| Phenoloxidase (EC.1.14.18.1, PO), also known as tyrosinase, is a key enzyme of insects, which can take part in many physiological process, such as cuticle hardening, stabilizing and staining. Moreover, PO plays an important role in innate immune system, also in wound healing of insect. To approach the characteristics and the physiological function of PO in the growth of insect is helpful to the development of the newly"insect controller", which act on phenoloxidase, a novel mode of action. Follows the previous work in the same lab, both the characteristics, physiological and immune functions of phenoloxidase of Tenebrio molitor and Plutella xylostella have been studied in present thesis.The results showed that:1. Phenoloxidase activity and protein content was assayed using microtitration and colorimetry experiments and the difference of the two methods was compared. Results indicated that there was no remarkable difference in the activity values using the two methods. However, microtitration was found to be the more rapid and convenient method as compared to traditionally used colorimetry, and costed less reagents.2. Study the PO activity from hemolymph and cuticle of T. molitor after it molted at different times (from 1 to 7 hours). Results showed that the hemolymph PO activity was slowly decreased during larvae molted for 0 to 3 hours, then the PO activity increased during 4 to 7 hours, after molted for 7 hours, the activity was near to the normal larvae; the cuticle PO activity has no remarkable difference during molted for 1 hour, while with the passage of time, the activity was also decreased till molted for 3 hours, then increased agagin. 6 hours later, the activity was near to the normal larvae. The Km value of PO extracted from hemolymph and cuticle were determined by Lineweaver-Burk plot, the former is 1.176mmol/L, and the latter is 0.881mmol/L, indicating that PO from cuticle has more high catalyze ability than PO from hemolymph.3. The Physiological effect of quercetin on phenoloxidase from Tenebrio molitor (in vitro or in vivo) was determined, and the results showed that the inhibition concentration showing 50% of the maximum inhibition (IC50) was to be 0.625 mmol/L. PO activity of hemolymph (in vivo) was increased when the quercetin-DMSO solution or quercetin-water suspension (5μL) with concentration of 0.1~1.0 mmol/L was injected into the larvae of T. molitor, but the compound would decrease the PO activity while the concentration of quercetin was going higher that is beyond the concentration of 2 mmol/L. Meanwhile, PO activity also was increased when DMSO was injected into the tested insect (5μL) (in vivo). During 2～4 hours after injection of quercetin into the body of the insect PO activity have been decreased rapidly but the PO activity went slowly up until at about 8 h after the compound injected. The PO activity reached its highest point after treatment 8h and then dropped down again except the low concentration treatment. The results above indicated that quercetin with low concentration can cause immune response to the tested insect. The present paper showed also that it have significant effectiveness while 0.5% BSA was added to the assay system in order to prevent quercetin sedimentating and the protein has no effects on PO activity as well.4. Investigated the phenoloxidase and the immune response of T. molitor after immune challenged by Escherichia coli (DH5αstrain). The results showed that when E. coli incubated with hemolymph at 0℃for 30 minutes, the PO activity increased 1.4 folds; after the hemolymph was centrifuged to remove the hemocytes, the supernatant was used as plasma to incubated with E. coli at 0℃for 30 minutes, the PO activity of plasma was as same as control; after the plasma incubated with trypsin solutions, the PO activity was also increased. The data above indicated that the recognition factor of T. molitor to E. coli may distributed on the hemocytes or other big molecules (such as fat body), not in the plasma.After the E. coli was injected to the body of T. molitor for 1hour, some black nodules were formatted, indicating that the immune system of larvae responded to invaders soon after the infection, and the numbers of nodule increased. About 10 hours later, the nodule formation has completed, sometimes many nodules adhered together to form patch structure. At the same time with the nodules formation, the PO activity decreased rapidly, 12 hours later reached the lowest point, and then slowly increased. The results indicated that the phenoloxdiase participated in the formation of nodules.5. Using microtitration, the relationship between PO activity and the resistance of the diamondback moth Plutella xylostella (Linnaeus) to the novel insecticide butane-fipronil was determined in vitro. The results indicated that PO activity was obviously enhanced by the application of butane-fipronil. Phenoloxidase activity of the resistant strain (POr) was 2.29-fold higher than the susceptible one (POs); but the Km and optimum pH values, 1.11 mmol/L and 6.5, respectively, were similar for both samples. Both POr and POs have maximum stability at pH values less than 7.0, although POs was less stable at lower pH values than POr. In addition, the thermal stabilities of the two samples were very similar. The data suggested that PO may play an important role in the increasing resistance of pests to pesticides.6. Effect of five different insecticides to phenoloxidase activity was studied with sub-lethal dosage. The results showed that methomyl and monosultap, which belongs to carbamates and nerestoxin insecticide respectively, have the same effect on PO activity. Both of them can increase the PO activity of hemolymph from T. molitor with higher concentration, while there was no effect with lower concentrations compared with control. Chlorpyrifos, an organophosphorous insecticide, can decrease the PO activity with sub-lethal dosage. Beta-cypermethrin, a member of pyrethroid family, had no effect on the PO activity with sub-lethal dosage. Diflubenzuron, belongs to the benzoylurea family, can also increased the PO activity with sub-lethal dosage.
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