Abstract:After six- generation selection successively , Fenneropenaeus chinensis showed better economical traits such as fast growth and disease- resistance and so on. The average body length and the weight of each selective population increased by 8. 4% and 26. 86%, respectively, compared to the control. The morbidity was less than 10%, while it was more than 40% in the control. Genetic structure of each generation was also analyzed in the paper, the results were as follows: ( 1) Isozyme analysis: thirteen loci were examined and five loci were polymorphic, i. e. , MDH-2, GPI , MPI , PGM- 2 and PGM- 3. There were significant differences at the locus PGM- 3, and the gene frequency showed a decreased trend. At MPI locus allele C was found at the cultured populations, and the frequency showed an increased trend. ( 2) RAPD analysis: RAPD technique was used to investigate the genetic structure of five generation successively. The proportions of polymorphic loci showed a decreased trend while there was no difference between fifth generation and sixth generation. The degree of genetic differentiation ( Gst ) was moderate between populations except third population and fourth population. ( 3) SSR analy sis: eight loci were analyzed among five generations, and 108 alleles were detected. The average heterozy gosities showed a decreased trend while sixth generation remained the level of fifth generation. The values of Gst were less than 0. 05, which showed that there was differentiation to some degree but it was weaker.

Abstract:Selective breeding on growth character and disease2resistant trait of shrimp Fenneropenaeus chinensis has been conducted since 1997, and six successively bred stocks were produced between 1997 and 2002 at breeding base of the Yellow Sea Fisheries Research Institute, Shandong Province, China. Challenge Test has shown their better growth rate and disease2resistant performance than controlled shrimp. Samples of CP1 and CP6 were randomly collected. Tail muscle tissue from shrimp of mostly adults and sub2 adults was collected. Total DNA was extracted from tail muscle. DNA quality controls were performed using agarose gel electrophoresis and only samples with good DNA quality were used for microsatellite analysis. Microsatellite technique was used to investigate the genetic variation between the cultured populations (CP1 and CP6) of F . chinensis. Ten microsatellite primers were designed according to small2size fractionated genomic libraries of F. chinensis. Optimal results were obtained by optimizing quantity of reagents and reaction conditions. Under the optimal conditions, PCR was performed for analyzing the genetic diversity of the two selected populations. PCR products were electrophoresed through 8% polyacrylamide gels. Samples were heated to 95 e for 5 min  before being loaded onto the polyacrylamide gels. The gels then were silver2stained. All samples were run next to a known sequence in order to determine size. To avoid inaccuracy in scoring due to differences in gels, a control DNA sample was included in each set of samples for each gel. To analyze the variation in microsatellite loci in the two populations, number of alleles per locus ( na ) , Polymorphism Information Content ( PIC) , number of genotypes per locus ( G ) effective number of alleles (Ae ) , observed heterozygosity (Ho) and expected heterozygosity (He) were estimated for each population at each locus. Allele frequencies for each population at each locus were calculated based on the number of alleles provided by the computer software TFPGA. A total of 74 different alleles were found over all ten loci. The total number of alleles per locus ranged from 3 to 13. In the two populations, the effective number of alleles was lower than the observed number. PIC per locus ranged from 0. 5567 to 0. 8877. The average heterozygosity of the first and sixth selected generation was 0. 6400 and 0. 6300, respectively. To access departures from HWE, P2 values of genotypic frequencies were calculated. Furthermore, five cases of observed heterozygosity excess ( Fis< 0 ) were observed in the two populations. Shannon genetic diversity index revealed that the average variation level of the two populations was 1. 7742, and the variation level of them was 1. 6830 and 1. 7382, respectively. Partitioning of the genetic variation revealed that 96. 415% is distributed within populations and 3. 585% among populations. Nei. s genetic identity and genetic distance between the two populations were 0. 9187 and 0. 0848, respectively. In summary, data showed low genetic diversity of F. chinensis and difference over the levels of genetic variation between the two populations. There was great potential of selective breeding for the sixth generation.

Abstract:Amplified fragment length polymorphism (AFLP) was used to detect the genetic variation of four successively selected specific2pathogen2resistance (SPR) generations. Seven EcoR Ⅰand Mse Ⅰprimer combinations produced 202 polymorphic markers out of the total of 350 bands amplified. The proportions of polymorphic loci of the first , second , third and fourth generations were 39. 4286 % ,41. 4286 % , 33. 4286 % and 39. 1429 % , respectively. The Nei genetic diversity was 0. 1197 , 0. 1259 , 0. 1133 and 0.1249,respectively. Shannon genetic diversity index was 0.1831,0.1917,0.1702 and 0.1896,respectively. The genetic diversity index of the third generation was low , while the index of the other three generations remains at a relatively constant level. The analysis showed that the SPR population has great potential in genetic breeding program. The genetic distance of four generations based on the Nei analysis ranged from 0. 0282 to 0. 0458. Partitioning of the genetic variation revealed that 80. 54 % is distributed within populations , which is similar to the value (86. 72 %) derived from AMOVA. Mantel tests showed good correlation of different genetic distance matrices. By comparing the genetic parameters obtained from different statistical methods , we recommended AMOVA the first choice in population genetics analysis with AFLP markers. Co2dominant loci were found in AFLP fingerprinting. Two fragments adjacent in gel positions were recovered , cloned and sequenced. The sequence analysis showed high similarity between them , which indicated that they belong to the same locus. Our results proved that AFLP markers were not complete dominant markers. Our study suggests that AFLP is sensitive to detect genetic variability and effective to find markers and it is useful in marker assisted selection.

