that,the average fertilizing rate,hatching rate and survival rate in the cross group(2n ×4n) were 54.69%,80.64% and 56.64%,respectively,and those in the control group(2n ×2n) were 76.23%,82.97% and 79.44%,respectively.The chromosome number of the early embryos of the progeny was 71-75,which could be considered as 100% triploidy.The result of DNA-content flow cytometry on the 6 months old progeny was also conducted to confirm the ploidy status,showing 100% triploidy.The result of the present research showed that the cross between nature tetraploid loaches from Honghu,Hubei and nature diploid loaches from Dalian could produce 100% triploid loaches.This cross method was simple to operate,reliable,and the best way to produce all-triploid loaches.