The Mechanism Of Trace Acetamipird-caused Reproductive Disorders In Silkworm,Bombyx Mori

Silkworm(Bombyx mori)is an important economic insect which belongs to Lepidoptera,and it has a history of more than 8,500 years of domestication.Silkworm is an insect with complete metamorphosis,which develops through four stages:egg,larva,pupa and adult(moth).The number of eggs laid by a silkworm moth is about 500 eggs.The main factors that affect the amount of oviposition include varietal characteristics,nutritional conditions and environmental conditions,etc.The growth,development and oviposition performance of silkworm will be affected by the addition of sublethal dose of chemical pesticides.Acetamiprid is a new and efficient nicotinamide chloride insecticide and it is not cross-resistant to organophosphorus and pyrethroid insecticides.At present,it has been widely used in the control of agricultural and forestry pests.After exposure to the sublethal dose of acetamiprid,the oviposition of silkworms will decrease significantly,which may affect the development of silk industry.The oogenesis of silkworm is mainly regulated by two internal hormones(ecdysone and juvenile hormone).In order to study the effect of acetamiprid on relations between the oogenesis and hormones,a sublethal dose of acetamiprid(0.01 mg/L)was exposed to the silkworm in this study to investigate the pathological changes of gonadal characterization,oviposition performance,ovarian-related gene transcription and the levels of hormone metabolism.The main results of this study are shown as follows:1.Quantification of acetamiprid residue in hemolymph of silkwormIn order to study the changing characteristics of residual quantity in silkworm after exposure to trace acetamiprid,the level of acetamiprid in hemolymph was determined by high performance liquid chromatography(HPLC)in different time after treatment.The results showed that the acetamiprid reached its highest value(2.278 μg/mL)at 24 h after acetamiprid exposure;decreased slowly to a stable range at 48 h;and finally decreased to a minimum value(0.477 μg/mL)at 96 h after treatment.The results showed that the residual amount is enriched in silkworm after exposure to trace acetamiprid and it may have a chronic toxic effect on silkworm.2.Effects of trace acetamiprid on growth and reproduction of Bombyx moriIn order to study the toxic damage caused by trace acetamiprid on silkworm,the characterization was observed after continuous exposure to trace acetamiprid.The results showed that the treatment group appeared slight poisoning symptoms after 48 h exposure to acetamiprid,such as eating mulberry slowly,average weight loss of 8.65%,and the survival rate was 99.0%.The silkworm refused to feed 96 h later;the body weight decreased by 7.67%as compared to the control group;the survival rate was 98%and the survival rate decreased to 94%at 120 h after treatment.The female pupae in acetamiprid treatment group were significantly smaller than that in control group,with some molted pupae.The whole cocoon weight was 82.43%of that of the control.Results suggested that the trace acetamiprid has a slight toxic effect on silkworm and affects its growth and development.After 96 h of trace acetamiprid exposure,there was a downward trend in gonadosomatic index(GSI)compared with the control group,decreased by 2.31%in males and 9.38%in females.The results showed that trace acetamiprid inhibited the growth and development of the gonadal gland of silkwom,and had a great effect on the female moths.The development of ovarian tube was affected.The average number of eggs laid in the treatment group was reduced by 197 eggs,the single egg weight was decreased by 0.52 mg and the unfertilized rat increased by 8%.The results showed that trace acetamiprid exposure has affected the ovary development of silkworm,resulting in a decrease of oviposition.3.Damage of trace acetamiprid to ovaries of bombyx moriIn order to study the damage of ovary of silkworm after trace acetamiprid exposure,pathological section and electron microscope technology were used to observe the pathological damage of ovary tissue of silkworm.Pathological section analysis showed that the development of ovarian germ cells was slower than that of the control group after acetamiprid exposure,and the number of oocytes in the ovary decreased obviously and the vacuoles increased.After exposure to acetamiprid,there was more oogonia in the ovary and relatively less early mature oocytes.Electron microscopic results showed that there were incompletely degenerated trophoblasts and immature oocytes turned up in the ovarian canal after the exposure to trace acetamiprid,and the number of follicular cells decreased and the arrangement was uneven.These results suggested that trace acetamiprid has caused the damage of ovarian tissue and affected the normal development of oocytes.4.Effects of trace acetamiprid on transcription of ovarian development-related genesIn order to study the transcription characteristics of ovarian development-related genes after trace acetamiprid exposure,the transcription levels of ovarian development-related genes were detected.The results of qRT-PCR analysis showed a downward trend in the transcription levels of Vg,Ovo,Otu,Sxl-S and Sxl-L after 96 h of treatment,and down-regulated by 0.71,0.77,0.47,0.67 and 0.88-fold,respectively.The results suggested that trace acetamiprid has caused the damage to silkworm ovaries and inhibit the transcription of development-related genes.5.Effects of trace acetamiprid on hormone levels in bombyx moriIn order to study the effects of trace acetamiprid on hormone metabolism in silkworm,the content of ecdysone and juvenile hormone was detected by qRT-PCR,and the transcription levels of related genes were detected by ELISA.The results of qRT-PCR showed that the expression of ecdysone-associated gene EcR was down-regulated by 0.46-fold after 96 h treatment with trace acetamiprid,while the transcription level of juvenile hormone-associated gene JHBP2 was up-regulated by 1.36-fold.The results of ELISA showed that there was a rising trend in the level of ecdysone in the control group in the period between 24 h and 96 h,and reached a maximum value of 2.084 U/mg prot at 96 h.After 72 h exposure to trace acetamiprid,the level of ecdysone increased to the highest value of 1.770 U/mg prot,and decreased at 96 h and maintained at a relatively high level.After 24 h treatment with acetamiprid,the juvenile hormone level was lower than that of the control group(1.849 U/mg prot),and increased to a certain extent at 96 h and higher than that of the control group.Hormone levels were consistent with the results of gene transcription.The results suggested that trace acetamiprid treatment has influenced the hormone production in silkworm.This study indicated that acetamiprid is enriched in silkworms and causes the ovarian injury.At the same time,acetamiprid exposure may also lead to abnormal metabolism of endogenous hormone;affect the development of silkworm ovary;and lead to the decrease of ovum production in silkworm.This paper provides an important reference for studying the mechanism of insect reproductive disorder caused by acetamiprid.