KK-42对感染嗜水气单胞菌的日本沼虾成活率的影响及其可能的机制
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摘要
日本沼虾（Macrobrachium nipponense）是我国重要的淡水甲壳类，随着养殖规模的扩大以及发病率的逐年增加，对其自身免疫机能的研究越来越受到关注。我们前期研究发现，咪唑类物质KK-42能提高凡纳滨对虾（Penaeus schmitti）虾苗的成活率。为了探究该类物质是否对日本沼虾也有类似的作用，本文首先研究了KK-42处理对日本沼虾感染嗜水气单胞菌（Aeromonas hydrophila）后成活率的影响，进一步观察肝胰腺组织形态变化，分析体液免疫因子α2-巨球蛋白（alpha2-macroglobulin, α2M）和几丁质酶（chitinase,Chi）的表达变化，为阐明KK-42的作用机制提供资料。
将体长3.5~5.0cm的日本沼虾600尾随即分成3组：实验组、对照组和空白组，其中，实验组用1.95×10~(-4)mol/L的KK-42溶液浸泡处理1min，对照组用不含KK-42溶液做相同的处理，空白组不做处理；12h后向腹部第二肌节注射嗜水气单胞菌，空白组注射等量的生理盐水，观察48h内日本沼虾的死亡数量，计算成活率；同时，于不同时间分别从实验组和对照组抽取血淋巴、解剖肝胰腺等组织，用于后续实验。采用常规石蜡切片技术观察肝胰腺的组织学变化，Real-time PCR方法测定α2M、Chi mRNA水平，分光光度法测定α2M、Chi活性。结果如下：
实验期间，空白组成活率在90%以上（个别虾因蜕皮死亡）。注射嗜水气单胞菌后，对照组成活率迅速下降，在24h成活率最低并趋向稳定；实验组成活率在6、12、24h内比对照组增高了25%、141%和133%，呈显著或极显著差异（P<0.05, P<0.01）。
组织学观察显示，对照组在0h肝小管轮廓清晰，F-细胞易分辨；在6h肝小管管腔增大，可见较多絮状物；12h肝小管受损较重，空泡较多且体积较大；24h肝小管轮廓相对清晰，部分有空泡。实验组在6h肝小管管腔体积相对较小；在12h部分肝小管受损；在24h肝小管形态和0h基本相似。
首次从日本沼虾克隆出的α2M的部分cDNA序列，经在线BLAST比对，与罗氏沼虾（Macrobrachium rosenbergii）a2M具有90%以上同源性。Real-time PCR结果显示，α2M mRNA在血细胞中的表达量最高；在蜕皮周期过程中，血细胞α2M表达水平在蜕皮前期（D）最高，蜕皮后期（A、B）和蜕皮间期（C）相对较低。KK-42处理可显著诱导肝胰腺α2M mRNA的表达，在处理后6~48h，mRNA水平与对照组相比极显著升高（P<0.01），但血细胞α2M mRNA水平与对照组相比无显著性差异。注射嗜水气单胞菌后，对照组血细胞和肝胰腺α2M mRNA水平分别在3h和3、6、12、48h显著升高（P<0.05）；实验组血细胞和肝胰腺α2M mRNA含量分别在6、12、48h和12h显著高于相应对照组。血淋巴中α2M活力与其mRNA表达具有相似的模式。
首次从日本沼虾克隆出几丁质酶（Mnchi-3）的部分cDNA序列，经氨基酸序列分析发现，其序列为几丁质酶GH18催化域中的一部分，含活性催化中心-LDGLDMDWE-及保守性的谷氨酸残基，与其它物种相似性达90%。Real-time PCR结果显示，Mnchi-3mRNA在肝胰腺的表达量最高，表皮也有微量表达；在蜕皮周期过程中，Mnchi-3mRNA水平在D2/D3期最高。KK-42处理后12h，Mnchi-3mRNA表达量比相应对照组增高了150%（P<0.05）。注射嗜水气单胞菌后，对照组Mnchi-3mRNA水平在3h时显著升高，实验组mRNA表达量在12、24和48h比相应对照组分别增高了127%、304%和120%（P<0.05），酶活力在24h时与相应的对照组比显著增高（P<0.05）。
结论：咪唑类物质KK-42能够提高日本沼虾感染嗜水气单胞菌后的成活率，降低嗜水气单胞菌对肝胰腺的损伤程度，可诱导α2-巨球蛋白、几丁质酶基因表达及其酶活力。
Macrobrachium nipponense is an important freshwater species for aquaculture in China. With therapid production and development of the prawn industry, prawn diseases have become severe in recentyears. The exploration of prawn immune response is therefore of great signifcance. Our previous study hasdemonstrated that the treatment of imidazole derivative KK-42can increase the survival rate of juvenileLitopenaeus vannamei. In order to research whether the similar effect of KK-42on M. nipponense existed,we firstly investigated the survival rate of M. nipponense challenged Aeromonas hydrophila after KK-42treatment; secondly we observed the histological changes of hepatopancreas of M.nipponense challengedA. hydrophila and analysed the expressions of humoral immunity factors such as α2M and chitinase. Theresults were as follows.
