Department of Zoology, University of Madras, Guindy Campus, Chennai, 600025, India.
Department of Zoology, Bharathiar University, Coimbatore, 641046, India.
J Comp Physiol B. 2023 Oct;193(5):495-507. doi: 10.1007/s00360-023-01503-7. Epub 2023 Jul 17.
In insects, enzyme phenoloxidase plays a critical role in cuticular sclerotisation and defensive functions. In the present investigation, haemolymph phenoloxidase activity from the grub of Zophobas morio was attempted to evaluate as a reliable predictor of insect's immunological response. Among the various substrates tested, L-DOPA was chosen as an appropriate substrate due to its high oxidation. The optimum pH and temperature for haemolymph PO activity was found to be 8 and 30 °C, respectively. The optimum substrate concentration of L-DOPA was found to be 7.5 mM for subsequent PO enzymatic characterisation. Among the various chemical inhibitors and copper chelators, PO activity was significantly reduced in the case of PMSF and thiourea. Preincubation of haemolymph with non-self-molecules showed enhancement of PO activity in the case of LPS from Serratia marcescens. In addition, exogenous proteases like α-chymotrypsin enhanced the PO activity of haemolymph and an increase in PO activity was demonstrated when haemolymph was preincubated with the anionic detergent, SDS and cationic detergent, cetyl pyridium chloride. Alteration of PO activity was observed under agonising conditions of starvation, ligation and microplastics injection at different time intervals. Interestingly, there were no correlation between PO and insect defence under live challenge of microbes. SDS protein profile revealed a significant increase in the 85 kDa and 55 kDa polypeptides in all the experiments over control after 24 h, 48 h and 96 h. Mass spectrophotometric analysis of the polypeptides revealed their homology to antimicrobial peptides for 55 kDa protein and 85 kDa protein. A significant increase in 85 kDa polypeptide was observed in the haemolymph of the grubs after 72 h in the case of starved and microplastics injected groups only. These results demonstrated that PO may not be a reliable benchmark of immunological response in this insect.
在昆虫中，酶酚氧化酶在表皮硬化和防御功能中起着关键作用。在本研究中，试图评估 Zophobas morio 幼虫的血淋巴酚氧化酶活性作为昆虫免疫反应的可靠预测指标。在所测试的各种底物中，由于其高氧化，选择 L-DOPA 作为合适的底物。发现血淋巴 PO 活性的最佳 pH 和温度分别为 8 和 30°C。发现 L-DOPA 的最佳底物浓度为 7.5 mM，用于随后的 PO 酶学特征分析。在各种化学抑制剂和铜螯合剂中，PMSF 和硫脲显著降低了 PO 活性。非自身分子的预孵育使 LPS 从粘质沙雷氏菌引起的 PO 活性增强。此外，α-糜蛋白酶等外源性蛋白酶增强了血淋巴的 PO 活性，并且当血淋巴与阴离子去污剂 SDS 和阳离子去污剂十六烷基吡啶氯化物预孵育时，PO 活性增加。在饥饿、结扎和微塑料注射的激动条件下，在不同时间间隔观察到 PO 活性的变化。有趣的是，在活体挑战微生物下，PO 与昆虫防御之间没有相关性。SDS 蛋白谱在 24 小时、48 小时和 96 小时后所有实验中均显示 85 kDa 和 55 kDa 多肽与对照相比显著增加。多肽的质谱分析表明，它们与 55 kDa 蛋白和 85 kDa 蛋白的抗菌肽同源。在饥饿和注射微塑料的组中，幼虫血淋巴中的 85 kDa 多肽在 72 小时后显著增加。这些结果表明，PO 可能不是这种昆虫免疫反应的可靠基准。
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