• ISSN 1000-0615
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Effect of KK-42 on the carapace ultrastructure in Macrobrachium nipponense during postmolt

  • Corresponding author: Qianji NING, nqjnqj1964@163.com
  • Received Date: 2019-03-02
    Accepted Date: 2019-06-04
  • The previous studies found that KK-42 treatment can significantly shorten the duration of molting cycle in juvenile prawn Macrobrachium nipponense, and increase the thickness of the carapace endocuticle in late premolt (D3 stage). In order to further investigate the effect of KK-42 on cuticle structure, the juvenile prawns in postmolt stage during which the formation of exocuticle gradually ends but that of endocuticle starts, were employed to study the structures of carapace exocuticle and endocuticle using the observation of paraffin section by scanning electron microscopy (SEM). Healthy intermolt juvenile prawns with body length of 3.5±0.1 cm were randomly divided into two groups. The prawns were soaked for 1 min in KK-42 solution at a concentration of 1.95×10-4 mol/L (treatment group) or 0 mol /L (control group), respectively. The carapaces of juvenile prawns at 1.5, 3, 6 and 12 h after molting were obtained to be used for ultrastructural observation. To observe directly the surface structure of endocuticle, the carapace obtained at 6 h after molting was scraped gently with an anatomical knife to remove tissues on the inner surface of carapace, and then examined using SEM. The results showed that the carapace was only composed of the epicuticle and exocuticle at 1.5 and 3 h after molting. The number of exocuticle lamellae derived from KK-42 treatment group rised significantly at 1.5 and 3 h after molting, and the thickness of exocuticle increased by 72.07% and 38.67%, respectively, compared with the corresponding control group. At 6 and 12 h after molting, the loose lamellaes in exocuticle tended to be dense, and there was no significant difference in thickness to be found between two groups. The number of endocuticle lamellar with a loose structure, being only one at 6 h, separately increased to two in control group and three in treatment group at 12 h after molting. In addition, pore canals within the endocuticle presented different sizes and head-tail orientation. No significant structural change was observed in the endocuticle after KK-42 treatment. The results reveal that KK-42 treatment has a significant effect on the carapace ultrastructure of juvenile M. nipponense during postmolt, and accelerate the formation rate of the exocuticle as well as endocuticle.
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    [12] 吕艳杰, 陈香丽, 郭爱莲, 等. KK-42对日本沼虾D3期头胸甲表皮结构的影响[J]. 水产学报, 2014, 38(12): 1964-1969.Lv Y J, Chen X L, Guo A L, et al. Effect of KK-42 on the carapace structure in Macrobrachium nipponense during premolt D3 stage[J]. Journal of Fisheries of China, 2014, 38(12): 1964-1969(in Chinese).
    [13] Ning Q J, Fu S G, Xu X J, et al. A new and practical application of JH antagonist KK-42 to promoting growth of shrimp Penaeus schmitti[J]. Aquaculture, 2007, 270(1-4): 422-426. doi: 10.1016/j.aquaculture.2007.04.078
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    [17] 王佩, 郭爱莲, 张宇, 等. 日本沼虾表皮几丁质合成酶基因克隆及表达分析[J]. 水产学报, 2015, 39(10): 1450-1458.Wang P, Guo A L, Zhang Y, et al. Gene cloning and expression analysis of cuticular chitin synthase from Macrobrachium nipponense[J]. Journal of Fisheries of China, 2015, 39(10): 1450-1458(in Chinese).
    [18] 苗泽龙, 黄亚龙, 吕艳杰, 等. KK-42对日本沼虾表皮蛋白基因MnCP-1表达的上调效应[J]. 淡水渔业, 2018, 48(3): 19-24. doi: 10.3969/j.issn.1000-6907.2018.03.004Miao Z L, Huang Y L, Lv Y J, et al. Effects of KK-42 on up-regulation of cuticle protein gene MnCP-1 of Macrobrachium nipponense[J]. Freshwater Fisheries, 2018, 48(3): 19-24(in Chinese). doi: 10.3969/j.issn.1000-6907.2018.03.004
    [19] 郑征帆, 吕艳杰, 王佩, 等. 日本沼虾表皮蛋白-5基因全长cDNA克隆及表达分析[J]. 水产科学, 2017, 36(6): 747-752.Zheng Z F, Lv Y J, Wang P, et al. Full-length cDNA cloning and expression of cuticular protein-5 in freshwater prawn Macrobrachium nipponense[J]. Fisheries Science, 2017, 36(6): 747-752(in Chinese).
    [20] 宁黔冀, 吕艳杰, 黄亚龙, 等. 咪唑类物质KK-42对日本沼虾头胸甲中MnCP-3表达的影响[J]. 河南师范大学学报(自然科学版), 2018, 46(6): 86-90.Ning Q J, Lv Y J, Huang Y L, et al. Effect of imidazole derivative KK-42 on expression of MnCP-3 in carapace from Macrobrachium nipponensis[J]. Journal of Henan Normal University (Natural Science Edition), 2018, 46(6): 86-90(in Chinese).
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Effect of KK-42 on the carapace ultrastructure in Macrobrachium nipponense during postmolt