Abstract:The c2type lysozyme cDNA of Penaeus monodon cloned previously in our lab (GenBank accession no. AF539466) was modified by PCR to delete the signal peptide and subcloned to pBV220 expression vector which contains PRPL promoter to construct the recombinant vector pBV2202lyz. pBV2202lyz was transformed to E. coli DH5αand then induced to express at 42 ℃. A specific band about 16kD in molecular mass was showed by SDS2PAGE analysis , which was larger than those from other species such as human , fish and insects (about 14kD) . The recombinant P. monodon lysozyme existed as inclusion bodies in the cells. The inclusion bodies were collected , washed and then dissolved. And finally the target protein was dialyzed in order to recover its biological activity. Scanned and quantified by the Analysis System of Biology Image showed that the recombinant lysozyme accounted for 32 % of the total cell protein , and was about 90 % after purification. The biological activity of the recombinant protein was evaluated by turbidimetric assay described by Hultmark. The optimum pH and temperature of the lytic activities of the recombinant lysozyme were 6.0 and40 ℃,respectively. The recombinant protein was found to possess potent lytic activities against Micrococcus lysodeikticus12634 , Vibrio alginolyticus As1. 1833 and V. anguillarum E3211. And it has some lytic activities against Edwardsiella tarda E 895205 but with little lytic activities against Aeromonas hydrophila AhG1 , V . parahaemolyticus As1. 1615 and E. coli DH5α. The standard lysozyme (lysozyme from chicken egg white) showed lower lytic activities against Micrococcus lysodeikticus12634 , Vibrio alginolyticus As1. 1833 and V. anguillarum E321 compared with that of the recombinant protein. The present study suggests the potential application of the recombinant P. monodon lysozyme in aqauculture.

Abstract:The histology, histo chemistry and ultrastructure of early development of digestive tract of phyllo soma larve ( .- . ) of Panulirus stimp soni have been studied with light microscope and electron microscope in this paper. The digestive tract can be divided into foregut, midgut and hindgut. Foregut includes mo uth, esophagus and stomach. The wall of digestive tract consists of mucous layer, connective tissue, muscular layer and outer membrane. The muco us layer, which is co vered by chitin except midgut, is generally compo sed of simple columnar epithelium and basal membrane. The chitin layer can derive the formation of toothed projection, thorn, ridge and setae, and the muscular layer which has three way s of arrangement including circular muscle, longitudinal muscle and radiant muscle has larger changes in different places of digestive tract; its outer membrane includes thin connective tissue and simple squamous epithelial cells. With the development of larval stages the structure of digestive tract becomes more and more complex. Mouthpart of phllo sonalarve is developed, it consists of a upper- lip, a pair of mandible and a pair of maxilla. The wall of esophagus protrudes into lumen and forms four ridges, that make the esophageal lumen a shape of / X0 , and there are glands in connective tissue of esophagus. The stomach forms the cardiac and pyloric stomach. The structure of cardiac stomach whose chitin layer forms a few setae and thorns is simple, only rudiment of the teeth of the gastric mill o ccurs at the fourth stages of larva; but the chitin and circular muscular layer of pyloric stomach is developed, there is gland filter that consists of comb- setaes in lumen of stomach, its structure and function of filtration become better and better with the development of larval stages, and the wall of posterior part of pyloric stomach protrudes into lumen and forms four ridges, that make this stomach lumen a shape of / 工0 . The epithelial cells of midgut which have microvilli and a large bloodsinus are distributed in connective tissue and musclar layer is thin. There are six longitudinal ridges in the hindgut who se connective tissue is crossed with radiant muscule. The observation of ultrastructure shows that mito chondria and endoplasmic reticulum are most of the organelles in the epithelial cells of wall of the digestive tract and the epithelial cells of the pyloric stomach and midgut also contain rich ribosome, and there are developed mitochondria in the muscular cell of  esophageal wall. In the digestive tract at all stages of larva, the study of histochemistry show s that the chitin layer contains many polysaccharides, but the quantity of glycogen of epithelial cells of midgut is less; the protein is rich in the muscular cell while the quantity of epithelial cells of cardiac stomach and midgut do secondly; the fat granules are not observed clearly in various cells and connective tissue. The relation between the structure and function of the digestive tract of the phyllosoma larve is also studied in this paper.

Abstract:The chromosome specimens of Epinephelus malabaricus (Bloch &Schneider) were obtained from metaphase of kidney cell by viv2injection of PHA and cultivation of colchicines , hypatoic2air drying technique , and then by studying their Giemsa stain , C2bands and Ag2NORs. The results were as follows : 1) E. malabaricus had a diploid chromosome number of 48 and its karyotype formula was 48t , NF = 48 ,sex chromosome was not found;2) There was a pair of chromosomes with secondary constriction close to the centromere of chromosomet24; 3)1-4nucleoliappearedinthenucleusofinterphase,55%nucleihadlnucleolusandonly2%for4nucleoli;4) Ag2NORs appeared in the chromosome t 24 of 50 %metaphase ,sometimes in the chromosome t5 ,but not in other chromosomes ;5) The Ag2NORs polymorphisms were individually specific ,1 -4 pairs of the number ,and the frequency of 4 Ag2NORs were lowest ; 6) The secondary constrictions and positive C2bands were coincident , close centromere the chromosome , and mass constrictive heterochromatinsappearedinthatregion;7)Allthecentromeresofchromoso(tothe) mesweredarkl(of) y stained C2bands ,and the whole arm of chromosome t 24 and its centromere were same positive C2bands. 8) The evolutive regulation of the karyotype and the developing mechanism of Ag2NORs and C2bands were discussed.

