Bioassay and expression alterations of acetyl cholinesterase enzyme gene to spinosad (bio-insecticides) on nontarget silkworm, Bombyx mori (Lepidoptera: Bombycidae)
Authors: Mohamed Shereen A.H. , El-Ashram Dina F. , Elyamani Enas M. 
Published: August 2023

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.