About600prawns with body length of3.5~5.0cm randomly were divided into three groups: KK-42treatment, control, blank. The prawns of KK-42treatment or control group were soaked for1min inKK-42solution at a concentration of1.95×10-4mol/L or0mol/L, respectively. After12h, the prawns ofthese groups or blank group were injected with20μL of A. hydrophila suspension or sterile saline solutioninto the abdomen muscle. The survival rate for48h after A. hydrophila-challenge test was calculated; thehepatopancreas and hemolymph were collected, and the histological changes of hepatopancreas ofM.nipponense was observed by paraffin section; and the expressions of α2M and chitinase genes wereassayed using Real-time PCR; and the activities of α2M and chitinase were monitored byspectrophotometer method.
The survival rate of blank group keeped upon90%although few prawns died because of molt. AfterA.hydrophila injection, the survival rate of KK-42treatment group respectively increased by25%,141%and133%at6,12,24h compared with control group which rapidly descended and retained lower level at24h.
The histology result revealed that the hepatopancreas tubules of control group had complete structureand clearly distinguished F-cell, the lumen with floccules extended, basement membranes fractured andmany big vacuoles observed, the structure restored and few vacuoles observed at0,6,12,24h, respectively.The hepatopancreas tubules’ lumen was relatively smaller at6h in KK-42treatment group, few hepatopancreas tubules were damaged at12h and the structure was similar to those of0h at24h.
A partial fragment of α2M gene was cloned from M. nipponense by PCR, Sequence comparisonshowed that the fragment shares90%identity with that of Macrobrachium rosenbergii. The quantitativereal-time PCR analysis showed that α2M was mainly expressed in haemocytes; a2M mRNA level was thehighest in stages D, and the lower in stages A, B and C stages. After KK-42treatment, the hepatopancreasα2M mRNAlevel significantly increased compared with the corresponding controls at6~48h（P<0.01）,but the haemocyte α2M mRNA level couldn’t showed significant differentiation. Atfer A. hydrophilainjection, the α2M mRNA level of hepatopancreas or haemocyte significantly increased at3,6,12,48h or3h, respectively（P<0.05）.However, the haemocyte or hepatopancreas α2M mRNA level significantlyincreased at6,12,48h or12h, respectively, compared with the corresponding control in KK-42treatmentgroup（P<0.05）. The haemolymph α2M activity pattern was similar to α2M mRNA level.
A partial fragment of chitinase gene（Mnchi-3）was obtained, sequence comparison showed that thededuced amino acid of Mnchi-3cDNA fragment involved chitinase catalyticactive site‘-LDGLDMDWE-’and the fully conserve glutamate residue ‘-G-’, and which revealed homology around90%compared to those in other species. The real-time PCR analysis analysis showed that Mnchi-3wasexpressed mainly in hepatopancreas, and Mnchi-3mRNA level was the highest in stages D2/D3. AfterKK-42treatment, the hepatopancreas Mnchi-3mRNA contents significantly increased by150%comparedwith the corresponding controls at12h（P<0.05）. Atfer A. hydrophila injection, the Mnchi-3mRNA levelsignificantly increased at3h; the Mnchi-3mRNA contents increased by127%,304%,120%, respectively,compared with the corresponding controls at12,24,48h in KK-42treatment group（P<0.05）. Thehepatopancreas chitinase activity significantly increased at24h in KK-42treatment group（P<0.05）.