Abstract:In this paper, the distribution of sponge Stelletta tenui associated microorganisms was investigated by transmission electron microscope and the characteristics were discussed on the basis of sponge structure and nutrition ingestion. It was proved that there are abundant microorganisms in sponge mesohyl, sponge cells and the inner cavity except for the sponge bone, in which sponge mesohyl is the main site for symbiotic microorganisms. It was suggested that the microbial distribution characteristics are due to the cavity structure and the nutrition ingestion of sponge by water filtration to some extent, which is useful for us to understand the mechanism of microbial accumulation in sponge and the relationship between sponge associated microorganisms and the w ater environment in the future.

Abstract:Beibu Bay , located at the north-west part of South China Sea, is an important fishing area in China. From Feb. 1998 to May 1999 and Nov. 2001 to Jun. 2002, six cruises for the ecological survey were carried out in this area. Samples of zooplankton were co llected to study their distribution and the other biological and ecological aspects. In order to understand the Chaetognatha community in Beibu Bay, the cluster analysis was used in our data analysis. Using the sampling stations as the variable of cluster, the Chaetognatha of Beibu Bay could be treated as one stable community . To further understand the changing pattern of the Chaetognatha community, the species of Chaetognatha was also used as the variable of cluster . The results showed that the species of Chaetognatha o ccurred in Beibu Bay could be divided into three groups, with two main and one subordinate group. The group I was compo sed of Sagitta bedoti , Sagitta enflata, Sagitta neglecta, Sagitta ferox and Krohnitta pacif i. These species could be categorized as the warm water coastal and warm water eurytopic species. They were widely distributed in this area with high o ccurrence and abundance. However, the suitable ranges of salinity and temperature for this gro up species were relative narrow . The occurrence of these species was probably closely related to the existing of the co astal water mass ( with high temperature, low salinity ) . The group ? was composed of Sagitta regularis , Sagitta p acifica, Sagitta robusta and Pterosagitta draco , these were ocean warm-water and warm water eurytopic species. However, the ocean warm-water species are more common in this area. Compared with group . species, the group ? species had relatively wider ranges for salinity and temperature. Their distribution was probably related to water mass in the offshore area ( with high temperature, high salinity) . The group ? species was not widely distributed as the group . did and their o ccurrence and abundance was low. Sagitta delicata, Sagitta minima and Krohnitta subtilis were classified as group ? . They were o cean warm-water and warm water coastal species. However, the former was more important in this area. The distributed area of group ? was small and scattered. The o ccurrence and abundance were the lowest among all groups.

Abstract:The diurnal variations of standard metabolism ( SM ) and routine metabolism ( RM ) of Perinereis aibuhitensis were determined in feeding commercial Paralichthys oliveaceus diet at 20 ℃. The worms used in this experiment were collected from seaside of Zhuanghe , Dalian , Liaoning province. Worms were grouped according to the wet body weight [S 0. 50 ?0.05g,M 0.90 ?0. 05g , L 1. 50 ?0. 05g ] . The relation between dry body weight ( W , g) and wet body weight ( W , g) is equivalent to : W = 0. 2168 wt 0. 0548 ( R2 =0.9833, n = 43) . SM and RM were determined under the oxygen determining apparatus by flowing water system whose water was filtered with a salinity of 31 -32. The Winkler method and Nestle method were adopted to study the oxygen consumption rate (R) and the ammonia excretion rate (AE) . By comparing RM with SM , the specific dynamic metabolism (SDA) of P. aibuhitensis was studied. The results were as follows : (1) SM and RM of P. aibuhitensis decreased with the body weight increasing. It can be described as R = aWb , where W is dry body weight , R is oxygen consumption rate , a and b were parameters ( a were 0. 3725 ,0. 2743 , b were -0. 4817 , -0. 5988 respectively) . SM and RM were significantly affected by dry body weight ( F = 16. 449 , P < 0. 001; F = 19. 327 , P < 0. 001) . And AE were significantly affected by dry body weight too ( F =4. 855 ,P = 0. 014 ;F =4. 003 ,P = 0. 028) . 4 (2) Each group had the same changeable rule of standard metabolism. The averages of SM in S 、 M and L groups were 1. 0248 mg?(g?h) -1,0.7285 mg?(g?h) -1 and 0.5019 mg?(g?h) -1 respectively. But it was higher in night than that at day. SM in night of three groups were as 2. 17 ,1. 42 and 1. 87 times as at day. This might be explained as the activity of P. aibuhitensis was correlated with illumination. The AE of S , M ,L in starved state changed little too , because of the starvation threaten the protein in worm tissue was consumed in metabolism at the end of experiment , so the AE was increasing slowly after experiment beginning 18h ,24h ,24h respectively. (3) RM in each group changed more than that of SM in this experiment. The RM of S ,M and L went to the peak after experiment beginning 21 -24h. The peak values of SDA in S ,M ,L were 3. 936 mg?(g? h) -1,1.5222 mg?(g?h) -1and1.2853mg?(g?h) -1 respectively , as 4. 84 ,1. 71 and 2. 29 times as their SM. By comparing the diurnal variations of SM with RM metabolism of three groups of P. aibuhitensis , results showed that SDA of the three groups , started at the same time , namely after feeding 15h , persisted about 12h. The energy expenditure of SDAwere 271. 62 J ?g 1,73.56J ?g 1 and - 70.56 J ?g 1 respectively. The ammonia excretion rates of S ,M ,L in satiation state changed significantly during the experiment. The peak values of the wave of satiation state were 9. 2176μmol?(g?h) 1 ,6. 5935μmol?(g?h) 1 ,2. 6866μmol?(g?h) 1 respectively , as2.82,4.15and1.67times astheirstarvationstate,accordinglythe durations were9h,9h,12h. Thewave of eachgroup were started later than its oxygen consumption rate , and that of S group started after feeding 24h , and M group was 21h , L group was 15h. The wave energy expenditure of S ,M ,L were 0. 9729 J ?g 1 ,4. 9600 J ?g 1 and 4. 0773 J ?g 1 respectively. This paper preliminarily concluded that SDA of P. aibuhitensis decreased with dry body weight increasing , that is , small worms metabolize much more actively than large worms.

