Isolation, cloning and sequencing of AFLP markers related to disease-resistance traits in Fenneropenaeus chinensis

Amplified fragment length polymorphism (AFLP) technique was used to analyze the fingerprinting of four successive generations ofFenneropenaeus chinensis to reveal their disease-resistance traits. Some loci showed quite different genetic frequencies due to artificial selection, which implied that these fragments were putative markers related to the disease-resistance trait. We developed a simple and effective method to further characterize these AFLP fragments. Specific AFLP bands were cut directly from polyacrylamide gels, re-amplified, cloned and sequenced. Eight putative genetic markers were sequenced and their sizes ranged from 63 to 209 bp. The sequences were submitted to dbGSS (database of Genome Sequence Survey); and the BLAST analysis showed low similarity to the function genes, indicating these markers were tightly linked to a disease-resistance trait but were not functional genes. This research was supported by special funds from the National Key Basic Research Program (G1999012007) and the National High-Tech Research and Development Program of China (863 Program. 2001AA620105).     

Distribution and elimination of Norfloxacin in Fenneropenaeus chinensis larvae

This study examined the distribution and elimination of Norfloxacin(NFLX) in Fenneropenaeus chinensis ovary and egg and newly hatched larvae.Mature parental shrimp were exposed to 4 or 10 mg L 1NFLX for 2 or 5 d.Ovary and eggs of the shrimp were sampled after spawning in order to detect NFLX residue using high-performance liquid chromatography(HPLC).Results showed that NFLX residue accumulated in F.chinensis eggs after the parental exposure,with the highest residue detected in ovary.To examine the fate of NFLX residue in larvae,we further determined the concentration of NFLX residue in F.chinensis eggs and larvae at 4 different developmental stages after 24-h exposure.From the newly metamorphosed larvae(0 h post-metamorphosis,h.p.m),samples were taken at different time intervals to 72 h.p.m.HPLC assay showed that the concentrations of NFLX residue in zoea exposed to 4 and 10 mg L 1NFLX were the highest at 1.5 h,i.e.,0.332 and 0.454 μg g 1,respectively.At the two NFLX exposure levels,the elimination time of half NFLX(half life) in nauplius was 45.36 and 49.85 h,respectively,followed by that in zoea(31.68 and 33.13 h),mysis larvae(42.24 and 47.28 h) and postlarvae(24.48 and 30.96 h).Both NFLX exposure levels had a germicidal effect.The distribution and elimination of NFLX residue in F.chinensis tissue,eggs and larvae correlated well with the drug exposure level.The disappearance of NFLX residue coincided with the larval growth,and the half-life of NFLX decreased with the larval development.     

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.     

Cloning,characterization,and expression analysis of the DEAD-box family genes,Fc-vasa and Fc-PL10a,in Chinese shrimp (Fenneropenaeus chinensis)

RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication,DNA repair,and RNA processing.However,the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level.We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins,Fc-vasa and Fc-PL10a,from the testes of Chinese shrimp,Fenneropenaeus chinensis.The two predicted amino acid sequences contain all the conserved motifs characterized ...     

Heat-shock protein 70 expression in shrimp Fenneropenaeus chinensis during thermal and immune-challenged stress

Using western immunoblotting, we obtained heat-shock protein 70 (HSP70) induction data and distribution in different tissues from shrimp Fenneropenaeus chinensis during thermal and immune-challenged stresses. This is probably the first report of the effects of various stressors on the expression of HSP70 in shrimp.HSP70 was prominently induced in hepatopancreas and gills, but not in muscle, eyestalk and hemolymph, when the shrimp were exposed to heat shock and Vibrio anguillavium-challenged stresses. Cold shock and WSSV treatment had no significant effects on the levels of HSP70 expression in all tissues examined. HSP70 induction was greatest after 2 h exposure to heat shock stress, which was elevated after acute heat shock exposure of 10℃ above ambient temperature.     

