Data_Sheet_1_Aspergillus terreus (Trichocomaceae): A Natural, Eco-Friendly Mycoinsecticide for Control of Malaria, Filariasis, Dengue Vectors and Its Toxicity Assessment Against an Aquatic Model Organism Artemia nauplii.doc

Vector-borne diseases like malaria, filariasis, and dengue are transmitted by mosquitoes and they cause global mortality and morbidity due to an increased resistance against commercial insecticides. The present study was aimed to evaluate the neurobehavioral toxicity, knock-down effect, histopathology, ovicidal, adulticidal, and smoke toxicity effect of Aspergillus terreus extract against three mosquito species, namely Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti (Diptera: Culicidae). The isolated fungal strain was identified as A. terreus (GenBank accession no: KX694148.1) through morphological and molecular (phylogenetic) analysis. The morphological changes in the treated fourth instar larvae shown the demelanization of cuticle and shrinkage of the internal cuticle of anal papillae. The time duration of extract exposure against the larvae determines the level of toxicity. The extract treated larvae were displayed excitation, violent vertical and horizontal movements with aggressive anal biting behavior as the toxic effect on the neuromuscular system. The results of the biochemical analysis indicated that a decrease in the level of acetylcholinesterase, α-carboxylesterase, and β-carboxylesterase in extract treated fourth instar larvae of all tested mosquito species. The findings of histopathological investigation shown the disorganization of the abdominal region, mainly in mid, hindgut, and gastric caeca, loss of antenna, lateral hair, caudal hair, upper and lower head hairs in the mycelium extract treated An. stephensi, Cx. quinquefasciatus, and Ae. aegypti. The ovicidal bioassay test results showed the mosquito hatchability percentage was directly related to the concentrations of mycelium extract. Nil hatchability of mosquito eggs was noticed at 500 μg/ml concentration. The adulticidal activity of fungal mycelia ethyl acetate extract resulted in a dose-dependent activity (15 and 30 min recovery periods). The higher concentration of extract (1000 mg/L) acted as a repellent, the adult mosquitoes showed restless movement, uncontrolled/anesthetic flight at last died. The better adulticidal activity was observed in the ethyl acetate extract against An. stephensi, Cx. quinquefasciatus followed by Ae. aegypti with the best score of LD50 and LD90 values and nil mortality was found in the control. The results of smoke toxicity assay of the mycelia extract exhibited significant mortality rate against Ae. aegypti (91%), Cx. quinquefasciatus (89%), and An. stephensi (84%). In addition, the present investigation reported the stability and toxic effects of A. terreus mycelium ethyl acetate extract on Artemia nauplii. The swimming speed (0.88 mm s-1) of A. terreus was reduced with ethyl extract 24 h treatment whereas, the control A. nauplii showed the normal speed of 2.96 mm s-1. Altered behavior and swimming movement were observed in the 8 h A. terreus mycelium extract treated A. nauplii. A pale yellow color substance (metabolites) was found in the mid-gut region of the mycelial extract exposed A. nauplii. The outcome of the present study, suggest that the A. terreus metabolites might serve as an alternative, cost-effective, eco-friendly, and target specific mosquitocidal agent in the future.

以疟疾、丝虫病和登革热为代表的虫媒传染病由蚊虫叮咬传播，且随着商用杀虫剂抗性的不断增强，这类疾病已造成全球范围内的高发病率与死亡率。本研究旨在评估土曲霉（Aspergillus terreus）提取物对3种蚊虫——斯氏按蚊（Anopheles stephensi）、致倦库蚊（Culex quinquefasciatus）及埃及伊蚊（Aedes aegypti，双翅目:蚊科）的神经行为毒性、击倒效应、组织病理学影响、杀卵活性、杀成虫活性与烟熏毒性。通过形态学与分子（系统发育）分析，将分离得到的真菌菌株鉴定为土曲霉（GenBank登录号：KX694148.1）。经提取物处理的四龄幼虫出现表皮脱黑素、肛乳突内表皮皱缩等形态学变化，且幼虫暴露于提取物的时长与毒性程度呈正相关。提取物处理后的幼虫表现出兴奋、剧烈的垂直与水平运动，以及攻击性的肛刺行为，体现出其对神经肌肉系统的毒性作用。生化分析结果显示，经提取物处理的3种受试蚊虫四龄幼虫体内，乙酰胆碱酯酶、α-羧酸酯酶及β-羧酸酯酶的活性均显著下降。组织病理学观察结果表明，经土霉菌丝体提取物处理的斯氏按蚊、致倦库蚊及埃及伊蚊，其腹部组织出现紊乱，主要累及中肠、后肠与胃盲囊，且触角、侧毛、尾毛、头部上下毛均出现缺失。杀卵生物测定结果显示，蚊虫卵的孵化率与菌丝体提取物浓度呈直接负相关：在500 μg/ml浓度下，蚊虫卵完全无法孵化。土霉菌丝体乙酸乙酯提取物的杀成虫活性呈剂量依赖性（设置15 min与30 min恢复期）。当提取物浓度达到1000 mg/L时，其表现出驱避活性，成虫会出现躁动不安、飞行失控/麻醉状态并最终死亡。乙酸乙酯提取物对斯氏按蚊、致倦库蚊的杀成虫活性更佳，其半致死剂量（LD50）与90%致死剂量（LD90）值最优，对照组未出现蚊虫死亡。菌丝体提取物的烟熏毒性测定结果显示，其对埃及伊蚊、致倦库蚊及斯氏按蚊的致死率分别为91%、89%与84%。此外，本研究还报道了土霉菌丝体乙酸乙酯提取物对卤虫无节幼体（Artemia nauplii）的毒性与稳定性：经乙酸乙酯提取物处理24 h后，卤虫无节幼体的游泳速度从对照组的2.96 mm·s⁻¹降至0.88 mm·s⁻¹；经提取物处理8 h的卤虫无节幼体出现行为与游泳运动异常，且其中肠区域可观察到淡黄色代谢产物。本研究结果表明，土曲霉代谢产物有望成为一种经济实用、环保友好、靶标特异性强的新型杀蚊剂。