Induction Of Detoxifying Enzymes By Quercetin And Rutin In The Silkworm

Flavonoids are widely distributed in the plant kingdom with various biologicalactivities for organisms. Quercetin and rutin are abundant flavonoids and defensesecondary metabolites in plants, which have complex influences on the developmentand growth of insects. In the ecological interactions of plants and insects, herbivorousinsects have evolved a variety of mechanisms to adapt to their host plants. There are alot of detoxification enzymes in insects including cytochrome P450monooxygenase(P450), UDP-glucosyl transferase (UGT), glutathione-S-transferase (GST) andcarboxylesterase (COE). These enzymes were overexpressed in insect, which coulddetoxify the secondary compounds, such as quercetin and rutin.Recent researches suggested activities of some detoxification enzyme weresignificantly induced by quercetin and rutin. But these investigations were focused onthe effect of quercetin on insects at the physiological and biochemical levels, and thestudies at the molecular level are commonly limited to identify the induction of a classof detoxification enzymes. As an important model organism for Lepidoptera, thesilkworm, Bombyx mori (L.), specialized on mulberry leaves, which contains relativelyhigh content of flavonoids. This indicates that the silkworm has evolved somemechanisms to eliminate the toxic effects of secondary metabolites including quercetinand rutin in the adaptive evolutionary development. Therefore the silkworm could be amodel to study the detoxification mechanisms of quercetin and rutin in Lepidoptera.In this study, silkworm larvae were exposed to four different concentrations ofquercetin and rutin for different time durations to investigate the effect of quercetin andrutin on larval growth and activities of the detoxification enzymes. We measured theactivities of cytochrome P450monooxygenase (P450), glutathione-S-transferase (GST)and carboxylesterase (COE) after exposure. For P450and GST，activities of whichinduced by quercetin higher，the responses of eight CYP genes and seven GST genes inthe silkworm after quercetin exposure were examined by reversetranscriptase-polymerase chain reaction (RT-PCR) and quantitative real-timepolymerase chain reaction (qRT-PCR). To determine the phylogenetic relationship ofthe CYP6sequences related to secondary compounds detoxification, the phylogenetictree of23CYP sequences related to xenobiotic metabolism from11insect species and two induced genes CYP6AB5, CYP6B29by quercetin was reconstructed. Results are asfollows:1. The results showed that the weight gain of the silkworm larvae significantlydecreased after treatment by different concentrations of quercetin except for thetreatment with0.5%quercetin. Rutin did not inhibite the growth of silkworm obviously.2. Activities of three detoxification enzymes were induced by0.5%or1%concentration of quercetin. In the midgut, the induction activity of P450s reached to thehighest level (2.3-fold) by1%quercetin after seven days exposure, the highestinduction activities of GST toward CHP (Cumene hydroperoxide) and CDNB(1-chloro-2,4-dinitrobenzene) were4.1-fold and2.6-fold of controls by1%quercetinafter seven days exposure, respectively. For COEs, the highest activity (2.3-fold) wasinduced by0.5%quercetin after seven days exposure.3. The responses of eight cytochrome P450(CYP) genes belonged to CYP6andCYP9families and seven GST genes to quercetin were detected with RT-PCR. And thegenes induced by quercetin significantly were confirmed by qRT-PCR. CYP6AB5,CYP6B29and GSTe8were identified as inducible genes, of which the highest inductionlevels were10.9-fold (0.5%quercetin for seven days),6.2-fold (1%quercetin for sevendays) and7.1-fold (1%quercetin for seven days), respectively.4. The obtained phylogenetic tree indicated that the Bombyx mori CYP6AB5wasclosely related to the Depressaria pastinacella CYP6AB3, which shared56%aminoacid identity. Furthermore, the B. mori CYP6B29was closely related to4CYP6Bsequences: CYP6B27, CYP6B9, CYP6B8and CYP6B28of Helicoverpa zea andCYP6B6of Helicoverpa armigera, which shared58%-61%amino acid identities.5. The metabolism mechanism of quercetin in the silkworm was proposed based onthese results.In this research, the induction pattern of3detoxification enzymes in silkworm byquercetin and rutin were firstly investigated, and the molecular mechanisms of quercetindetoxification metabolism in silkworm were studied initially. These results will help usunderstand the mechanisms of mulberry-silkworm interaction and provide thefoundation for further RNA interference research aimed at pest control.