Metabolism Of Spirotetramat In Silkworm

Spirotetramat is a insecticide which can effectively control piercing-sucking mouthparts pests such as aphids,thrips and whiteflies.At present,the researches on spirotetramat in domestic and foreign countries mainly focus on the synthesis process,the efficacy,the residue in plants and the migration and transformation in the environment.It has been reported that the field efficacy of spirotetramat declined,however,there is currently no metabolic study of spirotetramat in insects and mammals to guide its rational use.In order to study the metabolic characteristics of spirotetramat in different tissues in silkworm,this paper used spirotetramat as the research object,and established the residue analysis method of spirotetramat and four metabolites in the main parts of silkworm.This paper also studied the toxicity of spirotetramat on silkworm and the distribution of spirotetramat and metabolites in silkworm midgut,silk gland,fat,blood and feces after adding mulberry leaves;the activity of three major detoxification enzymes of silkworm,treated with spirotetramat;the expression of the enzyme-related genes was verified by RT-PCR.The main findings as follows:(1)The paper used QuEChERS sample pretreatment method,combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)detection technology,negative ion scanning and multiple reaction detection mode(MRM),established residue analysis method of spirotetramat and metabolites in silkworm midgut,silk gland,fat.The results showed that with methanol as extraction solvent and C18 purification,the recoveries rate of spirotetramat and metabolites was 80.2-108.7%,which met the requirements of pesticide residue detection and was suitable for simultaneous detection of various parts of silkworm.(2)The distribution and the changes over time of spirotetramat and two metabolites in various parts of silkworm were analyzed by LC-MS/MS.The results showed that the spirotetramat was mainly degraded into two metabolites(B-enol and B-keto)in the silkworm,which could be detected in all tissues;B-glu could be detected in the midgut and feces;B-mono could not be detected.From the trend of change,spirotetramat can be degraded into metabolites B-enol,B-keto and B-glu in the midgut,and B-glu can be quickly excreted through feces.Through the flow of blood,spirotetramat and B-enol can accumulate in the silk gland,resulting in increased content and not easily degraded and metastasized.In fat,B-enol content is continuously increased,indicating that spirotetramat is may be effectively degraded to B-enol in fat,and B-keto is declining in the midgut,silk gland and fat,indicating that the spirotetramat is rapidly degraded to B-keto and then slowly transferred and discharged.(3)The mulberry leaves soaked with 200 mg/L spirotetramat were fed to the silkworm,and the activity of three detoxifying enzymesn silkworm parts(middle intestine,blood,silk gland and fat)were measured.The study found that in the midgut,the three enzyme activities in the treatment group were higher than the control group.The midgut is the main digestive organ of silkworm,and spirotetramat can be rapidly metabolized in the midgut and quickly excreted from the body.All three enzyme activities increased,which indicated that the midgut may be the main site for the metabolism of spirotetramat and all three enzymes are involved in the metabolism of spirotetramat.In the silk gland,only GST enzyme activity increased,and the spirotetramat can be metabolized in a small amount in the silk gland,but a large amount of spirotetramat and metabolites accumulate in the silk gland.This may affect the quality of silkworm silk,which affects its economic value.In fat,P450 enzyme activity is 1.87-2.44 times of the control group.Spirotetramat is mostly metabolized in fat,but B-enol continues to rise,indicating that P450 enzyme may be involved in the metabolism of spirotetramat,but fat cannot metabolize B-enol and excrete it.In the blood,only the activity of GST enzyme increased,indicating that the smaller metabolism of blood to toxic substances may be related to GST enzyme.(4)Seven genes including YP9a19、CYP9a22、CYP6AB4、GST01、GSTe2、BmCarE-11 and BmCarE8-V1 were selected as the research objects to study the expression of detoxification genes in silkworm after treatment with spirotetramat.The results showed that in the midgut,Up-regulation of the three genes from the P450 enzyme is involved in the metabolism of the spirotetramat in silkworm.The GST enzyme related gene was up-regulated at a lower level,The GST enzyme was up-regulated by 1.7-1.8fold.This indicates that GST enzyme is not the main detoxification enzyme in the midgut.Compared with the higher relative expression of the seven genes in the midgut,the relative expression of genes in fat is lower,and there is no significant change with time.This may be due to the inhibitory effect of spirotetramat on fat synthesis,affecting the the expression of the enzyme gene,thereby regulating the detoxification enzyme activity.Through the metabolism of spirotetramat in the silkworm,the metabolites in the silkworm can be identified;the main metabolic pathways of the silkworm can be understood and the role of detoxification enzymes and the regulation of related genes can be clarified.Therefore,this study provides data support for the rational use and safety evaluation of spirotetramat.