The Study Of Plankton In WSSV Transmission

Though penaeid shrimp farming has undergone rapid development in the world during last four decades, successful production is hampered by White Spot Syndrome (WSS) since 1993. One remarkable characteristics of White Spot Syndrome Virus (WSSV) is its wide host range, which contributes to its wide geographical distribution. In epizootiological survey, there are lots evidences of WSSV-positive zooplankton found in shrimp farming ponds, therefore, plankton species are suspected to be the host of WSSV. The objective of the present study is to assess the possibility that plankton could serve as a vector in WSSV transmission.Phytoplankton are the base of the food web in pond cultures. In addition, they remove small particles in culture water, including viruses which have been excreted by infected animals, to maintain stable environment condition for culture. In present study, we investigated the possibility that four microalgea ( Isochrysis zhanjiangensis, Platymonas subcordiformis, Dunaliella salina and Skeletonema costatum) may act as a carrier or vector of white spot syndrome virus. After mixed with the WSSV inoculum, the microalgae were found that they could adhere WSSV within 96 h. The harpacticoid copepod Nitocra sp. could infect WSSV by filtering phytoplanktons which were carrying WSSV. The results indicated that phytoplankton could be a carrier and vector of white spot syndrome virus.The pathogenicity of WSSV to three zooplankton species, Brachionus urceus (Linnaeus, 1758), Acartia clausi (Giesbrecht, 1889) and Neomysis awatschensis (Brandt, 1851), was estimated by virus–phytoplankton adhesion route to investigate a potential new transmission route of WSSV to zooplankton. WSSV succeeded in infecting these zooplankton species and nested-PCR revealed positive results for the virus–phytoplankton adhesion route, indicating a successful new transmission route of WSSV to zooplankton and implying that phytoplankton could be a carrier of WSSV.The rotifer Brachionus urceus (Linnaeus, 1758) was experimentally infected with WSSV by the virus–phytoplankton adhesion route in order to assess the possibility of rotifer acting as a vector of WSSV to infect the shrimp Fenneropenaeus chinensis (Osbeck, 1765) larvae at zoea stage III. The nested-PCR test revealed WSSV-positive results in the rotifers exposed to WSSV by the virus–phytoplankton adhesion route. The same positive results also showed in the resting eggs and neonates acquired from the WSSV-positive rotifers. Among 10 replicates in the infection treatment, 40 % of F. chinensis larvae became WSSV-positive when fed with WSSV-positive rotifers, whereas all were WSSV-negative for F. chinensis when fed with WSSV-free rotifers. Though the mortality of shrimp larvae in the infection treatment (39.47±15.44 %) was higher than that in the control treatment (34.67±15.11 %), there was no significant difference in the mortality between them (P > 0.05). However, the growth and the metamorphose were slackened in infection group. In addition, there was a significant difference in metamorphose rate between the two groups ( P < 0.05). These results indicated that the rotifer could serve as a vector in WSSV transmission when ingested.In our epizootiological study, 14.67 % of copepod resting egg specimens (20-30 resting eggs in each specimen), which were separated from sediments of shrimp farming ponds, were found WSSV-positive using a nested-PCR technique. In addition, of the neonates hatched from copepod resting eggs, 6 % of specimens (10-15 neonates fixed together in each specimen) were positive for WSSV. However, the WSSV prevalence was significantly high (36.37 %) in neonate specimens hatched from virus positive resting egg specimens. An artificially infectional experiment was also carried out in laboratory to validate the study results. The nested-PCR test revealed that WSSV-positive resulted in calanoid copepods, Acartia sp., exposed to WSSV by the virus–phytoplankton adhesion route. The same positive results were also showed in the resting eggs and neonates reproduced from the WSSV-positive copepods. Although WSSV prevalence in copepod resting eggs and neonates was very low, the study results indicated that the copepod resting eggs could served as a reservoir or vector of WSSV, making it overwinter, leading to prevail in the following years.Nested-PCR analysis showed positive results in the harpacticoid copepod Nitocra sp. exposed to WSSV by virus–phytoplankton adhesion route, whereas negative results got in the control treatment. Then, oral route and intramuscular injection were used to test pathogenicity of WSSV isolated from the WSSV-positive harpacticoid copepods exposed to WSSV by virus–phytoplankton adhesion route. For the oral route of infection, Marsupenaeus japonicus postlarvae were fed with WSSV-positive copepods. Shrimp postlarvae in the infection treatment (PCO) became WSSV-positive and had 52.50±5.00 % mortality which was significant higher (P < 0.05) than that in the control treatment (NCO) (20.00±0.00 %) when postlarvae were fed with WSSV free copepods. In the intramuscular injection challenge, M. japonicus juveniles were injected with the copepod inoculum extracted from the WSSV-positive harpacticoid copepod showed 72.50±9.57 % mortality which was also significant higher (P < 0.05) than that in the control treatment (22.50±5.00 %) when juveniles were received mock injection of a tissue homogenate prepared from WSSV-negative Nitocraa sp.. In conclusion, based on these laboratory challenge studies, we confirmed that harpacticoid copepod can serve as a vector in WSSV transmission.Challenge tests of WSSV to Artemia four different development stages (nauplii,metanauplii,pseudoadults and adults) was carried out by immersion challenge and virus–phytoplankton adhesion route in order to asses the possibility of Artemia acting as a vector of WSSV to mysid shrimp Neomysis awatschensis and penaeid shrimp Litopenaeus vannamei postlarvae. The WSSV succeeded in infecting four stages Artemia , and nested - PCR detection for WSSV revealed positive results to virus–phytoplankton adhesion route. The RT-PCR analysis,detecting the Vp28 transcript from WSSV-positive Artemia,was negative,which indicated that WSSV did not propagate inside Artemia. An attempt was tried to find a viable surrogate to penaeid shrimp larvae as test animal for WSSV related experiment. No mass mortalities were observed in mysid shrimp and penaeid shrimp postlarvae fed with Artemia,whereas the nested-PCR detected WSSV DNA in mysid shrimp and penaeid shrimp postlarvae fed WSSV - positive Artemia exposed to WSSV by virus–phytoplankton adhesion route. No WSSV-positive was detected in any animal fed with WSSV-negative Artemia. However,mass mortalities showed in animals fed with WSSV infected penaeid shrimp mince tissue and nested-PCR released positive results. These results indicated that Artemia could serve as a vector in WSSV transmission,and it is feasible to use mysid shrimp as viable surrogate to penaeid shrimp larvae as test animal for experiment.