| The epidemic pathogenic of Fenneropenaeus chinensis is main caused by White spot syndrome virus(WSSV), no effectively technical measures could control the WSSV epidemic so far. For the purpose of prevention and decrease of WSSV loss to F. chinensis, first of all we compared the sensitivity of three PCR methods on detecting the white spot syndrome virus (WSSV); and then we focused on analysing the correlation of WSSV load and reproduction,survival and breeding value in F. chinensis. The results are as following:1,Sensitivity comparison of three different kinds of PCR-based methods in white spot syndrome virus (WSSV) testIn this part, we compared the sensitivity of several PCR methods, including TaqMan real-time PCR, Office International Des Epizooties (OIE) and Genetic resource and Our Lab-designed nested PCR (GB) and two corresponding one-step PCR methods, with standard samples of serial known WSSV load of F. chinensis. As a result, TaqMan real-time PCR could detect with certainty 10 WSSV copies per reaction while 10~4, 10~3 copies were needed for OIE and GB nested PCR; 5×10~4, 2.5×10~4 copies were needed for the outer primer and inner primer amplification separately of OIE nested PCR; and 10~4, 5×10~3 copies were needed for that of the GB nested PCR. 44 F. chinensis samples which we did not know the WSSV load were tested with these PCR methods to validate the sensitivity, the positive ratio of TaqMan real-time PCR was 84.09 %, and that of the OIE and the GB nested PCR were 18.18% and 27.27%, respectively; the positive ratio of the outer primer and inner primer amplification separately of OIE nested PCR both were 15.91%; and that of the GB nested PCR were 18.18% and 20.45%. According to the results, the sensitivity of PCR detected the WSSV from high to low are: TaqMan real-time PCR, nested PCR, one-step PCR.2,Effect of different diets on the WSSV loads and reproduction of F. chinensis over winterBased on the detecting result of WSSV loads of two kinds of prawn overwinter diets , Ruditapes philippinarum and clamworm, which were 17.79±3.71 copies/ng DNA and 19.06±4.31 copies/ng DNA, respectively, three kinds of diet combinations were designed as 1) disinfected Ruditapes philippinarum musle + disinfected clamworm + artificial diets (disinfected group); 2) R. philippinarum musle + clamworm + artificial diets (control group) ; 3) artificial diets (artificial group). After 60 days it was showed that the loss of weight of prawn in three groups was 1.48±0.86g,0.39±0.63g and 1.71±0.91g; and the survival rate of each group was 52.53%,79.75% and 46.01%. The WSSV loads of prawn before and after overwinter were 7.63±6.24 copies/ng DNA,10.58±7.34 copies/ng DNA,11.97±9.32 copies/ng DNA and 11.69±9.18 copies/ng DNA,9.31±6.95 copies/ng DNA,16.87±14.47 copies/ng DNA, respectively, showing no significant difference (P>0.05). The fecundity,the rates of fertilization and hatching of the brookstock from high to low were group of feeding control group,disinfected group and artificial group, showing significant difference (P<0.05). It was indicated that the R. philippinarum and clamworm can carry small amount of WSSV, Besides this, the brookstock can gain high fecundity,high rates of fertilization and hatching when feeding on control group diet combination over winter, the WSSV load of prawn feeding on three diet combinations is showing no significant difference overwinter.3,Effect by feeding different diets on the WSSV load and survival rate of F. chinensis.Based on single-cell algae (SA), artificial feed (AF), brachionus plicatilis (BP) and brine shrimp larva (BS), four groups of diet combination were designed as SA + AF, SA + AF + BP, SA + AF + BS, SA + AF + BP + BS , and used to the cultivation of F. chinensis larval, the oosperm which feeding on the group SA+AF divided into disinfected and undisinfected group, other groups'oosperm all were disinfected group. The survival rate of each stage was compared in different groups. It was showed that there was no significant difference from each other in the survival rate of N-Z by diet different diet combination (P>0.05). The survival rate of Z-P fed BP was higher than others (P<0.01) and the survival rate of M-P fed SA + AF + BS was significant difference (P<0.01) from others. The body weight of 10-day post larva by diet SA + AF + BP was less than that by diet SA + AF + BP + BS (P<0.01). The result of detecting WSSV load of 10-day post larva were disinfection Group SA + AF WSSV larva carrying the lowest load of 10.52±3.3 WSSV copies / ng DNA, which lower than other diet groups (P<0.05 ).During the stage of P11-P60, R. philippinarum foot muscle (CF) + artificial feed (AF), 60Coγradiated R. philippinarum foot muscle (RCF) + artificial feed (AF), potassium-permanganate-disinfected R. philippinarum foot muscle (DCF) + artificial feed (AF) and artificial feed (AF), which four diet groups were feeding to prawn. Shrimp of group CF + AF gained the largest increase of body weight and body length and showed no significantly difference (P>0.05) from group AF, but showed significantly difference (P<0.05) from the other two groups. The survival rate of group CF + AF was highest, (P>0.05) The result of WSSV artificial infection experiment showed that all groups of shrimp accumulated mortality were higher than 90% (P>0.05). It was concluded that shrimp larval feeding on 60Coγradiated R. philippinarum foot muscle (RCF) and potassium-permanganate-disinfected R. philippinarum foot muscle (DCF) could not caused significant effect of survival rate.4,The relationship between viral loads and growth & WSSV resistanceAfter testing the growth and WSSV resistance of 130 F. chinnensis families, According to the results of statistics software analysis, we choosed the five families each of largest and smallest growth breeding value, five families each of largest and smallest WSSV-resistance breeding value.We detecting the WSSV load of these broodstock, 50 d shrimp and 170 d shrimp of 20 families. The mean WSSV load of broodstock, 50 d shrimp and 170 d shrimp of 20 families were 0.1908, 0.28662 and 0.2329 copies / ng DNA, respectively, which showed no significant difference (P>0.05). The correlation coefficient between WSSV-resistance breeding value and WSSV load of broodstock, 50 d shrimp and 170 d shrimp were 0.021, 0.463 and 0.185, respectively; and that between growth breeding value and WSSV load of broodstock, 50 d shrimp and 170 d shrimp were 0.033, 0.048 and 0.019, respectively. The results shows that the relationship between growth breeding value, WSSV-resistance breeding value and the WSSV load of shrimp were not significant (P>0.05).5.Conclusion and research implicationsThe sensitivity of PCR detected the WSSV from high to low are: TaqMan real-time PCR, nested PCR, one-step PCR; The correlation of WSSV load and reproduction,breeding value in F. chinensis all showing no significant difference, The correlation of WSSV load and suivival is showing certain significant difference. This research provided the theoretical and practical basis in the prevention and management WSSV epidemic of F. chinnensis. |
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