Defensive Responses To The Prophenoloxidase Activating System Of Nilaparvata Lugens And Myzus Persicae Induced By Pandora Delphacis (Entomophthorales)

Prophenoloxidase-activating system (proPO-AS) is one of the most important humoral immunity systems in insects, which involves in three physiologically important processes, i.e., cuticular hardening (sclerotization), defense reactions (immune reaction), and wound healing. Most of proPO-AS studies focus on large-type insects whereas little attention is paid to small insects such as aphids and planthoppers. This thesis sought to understand the proPO-AS present in the rice planthopper Nilaparvata lugens and the green peach aphid Myzus persicae and develop a method for use in proPO-AS studies of small insects.Impact of various compounds on the PO activities in the crude extracts of N. lugens and M. persicae. When Ca~2+ concentration increased from 0.1 to 100 mM, the activities of phenoloxidase (PO) in the crude extacts of N, lugens and M. persicae were concentration dependent. The maximal activation of the proPO was observed at 30 mM Ca~2+. However, a further increase in Ca~2+ concentration up to 100 mM resulted in a decrease in proPO activation. Incubation of the extracts in conjunction with different concentrations of laminarin (from 10~-4 to 10 mg/ml) witnessed the levels of proPO activation entirely depending on the laminarin concentrations. When different glucans were used as activating agents, laminarin and zysoman were found significantly enhancing the PO activities whereas the effect of curdlan was not significant compared to a blank control. In contrast, the PO activities were reduced when mannan, dextran or cellulose was included. These results indicate that (3-1,3-glucan effectively activated the proPO present in the extracts of the two insect species with the proPO selectively responding to different glucans. Inclusion of Ca~2+ and phenylmethyl sulfonyl fluorid (PMSF, a protease inhibitor) in the extracts affected the PO activities. Adding Ca~2+ to N. lugens extract prior to a reaction with PMSF led to a reduction in PO activity. Adding Ca~2+ to the extact after the reaction did not cause such reduction. Inclusion of Ca~2+ in M persicae extract,followed by the reaction with PMSF, caused no decrease in the activation of proPO, which was even enhanced when Ca~2+ was added after the reaction with PMSF.Purification and immune function of pGBP. With zymosan affinity precipitationmethod, a (3GBP of 45.4 kDa was isolated from N. lugens extract while two PGBP molecules of 44.6 and 48.2 kDa were observed from M. persicae extract. However, no band was gained using laminarin or curdlan affinity precipitation. After the extracts were separately incubated with different glucans at 0°C, the resultant supernatants were assayed for PO activity at the presence of Ca2+ and laminarin. Laminarin-precipitated supernatant significantly (PO.01) enhanced the PO activity whereas mannan-, dextran- or cellulose-precipitated supernatants had significantly reduced PO activity. Zymosan-precipitated supernatant had a very low level of PO activity due to its lacking pGBP. Surprisingly, a minimal PO activity was observed in curdlan-precipitated supernatant perhaps because curdlan is an insoluble glucan and thus may combine with some important components that are necessary to activate the proPO-AS. Moreover, P. delphacis mycelia enhanced the PO activity more than its protoplasts that lack cell wall so as to escape from immune system of the host.