Conclusion：Administration of imidazole derivative KK-42can increase the survival rate of M.nipponense, reduce the degree of damage of hepatopancreas of M. nipponense challenged A. hydrophila,and induce the α2M, Mnchi-3gene expressions and enzyme activities.
将体长3.5~5.0cm的日本沼虾600尾随即分成3组：实验组、对照组和空白组，其中，实验组用1.95×10~(-4)mol/L的KK-42溶液浸泡处理1min，对照组用不含KK-42溶液做相同的处理，空白组不做处理；12h后向腹部第二肌节注射嗜水气单胞菌，空白组注射等量的生理盐水，观察48h内日本沼虾的死亡数量，计算成活率；同时，于不同时间分别从实验组和对照组抽取血淋巴、解剖肝胰腺等组织，用于后续实验。采用常规石蜡切片技术观察肝胰腺的组织学变化，Real-time PCR方法测定α2M、Chi mRNA水平，分光光度法测定α2M、Chi活性。结果如下：
实验期间，空白组成活率在90%以上（个别虾因蜕皮死亡）。注射嗜水气单胞菌后，对照组成活率迅速下降，在24h成活率最低并趋向稳定；实验组成活率在6、12、24h内比对照组增高了25%、141%和133%，呈显著或极显著差异（P<0.05, P<0.01）。
组织学观察显示，对照组在0h肝小管轮廓清晰，F-细胞易分辨；在6h肝小管管腔增大，可见较多絮状物；12h肝小管受损较重，空泡较多且体积较大；24h肝小管轮廓相对清晰，部分有空泡。实验组在6h肝小管管腔体积相对较小；在12h部分肝小管受损；在24h肝小管形态和0h基本相似。
首次从日本沼虾克隆出的α2M的部分cDNA序列，经在线BLAST比对，与罗氏沼虾（Macrobrachium rosenbergii）a2M具有90%以上同源性。Real-time PCR结果显示，α2M mRNA在血细胞中的表达量最高；在蜕皮周期过程中，血细胞α2M表达水平在蜕皮前期（D）最高，蜕皮后期（A、B）和蜕皮间期（C）相对较低。KK-42处理可显著诱导肝胰腺α2M mRNA的表达，在处理后6~48h，mRNA水平与对照组相比极显著升高（P<0.01），但血细胞α2M mRNA水平与对照组相比无显著性差异。注射嗜水气单胞菌后，对照组血细胞和肝胰腺α2M mRNA水平分别在3h和3、6、12、48h显著升高（P<0.05）；实验组血细胞和肝胰腺α2M mRNA含量分别在6、12、48h和12h显著高于相应对照组。血淋巴中α2M活力与其mRNA表达具有相似的模式。
首次从日本沼虾克隆出几丁质酶（Mnchi-3）的部分cDNA序列，经氨基酸序列分析发现，其序列为几丁质酶GH18催化域中的一部分，含活性催化中心-LDGLDMDWE-及保守性的谷氨酸残基，与其它物种相似性达90%。Real-time PCR结果显示，Mnchi-3mRNA在肝胰腺的表达量最高，表皮也有微量表达；在蜕皮周期过程中，Mnchi-3mRNA水平在D2/D3期最高。KK-42处理后12h，Mnchi-3mRNA表达量比相应对照组增高了150%（P<0.05）。注射嗜水气单胞菌后，对照组Mnchi-3mRNA水平在3h时显著升高，实验组mRNA表达量在12、24和48h比相应对照组分别增高了127%、304%和120%（P<0.05），酶活力在24h时与相应的对照组比显著增高（P<0.05）。
结论：咪唑类物质KK-42能够提高日本沼虾感染嗜水气单胞菌后的成活率，降低嗜水气单胞菌对肝胰腺的损伤程度，可诱导α2-巨球蛋白、几丁质酶基因表达及其酶活力。
Macrobrachium nipponense is an important freshwater species for aquaculture in China. With therapid production and development of the prawn industry, prawn diseases have become severe in recentyears. The exploration of prawn immune response is therefore of great signifcance. Our previous study hasdemonstrated that the treatment of imidazole derivative KK-42can increase the survival rate of juvenileLitopenaeus vannamei. In order to research whether the similar effect of KK-42on M. nipponense existed,we firstly investigated the survival rate of M. nipponense challenged Aeromonas hydrophila after KK-42treatment; secondly we observed the histological changes of hepatopancreas of M.nipponense challengedA. hydrophila and analysed the expressions of humoral immunity factors such as α2M and chitinase. Theresults were as follows.