Analysis of the genetic diversity and differentiation of <Emphasis Type="Italic">Fenneropenaeus penicillatus</Emphasis> populations using AFLP technology

Fenneropenaeus penicillatus (redtail shrimp) is an important marine commercial animal in China. Recently, its resources have been depleted rapidly as a result of, for example, over-exploitation and environmental degradation of spawning grounds. Therefore, we analyzed the genetic diversity and differentiation of nine wild populations of F. penicillatus of China (Ningde, Lianjiang, Putian, Xiamen, Quanzhou, Zhangpu, Dongshan, Nanao, and Shenzhen populations) by amplified fragment length polymorphism (AFLP) technology, to provide genetic information necessary for resource protection, rejuvenation, artificial breeding, and sustainable use of the resource. Eight AFLP primer pairs were used for amplification, and 508 bands were detected among the populations. The results show that the percentage of polymorphic loci (P) ranged from 41.34% to 63.58%; the Nei’s gene diversity (H) of the populations was 0.119 4-0.230 5; and Shannon’s Information Index (I) was 0.184 1-0.342 5. These genetic data indicate that the genetic diversity of F. penicillatus was high. The genetic differentiation coefficient (GST=0.216 2) and gene flow (Nm=1.812 4) show that there was a high level of genetic differentiation and a moderate level of gene flow among populations. More studies on the genetic differentiation mechanism of F. penicillatus along the south-eastern coast of China need to be conducted to find more effective scientific protection strategies for the conservation of F. penicillatus genetic resources.     

Effects of Salinity Fluctuation Frequency on the Growth, Molting Rate and Hemolymph 20-Hydroxyecdysone Concentration in Juvenile Chinese shrimp, Fenneropenaeus chinensis

This experiment was conducted to examine the effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone (20-HE) concentration for juvenile Fenneropenaeus chinensis with an initial wet body weight of 1.115 g±0.012 g. The salinity of the control treatment (represented by D0) was 28 throughout the experiment, whereas treatments D2,D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8d, respectively. The range of salinity fluctuation was 4. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8d, respectively; each original salinity was decreased abruptly to salinity 24, which first lasted for another 2d and was then raised to its initial value 28. This constituted a salinity fluctuation cycle and afterwards the cycle repeated. It was found that: 1) The weight gain of shrimp in treatment D4 was 202.9% and significantly higher than that in treatment D0 (P<0.05). The molting rate in treatment D2 was the lowest, while that in treatment D6 was the highest and significantly higher than those in treatments D0 and D2 (P<0.05). 2) The hemolymph 20-HE concentration of shrimps in all treatments was at low levels and increased first gradually during the inter-molt period and then increased sharply. It reached a peak value during the pre-molt stage, and then decreased abruptly during the post-molt stage when the lowest level occurred. 3) During the post-molt stage, the hemolymph 20-HE concentration in treatment D2 was 7.47pgμL-1 and significantly higher than that in treatment D0(P<0.05). There were, however, no significant differences in hemolymph 20-HE concentration among all five treatments during the inter-molt stage (P>0.05). During the pre-molt stage, the hemolymph 20-HE concentration in treatment D6 was significantly higher than that in treatment D0(P<0.05), whereas the hemolymph 20-HE concentration in treatment D2 was significantly lower than that in other treatments (P<0.05). Adequate salinity fluctuation promoted the molting rate through increasing hemolymph 20-HE concentration at the pre-molt stage.     

草鱼种质退化相关SRAP分子标记的筛选

采用相关序列扩增多态性(sequence-realted amplified polymorphism,SRAP)技术分析野生草鱼和家养草鱼,并筛选与草鱼种质退化相关的分子遗传标记。共进行88对引物组合的检测,产生标记数目共计905个。依据标记在群体中出现的频率和变化规律,共筛选出21个可能与种质相关的特异性标记,对这些特异性标记进行测序并将测序结果进行BLAST分析,发现测得片段中有8个片段能在GeneBank中找到同源性较高的序列,而其他片段与数据库中序列的相似性较低。     