    Corresponding author: Qianji NING, nqjnqj1964@163.com
  • College of Life Science, Henan Normal University, Xinxiang    453007, China

Abstract: The previous studies found that KK-42 treatment can significantly shorten the duration of molting cycle in juvenile prawn Macrobrachium nipponense, and increase the thickness of the carapace endocuticle in late premolt (D3 stage). In order to further investigate the effect of KK-42 on cuticle structure, the juvenile prawns in postmolt stage during which the formation of exocuticle gradually ends but that of endocuticle starts, were employed to study the structures of carapace exocuticle and endocuticle using the observation of paraffin section by scanning electron microscopy (SEM). Healthy intermolt juvenile prawns with body length of 3.5±0.1 cm were randomly divided into two groups. The prawns were soaked for 1 min in KK-42 solution at a concentration of 1.95×10-4 mol/L (treatment group) or 0 mol /L (control group), respectively. The carapaces of juvenile prawns at 1.5, 3, 6 and 12 h after molting were obtained to be used for ultrastructural observation. To observe directly the surface structure of endocuticle, the carapace obtained at 6 h after molting was scraped gently with an anatomical knife to remove tissues on the inner surface of carapace, and then examined using SEM. The results showed that the carapace was only composed of the epicuticle and exocuticle at 1.5 and 3 h after molting. The number of exocuticle lamellae derived from KK-42 treatment group rised significantly at 1.5 and 3 h after molting, and the thickness of exocuticle increased by 72.07% and 38.67%, respectively, compared with the corresponding control group. At 6 and 12 h after molting, the loose lamellaes in exocuticle tended to be dense, and there was no significant difference in thickness to be found between two groups. The number of endocuticle lamellar with a loose structure, being only one at 6 h, separately increased to two in control group and three in treatment group at 12 h after molting. In addition, pore canals within the endocuticle presented different sizes and head-tail orientation. No significant structural change was observed in the endocuticle after KK-42 treatment. The results reveal that KK-42 treatment has a significant effect on the carapace ultrastructure of juvenile M. nipponense during postmolt, and accelerate the formation rate of the exocuticle as well as endocuticle.

  • 甲壳动物的表皮(又称外骨骼)具有支持、保护机体的作用,但缺乏延展性,故动物生长发育的显著特征是周期性的蜕皮,一般分为蜕皮前期、蜕皮期、蜕皮后期和蜕皮间期[1]。对蜕皮周期形成机制的研究历史悠久,报道较多[2-5],各种分子的作用都是通过直接或间接影响旧表皮的降解或新表皮的形成来实现。目前,甲壳动物表皮结构在蜕皮周期中的变化规律已基本明确[6-8],其中,占据蜕皮周期大部时间的蜕皮间期和蜕皮前期的表皮结构研究最为深入,这是由于蜕皮间期表皮发育最为完善,由外而内的上表皮、外表皮和内表皮结构完整[9]。而蜕皮前期是新旧表皮更替、结构变化最为剧烈的时期,该时期旧表皮逐渐降解,新表皮中的上表皮和外表皮开始形成[10-12]。蜕皮后期持续时间相对较短,是外表皮的形成及钙化作用趋于完成、内表皮开始出现的重要阶段。

    本团队前期研究发现,咪唑类物质KK-42处理可以明显缩短日本沼虾(Macrobrachium nipponense)幼虾的蜕皮周期[5],增加蜕皮前期的晚期(D3)头胸甲内表皮的厚度[12],提示,表皮厚度的变化可能影响蜕皮周期的时程。本实验以蜕皮后期幼虾头胸甲表皮为材料,利用石蜡切片的扫描电镜观察方法,着重探究了KK-42对蜕皮后期外表皮、尤其是内表皮超微结构的影响,旨在为阐明KK-42缩短蜕皮周期的机制提供理论支持。

    • 日本沼虾捕捞于河南原阳黄寺渔场,饲养于水族箱中,每天早晚各投喂一次,一周后选取60尾处于蜕皮间期、体长(3.5±0.1) cm的健康幼虾,随机分为对照组和处理组,处理组用1.95×10-4 mol/L的KK-42溶液浸泡1 min[13],迅速取出,逐只放于水族箱的网格中,每3 h观察一次。对照组以不含KK-42的溶液处理,方法同上。

