【作者】 张家松；

【导师】 董双林；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2007， 博士

【摘要】 从上世纪六十年代以来,对虾养殖业在世界范围内得到了快速的发展,然而由于1993年对虾白斑综合症(WSS)的暴发,使该产业的发展严重受挫。该病1993年首次在日本囊对虾养殖场暴发,并立即引起了各界的密切关注,因为该病是严重危害所有对虾养殖品种的一种病毒性疾病,可以造成对虾极高的死亡率和重大经济损失。对虾白斑综合症病毒(WSSV)的宿主范围极其广泛,这也是该病毒得以在世界范围内迅速传播的原因之一。虽然浮游生物作为WSSV宿主进入研究人员的视野已经十年多了,但是直到如今仍然缺少浮游生物作为媒介在WSSV传播中的实验证据。因此,本论文的目的就是检验浮游生物是否可以成为WSSV的传播媒介并研究其传播机制。以对虾养殖池塘中常见的湛江等鞭金藻Isochrysis zhanjiangensis、亚心形扁藻Platymonas subcordiformis、盐藻Dunaliella salina和中肋骨条藻Skeletonema costatum四种浮游微藻为实验对象,在与病毒粗提液混合后0.5、1、2、4、8、12、24、48、72、96、120、144、168h采样,每个实验三个重复,研究其对WSSV的携带和传播。研究结果表明,这四种微藻和WSSV病毒粗提液混合后,经PCR检测发现,在混合后96 h内微藻都可以携带WSSV,其中湛江等鞭金藻和亚心形扁藻在144 h内为WSSV阳性,盐藻在120 h内为阳性,然后在不同时间之后呈现WSSV阴性,这说明浮游微藻可以携带WSSV。通过病毒-浮游微藻吸附法对对虾池塘中三种常见浮游动物――壶状臂尾轮虫Brachionus urceus、克氏纺锤水蚤Acartia clausi和黑褐新糠虾Neomysis awatschensis进行了WSSV的感染实验,实验分对照、浸泡法和病毒-浮游微藻吸附法攻毒组三个处理,每个处理三个重复。套式PCR检测结果表明,对照组和浸泡法攻毒组中的三种浮游动物均为病毒阴性,而病毒-浮游微藻吸附法则使WSSV成功感染这些浮游动物,攻毒组中三个重复的检测结果均为阳性,从而找到了一种新的浮游动物感染WSSV的方法;同时表明,微藻可以传播WSSV。采用病毒-浮游微藻吸附法使WSSV感染壶状臂尾轮虫,然后刺激其产生休眠卵,每个处理三个重复,套式PCR检测结果表明,轮虫的休眠卵和由此孵化出的幼体也为WSSV阳性,这表明,轮虫可以通过水平和垂直两种途径感染上WSSV。用WSSV阳性的轮虫投喂中国明对虾Fenneropenaeus chinensis第III期蚤状幼体(Z III),每个处理十个重复。实验表明,虽然幼体没有出现严重死亡,对照组的死亡率(34.67±15.11%)和攻毒组(39.47±15.44%)差异性也不显著( P >0.05);但是幼体的生长和变态却受到很大的影响,变态率(19.26±5.4 %)与对照组(74.22±10.18%)相比差异显著( P < 0.01)。经套式PCR检测表明,攻毒组中40%的对虾幼体为WSSV阳性,而对照组全为阴性。由此证明,轮虫可以成为WSSV传播中的媒介。通过套式PCR检测发现,环胶州湾对虾养殖池塘底泥分离出的150份桡足类休眠卵中14.67%为WSSV阳性。由这些休眠卵孵化而来的幼体中6%为病毒阳性,但是,从WSSV阳性休眠卵孵化而来的幼体病毒阳性率却高达36.37%。运用病毒-浮游微藻吸附法对桡足类的哲水蚤类Acartia sp.进行WSSV攻毒实验,套式PCR检测结果发现,攻毒组三个重复中的哲水蚤均为WSSV阳性,其所产的休眠卵以及由此孵化而来的哲水蚤幼体也为病毒阳性,而对照组中的哲水蚤成体、休眠卵和幼体均为阴性。这表明,桡足类也可以通过水平和垂直两种途径感染WSSV。通过病毒-浮游微藻吸附法使桡足类美丽猛水蚤Nitocra sp.感染上WSSV,然后用这些病毒阳性猛水蚤投喂日本囊对虾Marsupenaeus japonicus的仔虾,每个处理四个重复,经过15天的投喂,攻毒组(PCO)仔虾的感染率是100%,死亡率是52.50±5.00 %,投喂WSSV阴性猛水蚤对照组(NCO)的死亡率是20.00±0.00%,两组相比差异显著(P < 0.05)。为了确认WSSV阳性猛水蚤体内病毒的感染性,本实验还用病毒阳性猛水蚤的组织粗提液注射日本囊对虾稚虾,每个处理四个重复,攻毒20天后,对虾的死亡率是72.50±9.57%;一步PCR检测攻毒组的稚虾全部为病毒阳性,而注射病毒阴性猛水蚤组织粗提液稚虾的死亡率是22.50±5.00%,显著低于攻毒组(P < 0.05);并且套式PCR检测该组对虾为阴性。本研究结果证明,猛水蚤可以作为WSSV的宿主而保持病毒的感染力,并成为病毒的传播媒介。利用病毒-浮游微藻吸附法使不同发育阶段的卤虫感染WSSV,运用RT-PCR方法,以WSSV囊膜蛋白VP28的特异引物检测WSSV阳性的不同发育阶段卤虫,发现WSSV没有在卤虫体内复制,由此推断,卤虫可能只是WSSV的物理携带者,不是生物携带者或宿主。用感染WSSV的卤虫投喂实验动物,以检验卤虫传播WSSV的能力,同时尝试了寻找新的实验动物来代替对虾仔虾用于WSSV相关的实验。黑褐新糠虾Neomysis awatschensis和凡纳滨对虾Litopenaeus vannamei仔虾摄食WSSV阳性的卤虫15天后,经套式PCR可以检测出均呈现WSSV阳性;而摄食WSSV阴性卤虫的实验动物呈现WSSV阴性。但是,摄食卤虫的实验动物死亡率没有显著性差异(P>0.05)。这些结果说明,卤虫作为WSSV的物理携带者,可以通过食物途径传播病毒。另外,黑褐新糠虾和凡纳滨对虾仔虾摄食WSSV阳性中国明对虾肉糜后,全部死亡,套式PCR结果也为阳性。由此说明黑褐新糠虾是WSSV的一种易感动物,可以作为对虾仔虾的替代而用于WSSV的实验,充当实验动物。更多还原

【Abstract】 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.更多还原