Abstract:At present , cadmium2induced damage to fish immune system especially immune cells is hardly seen in references. In the paper , the auther studied for the first time the cadmiumly2induced apoptosis to peripheral blood mononuclear cell (PBMC) . In the paper , the auther studued for the first time the cadmiumly2induced apoptosis to peripheral blood mononuclear cell (PBMC) of crussian carp Carassius auratus (mainly T cell ,βcell and monocyte) by electron microscopy , single cell gel electrophoresis (SCGE) , DNA gel electrophoresis and flow cytometry (FCM) . At the beginning of our procedure , fish sample (400g even purchased from Suzhou Xiangcheng District aquafarm were randomly assigned to glass aquaria ( 100cm ?60cm ?50cm) at five of each. All the culture conditions were kept for 4 weeks including continuously aeration , twice diets daily (08 :00 and 14 :00) at the rate of 3 % body weight , half to two2thirds of daily water exchange , 23 to 27 ℃temperature range , and over 5mg?L -1dissolved oxygen content. Then , the test fish were intraperitonealy injected with Cd2 at 1. 25mg?kg 1 , and the control is Cd2 free. After injection on the 14th , 7th , 21st day , 5mL blood samples (within 150IU?mL -1 heparin) from the fish tail vein were mixed with equal volume PBS (0. 01mol?L-1, pH7. 2) , these duluted blood was gently covered by 5ml Ficoll2Paque , the band containing mainly PBMC between plasma and isolation solution was observed after being contrifuged at 1500r?min 1 for 10 min , then cellected the PBMC from this band , and washed twice in PBS , counted and adjusted to a density of 1 ?107 ind ?mL -1 in RPMI21640 plus 10 % FBS. Under electron microscope, the treated cells appeared chromatin marginating , condensating and extruding into cytoplasm , unclear structure of organelles and large vacuoles in cytoplasm , while the normal did evenly2distributed chromatin in U shape nuclei and intact organelles . By SCGE , the treated cells exhibited comet head of DNA margination and comet tails of DNA migration , while the normal did full nuclear skeleton ; the apoptotic rate was calculated according to the fraction of the apoptotic cells to the total cells at 14 , 17 and 21d were 19. 4 % , 16. 4 % , 5. 6 % respectively , which were significantly higher than that of the normal (1. 1 %) ( P < 0. 05) . By DNA gel electrophoresis , the treated cells had typical nucleosome bands (180 -220bp integer fold) 14 , 17 and 21d respectively , and these ladder bands at 14d and 17d were more apparent than those of 21d , while the normal had only an DNA band. In FCM , the treated cells showed aneuploid peaks , while the normal didn’t ; the nomber of cells in aneuploid peak were calculated aicording to the fraction of cells in aneuploid peak to the total cells at 14 , 17 and 21d were 17. 5 % , 23. 6 % and 8. 2 % respectively , which were significantly higher than that of the normal (1. 4 %) ( P < 0. 05) . The above evidences was suggested that cadmium could induce apoptosis of PBMC at this dose in vivo , and its apoptosis rate could reach summit valve in 14 -17d and then drop at 21d. So apoptosis of lower dose cadmium2stressed PBMC is likely to play a key role in the damage of cadmium to the immune system of C. auratus.