    • 取蜕皮后1.5、3、6以及12 h的幼虾,每个时间点取3只,将头胸甲背部剪成5~6 mm的小块,10%中性福尔马林溶液固定36 h,细水冲洗1 h,梯度酒精溶液脱水(在80%酒精脱水前进行伊红染色,便于后期包埋),正丁醇透明过夜,石蜡包埋,切片(厚度8 μm)烘干,60 °C的二甲苯中脱蜡3 d,放于玻璃真空干燥器备用。将干燥后的切片喷金,置于JSM-7800F扫描电镜下观察。

    • 取蜕皮后6 h幼虾头胸甲,体视显微镜下用解剖刀轻轻刮去内面组织,0.1 mol/L磷酸缓冲液冲洗,2.5%戊二醛固定12 h,后续处理参照杜娟等[8]方法,将处理好的材料直接喷金,JSM-7800F扫描电镜观察。

    2.   结果
    • 蜕皮后1.5和3 h,头胸甲只有上表皮和外表皮,上表皮是一匀质的蜡状层,其下方与之平行且排列较为疏松的多板层结构为外表皮(图版-1, 2);随着时间的延长,外表皮的板层由疏松变得致密(图版-3, 4),厚度先增加后逐渐降低(图1-a)。KK-42处理能明显加快外表皮的形成速率,蜕皮后1.5~3 h,外表皮的板层数量明显增多(图版-5, 6),其厚度分别比相应对照组提高72.07%和38.67%(P<0.01),蜕皮后6~12 h无统计学差异(图1-a)。

      Figure 图版.  Effect of KK-42 on the carapace ultrastructure in M. nipponense during postmolt

      Figure 1.  Effect of KK-42 on thickness of carapace exocuticle or endocuticle in M. nipponense during postmolt

    • 蜕皮后6 h,对照组和处理组头胸甲只有1层内表皮生成(图版I-3, 7);到12 h内表皮板层数量分别增加至2层(图版I-4)和3层(图版I-8),且板层均呈疏松状结构,两组间内表皮厚度无显著性差异(图1-b)。刮去头胸甲内面组织以暴露出内表皮,扫描电镜下观察到内表皮表面分布有大量孔道(pore canals),其形态多呈大小不等、头尾走向的梭形(图2),KK-42处理组内表皮结构未发现明显的变化(结果未示)。

      Figure 2.  The ultrastructure of carapace endocuticle from M. nipponense at 6 h after molting

    3.   讨论
    • 利用扫描电镜研究甲壳动物表皮结构,一般采用冰冻断裂技术制备材料,但该技术适用于体型较大的动物如中华绒螯蟹(Eriocheir Sinensis)[14]等。日本沼虾幼虾个体小、表皮薄,很难平行于表面断开暴露出外表皮和内表皮,故本实验采用石蜡切片的扫描电镜法。

      研究表明,甲壳动物外表皮的形成与钙化不同步,蜕皮后与钙化相关的表皮蛋白开始表达,钙盐在蜕皮后3 h发生沉积,一直持续到蜕皮后5~8 h[15, 16],这与日本沼虾蜕皮后外表皮板层由疏松变为致密(图版I-3, 4)的时间基本一致,推测钙盐的不断沉积使板层结构趋于致密。本文首次发现,KK-42处理明显加快了外表皮的形成速率,板层数量及厚度显著高于对照组(图1-a)。几丁质与蛋白质是组成外/内表皮的主要物质,本课题组前期研究显示,日本沼虾表皮几丁质合成酶基因在蜕皮前期、后期高表达[17]且KK-42能诱导该基因表达(结果未示);同时,KK-42能显著上调表皮蛋白基因在蜕皮间期[18]、蜕皮前早期[18, 19]的表达,这与新外表皮形成加快的结果相符。

      结果发现,在蜕皮后6 h内表皮开始生成(图版I-3, 7),构成内表皮的板层较为疏松,这与蜕皮间期内表皮的结构相似[8],提示,内表皮在形成过程中结构变化不大。KK-42处理同样加快了内表皮板层的形成速率(图1-b),这应该与KK-42能上调表皮蛋白在蜕皮前期[20]、蜕皮后期[21]的表达量有关。有趣的是,KK-42处理组内表皮厚度与对照组相比并无显著性差异,与前期结果不一致[12],原因可能是观测时间点不同造成的,本实验选择的是蜕皮后期内表皮刚开始形成,虽板层数增加但太薄,所以内表皮厚度的统计数据无明显差异;而前期研究选择的是蜕皮前晚期,内表皮已有一定的厚度,故两组间差异显著。孔道在物质运输和表皮的矿化作用中起重要作用[7],本文对内表皮的表面进行了观察,发现孔道多呈梭形,这与已报道的内表皮孔道形状基本一致[8, 22],推测与几丁质-蛋白质纤维的收缩[23]和表皮中钙盐的不断沉积[8]有关。

      综上,KK-42处理能显著影响蜕皮后期日本沼虾幼虾头胸甲的结构,加快外、内表皮的形成速率,这可能是KK-42缩短蜕皮周期的机制之一。

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