咪唑类物质KK-42提高日本沼虾成活率机制的研究

Effect of KK- 42 on survival rate of Macrobrachium nipponense and its potential mechanisms

目的：探讨KK-42提高感染嗜水气单孢菌后的日本沼虾成活率的机制.方法：首先将虾分为两组,分别用1.95×10^-4mol/L的KK-42溶液（处理组）或不含KK-42的溶液（对照组）浸泡1 min后取出,放置养殖水槽中饲养12h后,进行嗜水气单胞菌感染实验,每组又分别分为两个亚组（Challenged和Unchallenged）;于感染后的不同时刻,分别测定日本沼虾的成活率、细菌清除率及proPO基因（甲壳动物重要的免疫因子）表达水平和酶活力的变化.结果：KK-42预处理可以提高感染嗜水气单孢菌后的日本沼虾的成活率及细菌清除率;KK-42处理后3、12和24 h,proPO基因表达明显提高,PO活力也于6～48 h持续升高,在24 h达到最高值,是对照组的2.3倍.嗜水气单胞菌刺激也可诱导血细胞proPO mRNA水平及血清中PO活力的增加,但对照组分别在菌注射后3h和6 h,mRNA表达突然升高,而KK-42实验组则在整个时期处于缓慢升高趋势;PO活力变化趋势和基因表达相一致.结论：KK-42可以诱导proPO mRNA表达水平及PO活力的升高,并能提高沼虾细菌清除能力,可能是KK-42提高日本沼虾成活率的分子机制之一.

Objective：To explore the molecular mechanisms of KK-42 acts for increasing survival of the prawn infected with Aeromonas hydrophila.Methods：Prawns were divided into two groups.Two groups prawns were pre-administered as with KK-42 at a concentration of 0 （control） or 1.95 × 10^-4 mol/L （treatment）,respectively.After KK-42 treatment 12 h,the control prawns were injected individually with 20 μl of A hydrophila suspension （challenged control） or 20 μl of saline （unchallenged control） into the ventral sinus ; so did the KK-42 treatment group.Then the survival rate,bacterial clearance rate,the expression level of prophenoloxidase （proPO） and PO activity,a conserved copper-containing enzyme that plays an important role in defense against pathogens,were detected.Results：KK-42 pretreatment could promote the survival rate of Macrobrachium nipponense infected with Aeromonas hydrophila,and enhance the bacterial clearance efficiency.Real-time PCR analysis revealed that the proPO expression increased significantly at 3,12 and 24 h after KK-42 treatment,the PO activity also importantly rose from 6 to 48 h in KK-42-treated prawns and reached the maximum at 24 h with a 2.3-fold higher than that in control group.Injection of A hydrophila could stimulate the proPO transcription and PO activity whether or not the prawns were pretreated by KK-42,the mRNA level increased obviously only at 3 h and 6 h after the bacterium injection （challenged control）,but increased constantly during the phase of experiment except at 6 h under the condition of KK-42 pretreatment （challenged treatment group）.The change trend of PO activity was basically similar to that of proPO expression.Conclusion：ProPO expression as well as PO activity may be induced by KK-42,KK-42 pretreatment could enhance the bacterial clearance rate,which is likely one of the molecular mechanisms of KK-42 acts for increasing survival of the prawn infected with A hydrophila.