Abstract:A high performance liquid chromatography ( RT-HPLC) method for detection of chloramphenicol ( CAP) in the plasma of nile tilapia ( Oreochomia niloticus) was described and the effect of different water temperatures ( 18e and 26e ) and age of fish ( 1 year old and 2 years old) on the pharmacokinetics of CAP in nile tilapia after oral administration at a single dosage of 50 mg#kg - 1 body weight was examined in this study . The results showed that with RT-HPLC base line was smooth and the object peak of the drug was utterly separated from the impurity . The method was validated with blank samples fortified at 0. 25, 2. 5, 12. 5 Lg#mL- 1, and the mean recovery rate of CAP in plasma was ( 90. 47? 3. 42)% and the co efficient of variation was below 10%. The average intraday precision and inter- day precision were perfected, and the coefficient of variation were 1. 199% and 1. 770%, respectively. The pharmacokinetics of CAP had some relationship with water temperature and age. With the rise of water temperature, the speed of absorption, distribution and elimination of CAP in plasma accelerated. Not only the half- time of absorption ( T1/ 2kA) of the 26e group was about one third that of the 18 e gro up, but also the half-time of distribution ( T1/2A) and the half- time of elimination ( T1/ 2B) less than those in the 18e group. The time to peak concentration ( Tp) of the 26e group was less than half that in the 18e group, and the body clearance ( CLs) of the 26e gro up was about 1. 74 times greater than that in the 18e group, indicating that CAP was more rapidly cleared in higher water temperature. The speed of absorption in the 1 year old group was significantly higher than that in the 2years old gro up, and Tmax in the 1 year old group was significantly shorter than that in the 2 years old group, but T1/ 2kA and the area under concentration ( AUC) in the 1 year old gro up were close to that in the elder gro up. The speed of absorption in one years old group was much higher than that in tw o years old group, however, the speed of elimination of the former was much slower than that of the latter. Therefore, in view of the toxicity of CAP, it is important to be concerned about the pollution of CAP in aquatic animal food, and more attention should be paid to aquatic animals with relatively small age those live in relatively low water temperature in order to assure safety of aquatic food product.

Abstract:To study the stimulating mechanisms of immunostimulants in shrimp autogenous immunocompetence ,the immunostimulating effects ofβ2glucan ,lipopolysaccharide (LPS) ,inactivated Vibrio harveyi and Vibrio anguillarum on the hemocytes and phenoloxidase (PO) from shrimp , Penaeus chinensis , were investigated in this study. After shrimps stimulated with lipopolysaccharide (LPS) ,β2glucan ,inactivated Vibrio harveyi and inactivated Vibrio anguillarum ,the number of their overall hemocytes increased 83. 4 % ,52. 0 % , 73. 4 % and 111. 3 % respectively ,in which the number of semigranular cells increased 100. 4 % ,67. 3 % ,57. 2 % and 102. 9 % ,and the number of granular cells increased 47 % ,10 % ,127 % and 173 % ,respectively. And the amount of PO increased 104. 7 % ,81. 3 % 30.1%and40.4%,respectively,whereas the unit activity of PO remains almost the same before and after stimulation.Under the stimulation ofβ2glucan or LPS ,the amount of rough endoplasmic reticulum (RER) of semigranular cells and granular cells increased greatly and with swelled cristae. And the number of free ribosomes and mitochondria increased obviously and the number of secretary vesicles decreased greatly. However ,the ultrastructure of hyaline cells changed less before and after stimulation. Under the stimulation of inactivated Vibrio anguillarum ,the amount of RER in semigranular cells and granular cells increased greatly. And the number of free ribosomes increased obviously in granular and hyaline cells. The number of mitochondria increased obviously and its nucleoplasmic ratio decreased in hyaline cells. Under the stimulation of inactivated Vibrio harveyi ,the amount of RER and the number of mitochondria increased greatly and nucleoplasmic ratio decreased obviously in semigranular cells and granular cells. The amount of RER and the number of mitochondria increased obviously and nucleoplasmic ratio decreased in hyaline cells. Among the four kind of immunostimulants ,β2glucan and LPS acted mainly on semigranular cells and granular cells , whereas inactivated Vibrio harveyi and Vibrio anguillarum acted on semigranular cells , granular cells and also hyaline cells. Therefore , it can be concluded that 1) the enhancement of shrimp autogenous immunocompetence is finally realized via increase of OP production instead of PO unit activity ,2) the enhancing mechanisms of polysaccharides and inactivated vibros are different , 3 ) the enhancement of shrimp immunocompetence is finally realized via inducing RER hyperplasia in granular cells to increase PO production after stimulation byβglucan and LPS ,4) the enhancement of shrimp autogenous immunocompetence is finally realized via stimulating the proliferation of granular cells to increase PO production after stimulation by inactivated Vibrio harveyi and Vibrio anguillarum , and 5) the immunostimulating effect of polysaccharides is much better than that of inactivated vibrios.

Abstract:White spot syndrom (WSS) is a disease that has caused high shrimp mortality in a short period and severe damage to shrimp culture industry up to date. The purpose of this study was to comprehensively investigate the virus carrying conditions of wild Fenneropenaeus chinensis in Yellow Sea and Bohai Sea. Four spawning ground populations of P. chinensis collected from Yellow Sea and Bohai Sea in 2001 were examined by nested- PCR ( polymerase chain reaction) for the detection of white- spot syndrom virus ( WSSV) . The virus positive rate of the several populations were : the south coast of Korea population 55% ; Bohai Bay population 35%; Liaodong Bay population 94. 7% and Haizhou Bay population 47. 4%, respectively . As a result, the several populations were all carrying WSSV at different levels. Among those, Liaodong Bay population shows the highest WSSV infective rate, which is significantly higher than those of other populations. It may be due to the severe environmental problems and geographical conditions of the bay . Furthermore, the releasing of hatchery seedlings which may carry WSSV during culturing could be related to prawns. high WSSV infective rate. However, the effects of wild shrimp carry ing virus on the shrimp culture industry were obvious and sho uld not be ignored. Therefore, it can be concluded that the establishment of the specific pathogen free( SPF) and specific pathogen resistance ( SPR) cultured population was the only way to avoid the outbreak of the disease, which may be induced by the virus carried by shrimp. At the same time, something should be done to deal with the pollution in order to decrease the inducement that leads to the o utbreak of virus disease. In this study, a method for the detection of WSSV by PCR was developed, a high sensitivity about 1pg WSSV DNA could be seen with nested- PCR, which provided an effective method to diagnose WSSV ahead of time.

