The Response Of Tolerance And Reproduction After Lead Exposure In Spodoptera Litura Fabricus,and Its Molecular Mechanism

Adaptability to pesticides and high fecundity are the main reason of pest outbreak.The rapid development of industrialization and urbanization has generated heavy metalcontamination, and has become a global problem. In particular, the toxic heavy metallead (Pb) is found ubiquitous in the ecosystems. Pb can eventually reach to higher trophiclevels such as the arthropods including herbivorous insects. In this study, Spodopteralitura larvae were fed on the artificial diet containing a range of Pb concentrations asdetermine by its concentration in vegetables. We investigated the effects of Pb on thelarvae weight, pupae weight, mortality, oviposition duration, the number and hatchingrate of eggs. Its molecular mechanism on the tolerance to pesticides and reproduction of S.litura were also investigated. The main results were as following:1. The tissues and stages distribution of Pb accumulation in S. litura larvae wereexamined by flame atomic absorption spectrometry. Levels of Pb in midgut, fatbody and brain significantly correlated with increased exposure to high Pbconcentration in an artificial diet. Pb is initially accumulated in the intestine, andthen distributed in the fat body and brain by the transportation of hemolymph. The concentrations of lead in cuticle were not detectable, which indicated that Pbcan not be excreted through metamorphosis, so Pb concentrations in adults weresignificant higher than that in pupae.After exposure of Pb for5generations, Levels of Pb in midgut, fat body andbrain significantly correlated with also increased exposure to high Pbconcentration in an artificial diet. The accumulations by the midgut after5generations of Pb exposure were less than at96h, while Pb accumulations in thehemolymph and fat body of larvae significantly increased. The ultrastructure ofmidgut cell was observed by electron microscopy. The mitochondrias of hasswelled, deformed, voided and cristae fractured. After high concentrationexposure, Pb lead inclusion bodies occupied most of the space of the nucleus,which implied that most of Pb accumulated in nucleus and affected thephysiological responses of midgut cell.2. Larvae exposed to lead (Pb)(0-100mg Pb/kg in artificial diet) did not inhibittheir growth. Larvae tolerance to the alpha-cypermethrin, theta-cypermethrin andfenvalerate were improved after pre-exposure to100mg Pb/kg, however no effectof methomyl and chlorpyrifos of LC50was detected. After96h of Pb (0-100mgPb/kg) exposure, topical application and feeding of cypermethrin to S. lituradecreased their mortality and increased weight gain. Moreover, the mortality of S.litura treated with25and50mg Pb/kg of Pb for five generations wassignificantly lower than control. The results indicated short or long-term leadexposure improves the tolerance of S. litura. to cypermethrin.3. The overexpression of cytochrome P450is one of resistance mechanisms ofpyrethroid insecticides in lepidopteran insects. Three novel full-length P450cDNA were cloned from S. litura and named CYP6B47(465039.2), CYP6B48(GQ465038.1) and CYP9A39(GQ465040.1). The CYP6B47transcripts levelsappeared in midgut and fat body of larvae, while were absent in pupae and adults.The transcripts levels of CYP9A39in papae and adult were higher than that of inlarvae. CYP9A39in the ovary had a high level of transcription. Interestingly, theexpression of CYP6B47and CYP9A39significantly increased in the midgut and fat body after Pb exposure for5generations. In addition, we cloned a2141-bp5'-fanking regions and presented the basal luciferase activities of promoter. Wealso predicted multiple putative elements for transcription factors binding in the5'-fanking region.20E could increase the luciferase activities of promoter ofCYP9A39in Hi5. Pb²⁺could increase the luciferase activities of promoter ofCYP6B47in Hi5. Therefore, the tolerance of S. litura to cypermethrin isincreased by Pb exposure at certain concentrations through Pb accumulation inbody and the increase of CYP6B47and CYP9A39expression.4. Vitellogenesis plays a key role for maturate ovary of insect, which directly affectsthe reproduction. The reproductive success depends on the synthesis and uptakeof vitellogenin (Vg) during vitellogenesis. The element CACGTG in the5'-fanking regions of Vg was the binding sites of enhancer. The bHLHtranscription factor may regulate the Vg gene. Through ligation and injection20Eexperiment, it showed that ecdysone could affect the expression of Vg. Long-termlead exposure decreased the reproduction of S. litura, but Pb²⁺could not decreasethe luciferase activities of promoter of Vg in Hi5.Most of Pb accumulated in midgut and fat body, then increased the increase ofCYP6B47and CYP9A39expression. Low levels of Pb contamination could enhance thetolerance (not inherited) against cypermethrin, which possibly lead to resistance.This research is helpful to understand the impact of lead Pollution on the outbreaks ofherbivorous insects S. litura.