College of Aquaculture and Life Science, Dalian Ocean University, Dalian, 11026, China.
College of Aquaculture and Life Science, Dalian Ocean University, Dalian, 11026, China; Key Laboratory of Marine Bio-Resources Restoration and Habitat Reparation in Liaoning Province, Dalian Ocean University, Dalian, 116023, China.
Fish Shellfish Immunol. 2024 Oct;153:109842. doi: 10.1016/j.fsi.2024.109842. Epub 2024 Aug 15.
Molting is a crucial biological process of crustaceans. Crustaceans go through three separate stages throughout their molting process, including pre-molt, post-molt and inter-molt. However, the exact mechanism of immunological modulation during molting remains unclear. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been extensively documented to participate in immune defense. In the present study, a TRAF6 gene with two TRAF-type zinc finger domains was identified from Eriocheir sinensis (designed as EsTRAF6), and its role in regulating immune response during molting process was explored. The mRNA expression level of EsTRAF6 at pre-molt stage was higher than that at post-molt stage and inter-molt stage. After Aeromonas hydrophila stimulation, the expression levels of EsTRAF6, EsRelish and anti-lipopolysaccharide factors (ALFs) genes exhibited a considerable increase at three molting stages. Subsequently, the expression patterns of EsTRAF6 and EsRelish in response to the treatment with 20-hydroxyecdysone (20E) were examined. The mRNA expression of EsTRAF6 and EsRelish were significantly increased at 12 h after 20E injection. Additionally, the protein expression level of TRAF6 was also up-regulated in 20E group compared to control group. Furthermore, the role of EsTRAF6 in regulating the anti- ALFs expression at pre-molt stage post A. hydrophila stimulation was investigated. Following the inhibition of the EsTRAF6 transcript using RNAi or the injection of inhibitor (TMBPS), there was a notable decrease of the EsALF1, EsALF2 and EsALF3 transcripts. Moreover, a significant reduction in the phosphorylation level of NF-κB at pre-molt stage was observed after A. hydrophila stimulation in TRAF6-inhibited crabs. Collectively, our results suggest that EsTRAF6 could be induced by 20E and promoted the EsALFs expression by activating NF-κB at pre-molt stage, which provides a novel insight into the research of immune regulatory mechanism during the process of molting of crustaceans.
蜕皮是甲壳动物的一个重要的生物学过程。甲壳动物在蜕皮过程中经历三个不同的阶段，包括蜕皮前期、蜕皮期和蜕皮后期。然而，蜕皮过程中免疫调节的确切机制仍不清楚。肿瘤坏死因子受体相关因子 6（TRAF6）已被广泛证明参与免疫防御。本研究从中华绒螯蟹（设计为 EsTRAF6）中鉴定出一个具有两个 TRAF 型锌指结构域的 TRAF6 基因，并探讨了其在调节蜕皮过程中免疫反应中的作用。EsTRAF6 在蜕皮前期的 mRNA 表达水平高于蜕皮后期和蜕皮后期。在哈维弧菌刺激后，三个蜕皮阶段的 EsTRAF6、EsRelish 和抗脂多糖因子（ALFs）基因的表达水平均显著增加。随后，检测了 EsTRAF6 和 EsRelish 对 20-羟基蜕皮酮（20E）处理的反应模式。20E 注射后 12 h，EsTRAF6 和 EsRelish 的 mRNA 表达显著增加。此外，与对照组相比，20E 组 TRAF6 的蛋白表达水平也上调。此外，还研究了 EsTRAF6 在调节哈维弧菌刺激后蜕皮前期抗 ALFs 表达中的作用。使用 RNAi 抑制 EsTRAF6 转录或注射抑制剂（TMBPS）后，EsALF1、EsALF2 和 EsALF3 的转录明显减少。此外，在哈维弧菌刺激后，TRAF6 抑制蟹的蜕皮前期 NF-κB 的磷酸化水平显著降低。总之，我们的结果表明，EsTRAF6 可以被 20E 诱导，并通过在蜕皮前期激活 NF-κB 促进 EsALFs 的表达，这为甲壳动物蜕皮过程中免疫调节机制的研究提供了新的视角。