Abstract:Based on the published 16S rDNA gene sequence of Aeromonas spp. and aerolysin gene sequence of Aeromonas hydrop hila, the synthetic oligonucleotide primers were used in a polymerase chain reaction ( PCR) technique to detect the gene for specific 16S rDNA and aerolysin of patho genic A . hydr ophila. A. hydr ophila can be clearly discriminated from the other Aeromonas species by 16S rDNA gene PCR, the detection limit for the aerolysin gene by PCR amplification was 1fg DNA. 36 strains were tested for pathogenic A. hydrophila by PCR method and pathogenic aeromonads diagnosis kit and their coincident rate was 94. 4%. The PCR can clearly identify A. hydrophila from Aeromonas species, and can identify aerolysin- producing strain of A . hydrop hila. In conclusion, this PCR- based method is rapid, sensitive and specific for the detection of pathogenic A . hydrophila, and it is a practical method for pathogenic A. hydrophila detection.

Abstract:7 sem-i purified diets formulated with casein and white fish meal as protein sources to contain graded levels of protein ranging from 32. 07% to 45. 64% were fed to Erythroculter ilishaef ormis ( initial weight 2. 88? 0. 22g#ind- 1 ) in triplicate for 8 weeks. The feeding trial was conducted in fiberglass tanks in which the water temperature during the feeding was controlled from 25e to 29e . The results showed that the weight gain, specific growth rate, feed efficiency and protein efficiency ratio of fish fed a test diet with 41. 05% cr ude protein on dry basis were significantly higher ( P < 0. 05) than tho se of the rest gro ups. Based on quadratic model regression analysis of SGR and FE, it was found that the optimal protein requirements were 40. 94% and 41. 35% of dry diet respectively. Moreover, there was no significant effect of dietary protein levels on who le body moisture, protein and ash( P> 0. 05) , but its body lipid decreased ( P < 0. 05) when the fish were fed 2 diets with high levels of dietary protein at the end of the feeding trial.

Abstract:The quality changes of Northern shrimp, Pandalus borealis , stored in ice, liquid- ice or sal-t water mixed with ice, either at ambient temperature of - 1. 5 or 1. 5 e , were evaluated by using sensory assessment, physical methods, chemical and microbial analysis. Storage in liquid ice was more effective than ice or sal-t water mixed with ice, in delaying spo ilage of the shrimp. It was observed that the total volatile nitrogen ( TVB-N) content decreased during the first day in shrimp stored in liquid ice at both - 1. 5bC and 1. 5bC and its formation was further delayed for 3 days in shrimp stored at - 1. 5bC. In other groups the TVB-N content increased w ith time. The trimethylamine ( TMA) value increased gradually with storage time, in all samples, except for the one stored in liquid ice at - 1. 5e during the first day of storage. Total viable counts ( TVC) showed that bacteria grew most quickly in shrimp stored in ice and in sal-t water ice, followed by those in liquid ice at 1. 5 e or - 1. 5 e respectively throughout the storage period. Lowest counts were observed in sample stored in liquid ice at - 1. 5e where the lag phase of growth of bacteria was apparently extended at the beginning of storage. Principal component analysis ( PCA) and analysis of variance ( ANOVA) were performed to analyse the variation and correlation of indicators. The results show good correlation between some of the quality indicators, TVB-N, TMA, TVC, pH, NH3 response of electronic nose and sensory evaluation.

Abstract:Preparing angiotensin I2converting enzyme inhibitors derived from the enzymatic hydrolysate of Acetes chinensis was discussed in this paper. To get the optimal conditions of peptic hydrolysis we did orthogonalty trials with the ACE inhibitory ratio in vitro being the index. The hydrolysate with IC50 being 0. 65 mg?mL -1 was gained under the conditions of pH 2. 4 , temperature 41 ℃, enzymatic hydrolysis time 3 hours , enzymatic concentration 900 U?g 1 substrate and substrate concentration 8 %. The hydrolysate was filtrated with a Sepahdex G225 column , the fraction with the highest ACE inhibitory activity was collected and filtrated with a Sephadex G215 column , and the fraction with the highest activity was collected again , their IC50 were found to be 0. 084 mg?mL -1 and 0. 046 mg?mL -1 , respectively. After purification the content of hydrophobic amino acids tended to increase in the amino acids composition. Molecular weight distribution of the highest ACE inhibitory activity fraction of Sephadex G215 gel chromagraphy was located between 700 and 1900.

Abstract:The effects of sample pretreatments on chloramphenicol residue measurement in aquatic products by gas chromatography was studied. Such factors as extraction solvents used , method of extraction , sample cleanup , SPE activation and the derivatization time for chloramphenicol were studied in the sample pretreatment , and also their effects on chloramphenicol recovery were compared in this paper. The experimental results showed that ethyl acetate was the best solvent used for chloramphenicol extraction and n2hexane for lipids removal. The sample cleanup procedure was preferably conducted on a ENVI218 SPE cartridge with water as solvent. The optimal time for derivatization with BSTFA2TMCS was 20 to 30 min. Subsequently , the derivatized chloramphenicol was measured by capillary GC with ECD. The average recoveries ranged from 67 % to 115 % at spiking levels of 0. 1μg?kg -1to20μg?kg -1.

