Virulence changes in Vibrio parahaemolyticus during the freezing of Penaeus chinensis

Although Vibrio parahaemolyticus has become the most common pathogen in fresh and frozen seafood,its virulence changes have often been ignored during the processing of seafood.To investigate these potential risks,we used frozen Penaeus chinensis as examples,and the most virulent factors of V.parahaemolyticus,including amounts,viable but nonculturable(VBNC)status,toxins TDH and TRH,and virulence genes tdh and trh,were determined.Bacterial quantities were signifi cantly reduced during drain and sprinkling phases,but caused by different factors.By SYTO9 and PI staining showed that washing was the main reason for the bacterial reduction at the drain phase,while the strain entering VBNC state was another reason at sprinkling phase.Their hemolysis toxicity,produced by TDH and TRH,became stronger after inoculation on shrimp,and could be detected throughout the process.Moreover,tdh and trh also exhibited trends similar to that of the hemolysis toxicity test.tdh was almost to a two-fold expression level during ice-glazing phase,while trh only express at a low level,less than half of the expression level before inoculation.These results demonstrated that the strains were not dead during freezing process,but became VBNC cells,which still produced and accumulated toxins,especially TDH,the most virulent factor.

RAPD analysis of wild stock of penaeid shrimp (Penaeus chinensis )in Chinese coastal waters of the Huanghai Sea and coastal waters of the Bohai Sea

Genetic diversity of 32 individuals of P. chinensis in the Chinese coastal waters of the Huanghai and Bobal Seas was detected by RAPD technique. Twenty decamer primers of OPI were used for DNA amplification for each individual. The results showed that: Seventeen primers obtained reproducible fingerprints, and the bands were clear. Thirty--nine of 106 loci detected were polymorphic, amounting to 36. 8 %. Mean genetic distance was 0. 094 1 0. 020 6; 68 markers (63. 2 % of the total) showed stable homogeneity in all of the 32 individuals.

Application of RAPD technology for identification in three different stocks of Penaeus chinensis

The genetic structure of three stocks (K, H and S) of Penaeus chinensis was detected by RAPD method which useed 41 random oligonucleotide primers with 10 bases. Stock K was from the spawning stock of the western coastal waters of the Korea Peninsula, H was from the Huanghai Sea-Bohai Sea stock and S was from the health cultured stock in Jimo, Shandong Province, China. Three genetic markers, F-02(540), OPN-13(800) and OPN-20(2200), which could differentiate the three different stocks, have been reported. The basic data for marker assisted selection (MAS) on genetic breeding of Penaeus chinensis are provided.

Study on purification and ultrastructure of a baculovirus in Penaeus chinensis

A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathin section of the gills, stomach and mid-gut tissues also revealed the presence of rod-shaped baculoviral particles with the same size in the affected cell nuclei, where most of the virions arranging in cluster assembled and caused a series of cytopathic changes. The virion covered with bilaminal envelope was 320 ~ 400 nm × 100 ~ 130 nm in size, whereas the nucleocapsid ranged in size of 250~ 300 nm in length and 70 ~ 100 nm in breadth respectively. No nuclear polyhedron or granulin occlusion theies have been found in cells. According to the principle of viral classification, this newly found virus could probably belong to the non-occluded subgroup of insect baculoviridae, i. e., C subgroup baculovirus.

Effects of heavy metal ions on larval Penaeus chinensis and comparative study on their elimination methods

In order to establish a new method to eliminate toxicities of heavy metal ions to larval penaeid shrimp, the effects of copper, zinc, lead and cadmium ions on larval metamorphosis of Penaeus chinensis at each stage were determined in detail, and two methods eliminating toxicities were compared. The results indicate that sensitivity of larvae to heavy metal ions decreases in the order of nauplii, protozoea, mysis and postlarvae. The phytoplankton food can reduce, by chetating, the toxicities of heavy metal ions. Both EDTA chelation and PHMA absorption can be used to eliminate the toxicities effectively and there is no significant difference between the two methods. EDTA is harmful to larval P. chinensis at high concentration but affects neither survival nor metamorphosis at below 10 mg/dm3. It is preliminarily observed that copper and zinc ions at low concentration are beneficial to the development of larvae after protozoea stage. It is suggested that it is unnecessary to e-liminate the effects of heavy metal ions in the culture of penaeid shrimp, under normal conditions after protozoea stage.

MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

Total RNA was extracted from eyestalks of shrimp Penaeus chinensis . Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase polymerase chain reaction (RT PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt inhibiting hormone from shrimp Penaeus japonicu s. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt inhibiting hormones of P. japonicus . The cDNA could be a partial gene of molt inhibiting hormones from P. chinensis . This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis .

FOOD SOURCES AND CARBON BUDGET OF CHINESE PRAWN PENAEUS CHINENSIS

This study deals with contribution of artificial food pellet and natural food to Chinese prawn (Penaeus orientalis) growth in a semi-intensive culture pond. The prawn carbon consumption, budget, and the effects of some factors on the budget were investigated. The results showed that 26.2% of P. orientalis growth carbon came from formulated feed at the initial culture stage (when the prawns were 0.06±0.01 g in wet weight), and was 62.5% when the prawns were 9.56±1.04 g. The remaining part of the growth carbon was derived from organic fertilizer and natural food. The highest growth rate occurred at 20×10 -3 salinity. Suitable salinity for culturing Chinese prawn was (20-28)×10 -3 .

Cytochalasin B induced triploidy in Penaeus chinensis

Feartilized eggs of Penaeus chinensis were treated with cytochalasin B (C. B ) to induce triploid.A number of them were cultured to the stage of on larvae. The C. B concentrations employed were 0. 1～2. 5 mg/dm3. The treatments started at 5～20 min after fertilization and lasted for various periods of time from 10 to 40 min. The highest inducing rate was 62. 5%.It was obvious that haaer C. B concentration gave stronger inhibition on the releas of polar bodies,and the higher the C. B concentration was, the more abnormal and aneuploid embryos were obtained. The success in inducing tripled in P.chinensis provided a posibility of polyploid breding of this species.

Effects of Lanthanum-Amino Acid Complexes on Egg Hatching and Nauplius Metamorphosis of Penaeus chinensis

Studies have been carried out on the effect of lanthanum-amino acid complexes on embryo development and nauplius growth ofPenaeus chinensis. The experimental results indicate that: (1)The optimum concentrations of lanthanum-proline and Lanthanumphenylalanine for the development of eggs in monocell and dicell stages are 1.50～ 4.00mg/L and 0.50～3.00 mg/L, respectively, the egg hatching rates being raised by 21.0 ～ 46.0% and 23.0 ～42.8% ( P ＜ 0.05 ) respectively. (2)The optimum concentrations of lanthanum- proline complex and Lanthanum-phenylalanine complex for the growth ofnauplii are 1.50～4.00 mg/L and 0.50～3.00 mg/L, the metamorphosis rate from nauplius to protozoea being raised by 16.4 ～27.5% and 20.4～26.7% (P ＜ 0.05 ) respectively. (3)The positive effect of lanthanum-amino acid complexes on egg hatching and nauplius metamorphosis of Penaeus chinensis is better than that of lanthanum. With regard to the positive effect, lanthanum-proline complex is better than Lanthanum-phenylalanine complex.

Effects of Dietary Vitamin C on the Immune Function of Shrimps, Penaeus chinensis

Prepared in this experiment were six groups of diets, i.e. VC0, VC1, VC2, VC3, VC4 and VC5 with the contents of vitamin C (VC mg(100 g) -1 diet) of 0, 100, 200, 400, 800, and 1200 respectively. It was found that vitamin C increased the concentrations of immunoglobulin-like (IgG-like, IgA-like and IgM-like) substances in the serum of Penaeus chinensis after a feeding period of 3 weeks. The differences among groups were significant (P<0.01), but there was no diffe- rence in the contents of complement3-like and complement4-like substances in the serum (P>0.05). Phenoloxidase (PO) activity in the serum of VC3 group shrimps was higher than that of VC0 and other groups, but no significant difference was observed between VC0 group and other groups. Furthermore, bactericidal activity of the serum to Vibrio parahaemolyticus in shrimps fed with the VC1 diet was higher than that in the other groups (P<0.01), while no difference was demonstrated among all groups for the bactericidal activity to Vibrio alginolyticus (P>0.05). It is, therefore, suggested that vitamin C (100-400 mg(100 g) -1 diets) could be used as an immunostimulant of P. chinensis.