About600prawns with body length of3.5~5.0cm randomly were divided into three groups: KK-42treatment, control, blank. The prawns of KK-42treatment or control group were soaked for1min inKK-42solution at a concentration of1.95×10-4mol/L or0mol/L, respectively. After12h, the prawns ofthese groups or blank group were injected with20μL of A. hydrophila suspension or sterile saline solutioninto the abdomen muscle. The survival rate for48h after A. hydrophila-challenge test was calculated; thehepatopancreas and hemolymph were collected, and the histological changes of hepatopancreas ofM.nipponense was observed by paraffin section; and the expressions of α2M and chitinase genes wereassayed using Real-time PCR; and the activities of α2M and chitinase were monitored byspectrophotometer method.
The survival rate of blank group keeped upon90%although few prawns died because of molt. AfterA.hydrophila injection, the survival rate of KK-42treatment group respectively increased by25%,141%and133%at6,12,24h compared with control group which rapidly descended and retained lower level at24h.
The histology result revealed that the hepatopancreas tubules of control group had complete structureand clearly distinguished F-cell, the lumen with floccules extended, basement membranes fractured andmany big vacuoles observed, the structure restored and few vacuoles observed at0,6,12,24h, respectively.The hepatopancreas tubules’ lumen was relatively smaller at6h in KK-42treatment group, few hepatopancreas tubules were damaged at12h and the structure was similar to those of0h at24h.
A partial fragment of α2M gene was cloned from M. nipponense by PCR, Sequence comparisonshowed that the fragment shares90%identity with that of Macrobrachium rosenbergii. The quantitativereal-time PCR analysis showed that α2M was mainly expressed in haemocytes; a2M mRNA level was thehighest in stages D, and the lower in stages A, B and C stages. After KK-42treatment, the hepatopancreasα2M mRNAlevel significantly increased compared with the corresponding controls at6~48h（P<0.01）,but the haemocyte α2M mRNA level couldn’t showed significant differentiation. Atfer A. hydrophilainjection, the α2M mRNA level of hepatopancreas or haemocyte significantly increased at3,6,12,48h or3h, respectively（P<0.05）.However, the haemocyte or hepatopancreas α2M mRNA level significantlyincreased at6,12,48h or12h, respectively, compared with the corresponding control in KK-42treatmentgroup（P<0.05）. The haemolymph α2M activity pattern was similar to α2M mRNA level.
A partial fragment of chitinase gene（Mnchi-3）was obtained, sequence comparison showed that thededuced amino acid of Mnchi-3cDNA fragment involved chitinase catalyticactive site‘-LDGLDMDWE-’and the fully conserve glutamate residue ‘-G-’, and which revealed homology around90%compared to those in other species. The real-time PCR analysis analysis showed that Mnchi-3wasexpressed mainly in hepatopancreas, and Mnchi-3mRNA level was the highest in stages D2/D3. AfterKK-42treatment, the hepatopancreas Mnchi-3mRNA contents significantly increased by150%comparedwith the corresponding controls at12h（P<0.05）. Atfer A. hydrophila injection, the Mnchi-3mRNA levelsignificantly increased at3h; the Mnchi-3mRNA contents increased by127%,304%,120%, respectively,compared with the corresponding controls at12,24,48h in KK-42treatment group（P<0.05）. Thehepatopancreas chitinase activity significantly increased at24h in KK-42treatment group（P<0.05）.
Conclusion：Administration of imidazole derivative KK-42can increase the survival rate of M.nipponense, reduce the degree of damage of hepatopancreas of M. nipponense challenged A. hydrophila,and induce the α2M, Mnchi-3gene expressions and enzyme activities.
引文
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