Abstract:Osmoregulation involves many aspects of physiological function in crustacean. Crustacean species exhibit almost all possible patterns of osmotic regulation and they are widely distributed in most of known biotopes. With the changes of liquid environment , the structure and function of the osmoregulation organs (such as gills , antennary glands) ,haemolymph osmotic pressure and ionic transport will turn to maintain the well2balanced metabolism , which are under the neuroendocrine regulation. The current research status of physiological mechanism of crustacean osmoregulation was reviewed in the following aspects : 1) Structure and function of gills and antennal glands (maxillary glands) . The gills are very important organs and play a prominenet role in osmoregulation; 2) Regulation of ion transport in branchial epithelium. Ion transport enzymes (Na 2K 2ATPase , V2ATPase , HCO322ATPase and carbonicanhydrase) stimulated by bioamines and cAMP may participate in the ion transport of branchial epithelium in crustacean; 3) Haemolymph composition and osmoregulation. Haemolymph concentration of ion and free amino acid accompanied by metabolites of blood can contribute to the most of haemolymph osmotic pressure; 4) Neuroendocrine control. Many neuropeptides may regulate the osmotic pressure of haemolymph and proteinsases activity of epithelial gill cells. Bioamines , cAMP and CaM have been proved to stimulate the uptake of Na and transport of Cl .

Abstract:Interleukin21β( IL21β) is one of the most pleiotropic cytokines and a central regulator of the immune and inflammatory responses. Interleukin21βwas initially discovered within mice and humans and over the last 10 years has been characterized within a wide variety of animals. The IL21βplays a key role in the inflammatory process , enhancing cell2mediated immunity by inducing the growth and proliferation of lymphocytes , connective tissue cells , and by stimulating immune and inflammatory response effector cells. As an immunoregulatory cytokine , IL21βhas the potential to enhance the immune response induced by a vaccine and/ or to modulate the immune response leading to different effector mechanisms. It is produced by many cell types , including monocytes , macrophages , T and B lymphocytes , fibroblasts , and endothelial and epithelial cells. Expression is induced by a diverse range of stimuli , including mitogens , cytokines , and microbial products. There have been considerable evidences provided by biological cross reaction that fish produce IL21βduring immune responses , and the bioactivity of IL21 in fish has been known for over a decade. But only since 1999 , IL21βgene has been cloned from the rainbow trout. And from then on , IL21β gene has been cloned and expressed in many fish confirming that fish produces IL21β gene during immune responses. In mammals it is produced as an inactive precursor that is processed by interleukin converting enzyme (ICE) to give a biologically active‘mature’peptide. There is no signal peptide in IL21β and its mechanism is unknown. This is the special part of the gene structure of the IL21β. And through the program analyzing , we found this special structure in fish IL21βgene. This paper reviews the functions and structure of gene IL21βand the studies of the gene IL21βin fish recently.

Abstract:Gynogenesis is thought to be a useful method to generate fully inbred line and to initiate monosex culture in teleost fish. This article presents results of a study of gynogenesis of tench, Tinca tinca L. from Eltrix River of Xinjiang, China, induced by the common carp ( Cyprinus carpio ) sperm inactivated with UV irradiation. When cold shocking method was used to prevent exmasion of the second polar body in order to produce gynogenetic diploids, the optimum treated parameters were screened as cold shocking for 20min at 4℃, 5min after insemination. On this condition, 10.23% of the treated eggs survived at feeding stage. A comparative study between the gynogenesis and their parents was made by RAPD technique. 82 random primers of 10 nucleotide long sequence were used in RAPD analysis. The results showed that each primer gives 3- 12 fragments for each sample with the fragment length 330 - 2 460 bp, and the genetic similarity between gynogenesis and their mother was 97.4 %, while the genetic similarity between gynogenesis and their father was 27.4%, and there were no bands only shared by gynogenesis and their father, the common carp. It revealed that the chromosome of their father, the common carp, did not penetrate into the construction of gynogenetic tench.

