Molecular Mycopathology Lab, Biocontrol and Cancer Research Unit, PG Department of Botany, Ramakrishna Mission Vivekananda Centenary College (Autonomous), Rahara, Kolkata, 700118, India.
Sci Rep. 2021 Aug 6;11(1):16029. doi: 10.1038/s41598-021-95310-1.
Anopheline larvicidal property of T. asperellum has been found recently in medical science. The mechanism of actions exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some events of pathogenesis. We performed some experiments to find out the mechanisms of action of T. asperellum against anopheline larvae and compared its actions with other two well recognized entomopathogens like Metarhizium anisopliae and Beauveria bassiana. The methodology adopted for this includes Compound light and SE Microscopic study of host-pathogen interaction, detection of fungal spore adhesion on larval surface (Mucilage assay), detection of cuticle degrading enzymes (Spore bound pr1, chitinase and protease) by spectro-photometric method, Quantitative estimation of chitinase and protease enzymes, and determination of nuclear degeneration of hemocyte cells of ME (methanolic extract) treated larvae by T. asperellum under fluorescence microscope. Compound light microscopic studies showed spore attachment, appressorium and germ tube formation, invasion and proliferated hyphal growth of T. asperellum on epicuticle and inside of dead larvae. SEM study also supported them. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment indicating the presence of mucilage surrounding the attached spores. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline which indicates the presence of spore-bound PR1 protein (Pathogenecity Related 1 Protein) and it was highest (absorbance 1.298 ± 0.002) for T. asperellum in comparison with control and other two entomopathogens. T. asperellum exhibited highest enzymatic index values for both chitinase (5.20) and protease (2.77) among three entomopathogens. Quantitative experiment showed that chitinase enzyme concentration of T. asperellum (245 µg mL) was better than other two M. anisopliae (134.59 µg mL) and B. bassiana (128.65 µg mL). Similarly protease enzyme concentration of this fungus was best (298.652 µg mL) among three entomopathogens. Here we have detected and estimated fragmentized nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed that mosquito larvae exposed to 0.1 mg mL dose of ME showed maximum (100%) nuclear fragmentations of hemocytes and while 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL concentrations of ME. The knowledge of this work certainly will help in understanding of mechanism of action of T. asperellum for anopheline larval killing and consequently in eradication of malaria vector.
最近在医学科学中发现了粗毛盘尾丝虫对按蚊幼虫的杀幼虫特性。粗毛盘尾丝虫感染蚊子幼虫所表现出的作用机制是我们目前研究的关键背景。为了感染昆虫，昆虫病原体必须经历一些发病机制事件。我们进行了一些实验来找出粗毛盘尾丝虫对按蚊幼虫的作用机制，并将其与其他两种公认的昆虫病原体（绿僵菌和球孢白僵菌）进行了比较。采用的方法包括宿主-病原体相互作用的复合光和扫描电镜研究、真菌孢子在幼虫表面的黏附（黏液测定）、几丁质酶和蛋白酶的检测（分光光度法）、几丁质酶和蛋白酶的定量估计，以及用甲醇提取物处理的 ME 幼虫的血细胞核退化的荧光显微镜检测。复合光显微镜研究表明，粗毛盘尾丝虫在表皮和死幼虫内部附着、附着小体和芽管形成、入侵和增殖菌丝生长。扫描电镜研究也支持了这一点。在 3 小时的相互作用后，发现孢子附着在幼虫表面，附着部位呈现粉红色外层，表明附着孢子周围有黏液。对 4-硝基苯胺底物的酶切产生 4-硝基苯胺，表明存在孢子结合的 PR1 蛋白（致病性相关 1 蛋白），与对照和其他两种昆虫病原体相比，粗毛盘尾丝虫的 PR1 蛋白最高（吸光度 1.298±0.002）。粗毛盘尾丝虫在三种昆虫病原体中表现出最高的几丁质酶（5.20）和蛋白酶（2.77）酶指数值。定量实验表明，粗毛盘尾丝虫的几丁质酶酶浓度（245μg mL）优于其他两种绿僵菌（134.59μg mL）和白僵菌（128.65μg mL）。同样，该真菌的蛋白酶酶浓度在三种昆虫病原体中是最好的（298.652μg mL）。在这里，我们通过不同剂量的甲醇提取物处理的幼虫的荧光显微镜检测和估计了血细胞的片段化核，并观察到暴露于 0.1mg mL 剂量的 ME 的蚊子幼虫表现出最大（100%）血细胞核片段化，而在 0.02、0.04、0.06 和 0.08mg mL ME 浓度下，记录到 20%、45%、70%和 85%的核畸形。这项工作的知识肯定有助于理解粗毛盘尾丝虫对按蚊幼虫致死的作用机制，从而有助于消除疟疾媒介。