草鱼种质退化相关SRAP分子标记的筛选

采用相关序列扩增多态性（sequence-reared amplified polymorphism，SRAP）技术分析野生草鱼和家养草鱼，并筛选与草鱼种质退化相关的分子遗传标记。共进行88对引物组合的检测，产生标记数目共计905个。依据标记在群体中出现的频率和变化规律，共筛选出21个可能与种质相关的特异性标记，对这些特异性标记进行测序并将测序结果进行BLAST分析，发现测得片段中有8个片段能在GeneBank中找到同源性较高的序列，而其他片段与数据库中序列的相似性较低。     

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca²⁺, suggesting that the clotting reaction is catalyzed by a Ca²⁺-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.     

Partial Sequence Analysis of Mitochondrial COI Gene of the Chinese Shrimp, Fenneropenaeus Chinensis

Eight hundred and thirty eight base pair fragment of mitochondrial COI gene of wild and cultured populations (CP1, CP4, CP5 and CP6) of Fenneropenaeus chinensis was amplified and sequenced. The A, T, G and C contents of the sequence were 235bp (28.0% ),.307bp (36.6%), 138bp (16.5%) and 158bp (18.9%), respectively. Furthermore, 556bp fragment of the sequence was used to discuss the phylogenetic relationship among 14 Penaeidae species using Alpheus armillatus as the outgroup. From the molecular phylogenetic tree constructed by neighbor-joining method, we obtained three large shrimp groups: Farfantepenaeus, Litopenaeus and Fenneropenaeus group. The results also indicated that there were a closer genetic relationships between F. aztecus and F. paulensis, L. schmitti and L. setiferus, F. indicus and F. merguiensis, and the genus Farfantepenaeus was closer to Litopenaeus.     

Effects of Salinity Fluctuation Frequency on the Osmolarity, Na^＋-K^＋ -ATPase Activity and HSP70 Expression in Juvenile Chinese Shrimp, Fenneropenaeus chinensis

Experiments were conducted to examine the effects of salinity fluctuation frequency on the osmolarity, Na+-K+-ATPase activity and HSP70 of Chinese shrimp Fenneropenaeus chinensis with initial wet body weight of 1.460 g ± 0.091 g. The salinity in the control group (D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8...     

A genetic linkage map of marine shrimp Penaeus (Fenneropenaeus) chinensis based on AFLP, SSR, and RAPD markers

The Chinese shrimp Penaeus (Fenneropaeneus) chinensis is an important species in marine fishery and aquaculture in China. A female Chinese shrimp Penaeus (Fenneropaeneus) chinensis was captured from west coast of the Korean peninsula and mated with a “Yellow Sea No. 1” male to produce the first filial generation (F₁) 100 F₂ full-sib progeny from brother-sister crosses between F₁ families was used for the mapping study. A genetic linkage map of the Chinese shrimp was constructed, based on 354 markers, including 300 amplified fragment length polymorphism (AFLP) markers, 42 microsatellite (SSR) markers, and 12 randomly amplified polymorphism (RAPD) markers. Forty-seven linkage groups (LGs) were identified. The total map length was 4 580.5 cM, with an average spacing of 11.3 cM, covering 75.8% of the estimated genome size. The construction of this genetic linkage map was part of a genetic breeding program. This linkage map will contribute to the discovery of genes and quantitative trait loci (QTLs) in Chinese shrimp.     

Nitrogen and Phosphorus Budget of a Polyculture System of Sea Cucumber （Apostichopus japonicus）, Jellyfish （Rhopilema esculenta） and Shrimp （Fenneropenaeus chinensis）