Abstract:Probes are essential for study of gene expression and regulation. In this study, a method was established to prepare the biotin- labeled probe for 18S rRNA gene of freshwater prawn, Macrobrachium rosenbergii . And the labeled method was used to produce a lysozyme gene probe, then applied in analy sis of lysozyme gene expression. Primers were designed according to the nucleotide sequences of 18S rRNA of Decapoda in order to isolate the 18S rRNA gene sequences of M. rosenbergii. Total genomic DNA was isolated from hepatopancreas of the freshwater prawn. A specific DNA fragment with desired size was amplified by PCR using the total DNA as templates. The DNA fragment was inserted into pGEM- T Easy vector and sequenced. The result of BLAST and alignment analy sis confirmed that the DNA fragment iso lated was the 18S rRNA gene of M. r osenbergii, which was 418 nt in length. Biotin- labeled probe of the 18S rRNA was then produced by PCR using the recombinant plasmid as templates. The biotin-21- dTTP and the non- labeled dNTP were added to the PCR reaction system. Ratio of the biotin- 21-dTTP and the non- labeled dTTP was 3 to 1. The yield of the labeled probe is 300 ng#LL- 1. The detection limit of the probe is 60 pg. A biotin- labeled probe of ly so zyme gene was prepared by the same label method, and the yield of the lysozyme gene probe is 500 ng#LL- 1. These biotin- labeled probes were applied in Northern dot blotting analysis of tissue distribution of lysoyzme mRNA of M. rosenbergii. Signals were scanned and quantified by Analy sis System of Biolo gy Image. The signal intensity ratio of the lysozyme to 18S rRNA represents the relative expression level of lysozyme mRNA. The results showed that the lysozyme mRNA existed in all the tissues checked, including eye, muscle, gill, hepatopancreas, haemocytes and intestine. But lysoy zme mRNA levels varied among different tissues. The highest level was found in the intestine, and the second was in the hepatopancreas and the lowest was in the muscle. The signal intensities of 18S rRNA among tissues were consistent, which showed that the 18S rRNA gene expressed stably in different tissues and could be used as an internal standard for researches of specific gene expression in prawns.

Abstract:Natural resistance associated macrophage protein ( Nramp) is an innate resistance protein to intracellular parasites, which is expressed plentifully in macrophage cells. Nramp has been studied in mouse, human, cattle, rainbow trout and channel catfish. However, little was known about the structure of Pagrus maj or Nramp. In order to get the complete sequence of Pagrus major Nramp, a pair of primer is designed according to a 200bp known sequence of Pagrus major Nramp cDNA. By the use of SMART RACE, the full Nramp of Pagrus maj or cDNA about 5 000 bp was obtained, including about 200 bp 5c terminal region ( UTR) , complete encoding region and 3c terminal region. There were 3 ployA signals, which showed many possibilities of cutting at 3c terminal region. The character of Pagrus major Nramp nucleotide sequence and deduced amino acid sequence are analyzed. 12 putative transmembrane( TM) regions, a consensus transport motif ( CTM) , a predicted protein kinase C phosphroylation site and three predicted N- link glycosylation sites are indicated in its deduced amino acid sequence. The . consense transport motif. CTM is located between TM8 and TM9. Furthermore, a protein kinase C phosphroylation site and three N- link glycosylation sites were predicted. The alignment of amino acid sequences between Pagr us major Nramp cDNA and several animals is analyzed and the deduced amino acid sequence of Pagrus major Nramp had 77. 8%, 83. 0%, 82. 3%, 80. 0%, 81. 1%, 60. 4%, 70. 3%, 58. 5%, 69. 5% identity w ith rainbow trout A( AAD20721) , rainbow trout B ( AAD20722) , channel catfish ( AF400108) , fathead minnow ( AAF01778) , common carp ( CAB60196) , mouse 1 ( AAA39838) , mouse 2 ( AAC42051) , human 1 ( D50403) , human 2 ( NP - 000608) , respectively . The alignment reveals high conservation in TM and CTM regions. Analysis result makes us get familiar with the structure and character of fish Nramp, furthermore, offer s some information for the enhancement of immunity of fish and genetic amelioration on fish breeding.

Abstract:The variations of muscle tissus , peroxidase and Ca2 2ATPase activities and Vp (pasteurization value) were investigated with the fresh samples of grass carp ( Ctenopharyngodon idellus ) heated at different temperatures , thus revealing the mechanism of denaturation of muscle protein by heating. The experimental results showed the great difference in muscle tissue structure before and after the samples heated. In the initial stage of heating the water loss accounted for 50 % of the total. When the samples were separately heated at 75 , 85 , and 95 ℃, fh values (dehydration curve slope) were 5 , 4. 1 and 1. 7 , respectively , while Vp reaching 40 minutes were 22 , 8 and 5 min , respectively. As to the time for deactivation of peroxidase in the heated samples at the various temperatures , it varied from 38 , 10 and to less than 6 min , respectively and the deactivation rates of Ca2 2ATPase in 1 min were 50 % , 88 % and 100 % , respectively. Consequently , the higher temperature the sample was heated , the faster rate of thermocon ductivity , the lower rate of water loss , and the faster rate of peroxidase deactivation it was. Also , the Vp for the least requirement of pasteurization ( Vp ≥40min) was obtained.

Abstract:This present paper studied the technological characteristics of seawater treated by sand filtering , net filtering and reserve2water disinfection. The results showed that after treatment the average filtering ratio of phytoplankton was 89. 98 % , and that of zooplanktonwas89.5%; thatitcanisolatetheotherspeciesofshrimpandcrabbythefilteringnetof morethan40mesh,anditwas difficult for Copepoda to pass through the filtering net of more than 60 mesh ,so it can block the horizontal disseminate pathway of WSSV ; and that 6 ?10 6 of the efficient chlorine can kill Copepoda but most aquatic creatures were dead in the water treated by 10 ?10 6 of the efficient chlorine , and it can transfer algae after killing the harmful creatures by using bleaching powder when there was reserve water. The result of water culture for 4 days , in out2seawater and the water treated with sand filtering or net filtering of 60 mesh or 6 ?10 -6of efficient chlorine , showed that the species and quantity of plankton were the most by out2sea2water culture , and it reduced respectively in the water treated by filtering with 6 mesh ,sand filtering and 6 ?10 -6 of efficient chlorine.