Optimization of ultrasonic-assisted freezing of Penaeus chinensis by response surface methodology

Objectives:Optimization of ultrasonic-assisted freezing of Penaeus chinensis by response surface methodology was studied in order to(1)obtain frozen Penaeus chinensis of high quality and(2)provide practical guidance for the application of ultrasonic-assisted freezing in Penaeus chinensis.Materials and Methods:Three independent and major variables were selected,including initial ultrasonic temperature(℃),ultrasonic power(W)and ultrasonic time(s on/2 s off).On the basis of one-factor experiments,17 groups of experiments were established by response surface methodology according to Box-Behnken design.Using multiple regression analysis the experimental data were fitted into a second-order polynomial equation,which was tested by proper statistical methods.Results:The optimal ultrasonic conditions were as follows:initial ultrasonic temperature 0℃,ultrasonic power 180 W,ultrasonic time 5 son/2 s off.Under the optimization conditions,the time of passing through maximum ice crystal generation zone was 105.500 s,which was very close to the predictive passage time of 101.541 s.Conclusions:Initial ultrasonic temperature,ultrasonic time and ultrasonic power played an important role in the process of ultrasonic-assisted freezing of Penaeus chinensis.Response surface methodology was used to optimize the three factors in ultrasonic-assisted freezing,which could greatly shorten the time of passing through the maximum ice crystal generation zone and maintain the tissue structure of Penaeus chinensis well.

Effects of Lanthanum on Egg Hatching and Nauplius Metamorphosis of Penaeus Chinensis

Effects of lanthanum on egg hatching and nauplius metamorphosis of Penaeus chinensis(P. chinensis) were studied. The experimental results showed that the optimum concentration of lanthanum was 0 30～1 80 mg/L for monocell and dicell stage of embryo development, the egg hatching rates were raised by 33 5%～49 1%; and it was 0 90～1 80 mg/L for poly cell stage, the egg hatching rate was raised by 17 1%～23 5%. The optimum concentration was 0 90～1 50 mg/L for nauplius metamorphosis, the metamorphosis rate from nauplius(N 1) to protozoea(Z 1) was raised by 9 4%～11 7%. The promotion of lanthanum to egg embryo development was completed through absorption of lanthanum by egg in the water. The absorption capacity of one egg for lanthanum was 5 89 μg.

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.

Sea water environment copper requirement of egg hatching and naupliar metamorphosis of Peaneus chinensis

A new method was used to remove heavy metals in sea water. The requirement of copper in sea water ofegg hatching and naupliar metamorphosis of Penaeus chinensis was investored. A certain amount of copper ion andchelator nitrilotriacetic acid (NTA) were added in the de-heavy-metal-ionized sea water and then the cupric ion actvitywas calculated. The effects of copper ion on egg hatching and naupliar metamorphosis were observed. It was firstfound by experiments that trace ionic copper (10-10.80-10-8.80 mol/dm3) was essential to the growth and development of egg and nauplii of penaeid shrimp and chelated copper had no actions for these living processes,but in highconcentrations (>10 -7.80 mol/dm3) ionic copper was toxic. The nauplii were more sensitive to necessity and tokicityof ionic copper than eggs.

Studies on pathogen of explosive epidemic disease of shrimp Ⅱ. Purification of pathogen

The pathogen of explosive epidemio disease of farmed Chinese shrimp (Penaeus chinensis ) was isolated and purified from the cephalothorax and hepatopancreas of diseased shrimps by means of different speeds of centrifugation and sucrose gradient ultracentrifugation. The results showed that same virus isolated from the diseased shrimps in various regions of China was a kind of baculovirus covered with envelope, which was 95 ~ 125 nm in diameter and 360 ~410 nm in length. The size of the capsid was 80 ~ 90 nm × 330 ~ 350 nm. The nucleic acid of the virus was demonstrated as DNA. The result of artificial infection demonstrated that the virus was the pathogen of explosive epidemic disease of farmed Chinese shrimp.

A comparative study on the contents of vitamins in the natural and the cultured prawn

A systematically comparative study on the contents of vitamins in the natural and the cultured prawn, Penaeus chinensis, was made by using High-efficiency Liquid Phase Chromatography in 1989. The results show that the vitamin C (Vc) content in the natural prawn was 8. 19% higher than that of the cultured prawn.As the natural prawn's body length increasing, the Vc content in cephalothorax increased continuously, but after August 10, the Vc content in the cultured prawn fell down rapidly. No significant differences in vitamin B1, B2,B6, nicotinic acid (NAc), nicotinamide (NAm) and folio acid (FAc) were found between the natural and the cultured prawn. Therefore, Vc probably is one of major factors limiting the growth and development of Chinese prawn.