Bioassay and expression alterations of acetyl cholinesterase enzyme gene to spinosad (bio-insecticides) on nontarget silkworm, Bombyx mori (Lepidoptera: Bombycidae)

IF 0.7 Q4 PHARMACOLOGY & PHARMACY
Shereen Mohamed, Dina El-Ashram, Enas M. Y. Elyamani
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引用次数: 0

Abstract

Background Bombyx mori, the mulberry silkworm, feeds entirely on mulberry leaves and is extremely sensitive to agrochemicals, even in low doses. The mulberry plantations must be insecticide-free. However, contamination by pesticides from neighboring crops occurs indirectly and harms silkworm breeding. Spinosad, a neurotoxic insecticide that acts on the nervous system of insects through contact or feeding, is the most environmentally friendly suitable bioinsecticide. It has been used to control pests in field crops. Objective In this study, the insecticide, spinosad formulation was first tested for bioassay, utilizing three different concentrations of spinosad on B. mori larvae. Second, the total RNA was isolated (isolation of total RNA) from silkworm, B. mori larvae to study the spinosad effect on acetylcholinesterase (Ace) gene expression. Materials and methods The type of insecticide used in this study is the spinosad formulation. Spinosad is available under the commercial name, Biosad 22.8% SC; the recommended concentration is 0.1 ppm. Bioassay test was done with three different concentrations of spinosad (0.1, 0.05, and 0.025 ppm). Determination of the LC values of the toxicity of three concentrations of spinosad on the fifth instar larvae of B. mori was evaluated using the mulberry leaves dipping technique. The treated mulberry leaves were offered once on the first day of the fifth instar after morning feeds, then the fresh leaves were offered during the remaining days. After 24 h of treatment, the mortality counts were recorded. LC25, LC50, and LC90 values for spinosad were calculated by probit analysis using the Ldp line software. Total RNA was isolated from entire tissues of the fifth instar larvae of the silkworm, B. mori by the standard TRIzol reagent extraction method. The complete Poly (A)+RNA isolated from insect tissues was reverse transcribed into cDNA. The sequence of primers of apoptosis is used in real-time quantitative PCR reactions to determine the expression levels of Ace-related gene. Results and conclusion Spinosad is the most economically and ecologically recommended insecticide to be used to control the agricultural pests that attack different field crops in Egypt. The toxicological effects of spinosad and its effect on the Ace gene of mulberry silkworm, B. mori were studied in this study. The results showed that treatment with 0.1 ppm of spinosad caused the highest mortality (88.9%) to the fifth instar larvae of B. mori, followed by the spinosad concentrations 0.05 and 0.025 ppm. The results showed a significant difference in LC values of spinosad on the fifth instar of B. mori. LC25, LC50, LC75, and LC90 values were recorded to be 0.008, 0.0217, 0.0536, and 0.1969 ppm, respectively. The expression levels of Ace gene in the B. mori group treated with low (0.025 ppm) and medium doses (0.05 ppm) of spinosad were increased by 141 and 396%, respectively. However, the expression level of Ace gene was increased by 657% for the group exposed to high doses (0.1 ppm) of spinosad compared with the control group with highly significant differences (P<0.01). This study confirmed that using spinosad with the lowest concentrations and caused damage occurs to silkworms when feeding on sprayed mulberry leaves with a minimum of spinosad compound, so it is a better solution to not spray spinosad on mulberry trees and any neighboring fields.
非靶家蚕(鳞翅目:家蚕科)乙酰胆碱酯酶基因的生物测定及对生物杀虫剂spinosad的表达变化
背景家蚕是一种完全以桑叶为食的桑蚕,即使在低剂量下也对农用化学品极为敏感。桑园必须不含杀虫剂。然而,来自邻近作物的农药污染是间接发生的,并危害蚕种。Spinosad是一种神经毒性杀虫剂,通过接触或喂食作用于昆虫的神经系统,是最环保的合适生物杀虫剂。它已被用于控制大田作物中的害虫。目的利用三种不同浓度的多杀菌素对家蚕幼虫进行生物测定。其次,从家蚕幼虫中分离总RNA(总RNA的分离),研究刺突酸对乙酰胆碱酯酶(Ace)基因表达的影响。材料和方法本研究中使用的杀虫剂类型为多杀菌素制剂。Spinosad的商业名称为Biosad 22.8%SC;推荐浓度为0.1 ppm。用三种不同浓度的多杀菌素(0.1、0.05和0.025 ppm)。采用桑叶浸渍法测定了三种浓度的多杀菌素对家蚕五龄幼虫毒性的LC值。处理过的桑叶在5龄的第一天早上喂食后提供一次,然后在剩下的几天提供新鲜的桑叶。24小时后 治疗h,记录死亡率。使用Ldp线软件通过probit分析计算多杀菌素的LC25、LC50和LC90值。采用标准TRIzol试剂提取法从家蚕五龄幼虫的全组织中分离总RNA。将从昆虫组织中分离的完整的Poly(A)+RNA逆转录为cDNA。凋亡引物序列用于实时定量PCR反应,以确定Ace相关基因的表达水平。结果和结论Spinosad是埃及最经济、最生态的杀虫剂,可用于控制攻击不同农田作物的农业害虫。研究了多杀菌素的毒理学效应及其对桑蚕Ace基因的影响。结果表明,用0.1 ppm的多杀菌素对家蚕五龄幼虫的死亡率最高(88.9%),其次是0.05和0.025 ppm。结果表明,5龄家蚕多杀菌素的LC值存在显著差异。记录的LC25、LC50、LC75和LC90值分别为0.008、0.0217、0.0536和0.1969 ppm。用低浓度(0.025 ppm)和中等剂量(0.05 ppm)分别增加了141和396%。然而,高剂量组的Ace基因表达水平增加了657%(0.1 ppm)与对照组相比有非常显著的差异(P<0.01)。本研究证实,使用浓度最低且对蚕造成伤害的多杀菌素,在喂入含有最少多杀菌素化合物的桑叶时,最好不要在桑树和任何邻近的田地喷洒多杀菌素。
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来源期刊
Egyptian Pharmaceutical Journal
Egyptian Pharmaceutical Journal PHARMACOLOGY & PHARMACY-
CiteScore
1.10
自引率
0.00%
发文量
37
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