The nitrogen(N) and phosphorus(P) budget and the ecological efficiency of a polyculture system of sea cucumber(Apostichopus japonicus), jellyfish(Rhopilema esculenta) and shrimp(Fenneropenaeus chinensis) were studied in a cofferdam, 120.2 ha in size. The nutrients were supplied by spring tide inflow. In total, 139600 kg N yr-1 and 9730 kg P yr-1 input to the system; while 118900 kg N yr-1 and 2840 kg P yr-1 outflowed from the system concurrently, thus the outflow was 85.7%(N) and 29.2%(P) of inflow. The production of N and P was 889.5 kg yr-1 and 49.28 kg yr-1(sea cucumber) and 204 kg yr-1 and 18.03 kg yr-1(jellyfish and shrimp), respectively. The utilization rate of N and P by polycultured animals was 7.8‰ and 6.9‰, respectively, 21.9% and 38% higher than that of monocultured sea cucumber. Our results indicated that the polyculture system was an efficient culture system of animals and a remediation system of coastal environment as well; it scavenged 14.3% and 70.8% of N and P, respectively. Such an ecological efficiency may be improved further by increasing either the stocking density or the size of sea cucumber or both.     

Genetic diversity of populations and clones of Rhopilema esculentum in China based on AFLP analysis

Amplified fragment length polymorphisms(AFLP) markers were developed to assess the genetic variation of populations and clones of Rhopilema esculentum Kishinouye(Scyphozoa,Rhizostomatidae).One hundred and seventy-nine loci from 56 individuals of two hatchery populations and two wild populations were genotyped with five primer combinations.The polymorphic ratio,Shannon’s diversity index and average heterozygosity were 70.3%,0.346 and 0.228 for the white hatchery population,74.3%,0.313,and 0.201 for the red hatchery population,79.3%,0.349,and 0.224 for the Jiangsu wild population,and 74.9%,0.328 and 0.210 for the Penglai wild population,respectively.Thus,all populations had a relatively high level of genetic diversity.A specific band was identified that could separate the white from the red hatchery population.There was 84.85% genetic differentiation within populations.Individual cluster analysis using unweighted pair-group method with arithmetic mean(UPGMA) suggested that hatchery populations and wild populations could be divided.For the hatchery populations,the white and red populations clustered separately;however,for the wild populations,Penglai and Jiangsu populations clustered together.The genetic diversity at the clone level was also determined.Our data suggest that there are relatively high genetic diversities within populations but low genetic differentiation between populations,which may be related to the long-term use of germplasm resources from Jiangsu Province for artificial seeding and releasing.These findings will benefit the artificial seeding and conservation of the germplasm resources.     

Effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone concentration in Juvenile Chinese shrimp, Fenneropenaeus chinensis

This experiment was conducted to examine the effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone (20-HE) concentration for juvenile Fenneropenaeus chinensis with an initial wet body weight of 1.115 g±0.012 g. The salinity of the control treatment (represented by D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The range of salinity fluctuation was 4. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8 d, respectively; each original salinity was decreased abruptly to salinity 24, which first lasted for another 2 d and was then raised to its initial value 28. This constituted a salinity fluctuation cycle and afterwards the cycle repeated. It was found that: 1) The weight gain of shrimp in treatment D4 was 202.9% and significantly higher than that in treatment D0 (P<0.05). The molting rate in treatment D2 was the lowest, while that in treatment D6 was the highest and significantly higher than those in treatments D0 and D2 (P<0.05). 2) The hemolymph 20-HE concentration of shrimps in all treatments was at low levels and increased first gradually during the inter-molt period and then increased sharply. It reached a peak value during the pre-molt stage, and then decreased abruptly during the post-molt stage when the lowest level occurred. 3) During the post-molt stage, the hemolymph 20-HE concentration in treatment D2 was 7.47 pg μL⁻¹ and significantly higher than that in treatment D0 (P<0.05). There were, however, no significant differences in hemolymph 20-HE concentration among all five treatments during the inter-molt stage (P>0.05). During the pre-molt stage, the hemolymph 20-HE concentration in treatment D6 was significantly higher than that in treatment D0 (P<0.05), whereas the hemolymph 20-HE concentration in treatment D2 was significantly lower than that in other treatments (P<0.05). Adequate salinity fluctuation promoted the molting rate through increasing hemolymph 20-HE concentration at the pre-molt stage.