Studies On The Genotoxicity Of Low Doses Of Avermectin To Silkworm, Bombyx Mori

The silkworm, bombyx mori as an important economic insect of Lepidoptera, is more sensitive to most of pesticides than wild pests. In the sericultural regions in China, heavy lose of cocoon production frequently caused due to agricultural pest control by application of pesticides in the field, because trace pesticides can poison the silkworms directly or indirectly through contact, stomach toxicity, suffocating and etc.. Avermectin is a chemical compound of16-membered macrocyclic lactones derived from the actinomycete Streptomyces avermectinius. It stimulates the release of γ-aminobutyric acid (GABA) to affect the chloride channels of the central nervous system, which causes insect paralysis, hunger strike and finaly death. Avermectin has been used widely in agricultural pest control for its high efficiency and broad spectrum. Silkworm is very sensitive to avermectin, the accidents of avermectin poisoning caused decrease of cocoon productions happens every now and then.This study is focused on the negative effects of avermectin on larval development, hemocytes DNA damage, differential expression genes and differential expression proteins of Bombyx mori. The genotoxicity of low dosage of avermectin to silkworm is comprehensively evaluated in the present research and summarized as followings.1. Four concentrations of avermectin1、2、4、8μg/L were designed for treatments based on the result of96hr acute toxicity study in silkworm. The physiological processes of growth and development, food utilization, cocoon production were assessed after the treatments of designed low dosage avermectin. The results showed that no observable effect was found in silkworm larvae after treated by1μg/L avermectin. The body weight gain, relative growth rate, relative feeding amount, food utilization rate, food conversion rate of the larvae and single cocoon weight decreased in the treatments by2、4and8μg/L avermectin, and showed positive relationship with the concentrations of avermectin.2. DNA damage caused by low dosage of avermectin was evaluated in hemocytes of silkworm using the alkaline single-cell gel electrophoresis (SCGE). The fourth instar larvae of silkworm were exposed to avermectin by oral feeding with mulberry leaves treated by three different concentrations (1、2、4μg/L). The hemocytes were collected at day1,3,5,7,9for assessment of DNA damage with tail DNA percent. Results showed that there was positive correlation between avermectin concentration and DNA damage in three tested treatments. The maximum value of tail DNA percent and tail moment was observed at the third day, followed by a gradual non-linear decline. It indicated that the low dosage of avermectin had genetic toxicity on silkworm, and the degree of DNA damage was related to the dosage and time of avermectin treatment.3. The fourth instar larvae of silkworm were exposed to avermectin by oral feeding with mulberry leaves treated by four different concentrations (1、2、4、8μg/L). After96hours, the genomic DNAs of hemocytes were isolated for RAPD assay. By using24RAPD primers to amplify the genomic DNAs of five samples,143clearly distinguishable bands were obtained, among which19bands showed polymorphism, showing a polymorphism percentage of13.29%. Genetic similarity coefficient ranged from0.867-0.993among different samples. The result of dendrogram showed that1、2、4μg/L treatments and the control were classified into a group,8μg/L treatment was a separate one. It indicated that8μg/L avermectin caused the most serious effect on DNA integrality.4. Differentially expressed genes (DEGs) were explored in hemocytes of silkworm exposed to avermectin (1μg/L) using annealing control primer (ACP)-based PCR.15DEGs were screened and sequenced, among which Ribosomal protein S18(DEG4)、ALY(DEG5)、Ribosomal protein S8(DEG7)、 Ribosomal protein L6(DEG8)、Ribosomal protein L23A(DEG9)、ERp57(DEG10)、QM(DEG11)、Ribosomal protein L35(DEG13)、Rad23(DEG14) and Ribosomal protein L11(DEG15) were up-regulated, Polyubiquitin (DEG1)、ANT(DEG2)、Glycoprotein(DEG3)、Ribosomal protein L7(DEG6)、Y-box (DEG12) were down-regulated. Differential expressions of partial genes were related with DNA damage or DNA damage repair of hemocytes. Differential expressions of others were responses to avermectin pressure.5. The protein expression of hemocytes of silkworm treated by1μg/L avermectin was analyzed by2-DE and MALDI-TOF/TOF. The results showed that there were three down-regulated and one up-regulated differential protein in the treatment, and one protein expressed specifically in the control. The No.2protein, a kind of hemolymph protein with the molecular weight of29.7kD, is a 30K protein family member and supports energy for larval development. The No.3protein is Bombyrin precursor with the molecular weight of22.5kD and responsible for nervous system stability of larvae. The No.4protein is the promoting protein with the molecular weight of17.2kD and maybe related to stress physiology of silkworm.In conclusion, the present study explored the genotoxicity of avermectin to silkworm and the effects of low dosage of avermectin on silkworm Bombyx mori were observed in larval development, DNA integrality of hemocytes, genes expression and protein expression.