Effects Of Perilocosides Compounds On The Activities Of Midgut Protease Of Mythimna Separata And Agrotis Ypsilon (Lepidoptera:Noctuidae) Larvae

Periplocoside E, P, T, which were isolated from the root bark of the insecticidal plantPeriploca sepium Bunge, are analogical compounds in structure. But, periplocoside E has noinsecticidal activity, periplocoside P has high insecticidal activity, while periplocoside T hasstronger insecticidal activity than periplocoside P. To explore the insecticidal mechanism ofperiplocosides compounds, we compared different effects on the activities of trypsin-likeprotease, chymotrypsin-like protease in the midgut of the6th instar larvae of Mythimnaseparata and Agrotis ypsilon among the three kinds of periplocosides compounds, take serineproteinase inhibitor PMSF as agent control. To explore the insecticidal mechanism ofinsecticidal periplocosides compounds, the trypsin-like protease in the midgut of the6th instarlarvae of M. separata were purification, the related properties of purified trypsin-like proteasewere assayed, and the effects on purified trypsin-like protease of M. separata of the threeperiplocosides compounds were studied. The main results were as follows:1. Periplocoside P and T displayed strongly activated activity on trypsin-like protease,especially the weakly alkaline trypsin-like protease in the midgut of M. separata larvae. Theactivation ratio were167.78%and277.55%after2h of treatment, respectively, and theactivation time last up to8h and24h, respectively. Treatment by periplocoside P and T after2h, the trypsin-like protease activity in the midgut of A. ypsilon was weakly activated, whileother time after treatment, enzyme activity were inhibited, but the inhibition ratio were low.Periplocoside P and T showed no obvious effect on trypsin-like protease andchymotrypsin-like protease activity in the midgut of A. ypsilon.Periplocoside E displayed inhibition effects on the trypsin-like protease activity in themidgut of M. separata larvae, while no obvious effect on that of A. ypsilon. Periplocoside Eshowed no obvious effect on chymotrypsin-like protease activity in the midgut of M. separataand A. ypsilon. 2. The molecular masses(MM) of purified trypsin from M. separata midgut was25,078Da. IP of purified trypsin showed to be7.0. The optimum temperature of purified trypsin werefound to be45-50℃. The purified trypsin was found to be heat stable over a wide range oftemperature(<45℃). The maximum activity for purified trypsin-like of M. separata wasrecorded at pH10.0. The pH-stability were shown at the pH rang8.5-10.5. The Km value forBApNA hydrolysis was0.18±0.0045mmol/L for purified trypsin of M. separata, and the Kcatvalue was86.25s~-1.3. Periplocoside P and T displayed weakly activated activity on the purified trypsin-likefrom the midgut of M. separata larvae, which didn't show concentration dependence. Therewas no significant activity difference on purified trypsin-like from the midgut of M. separatalarvae between periplocoside P and T. Periplocosides E showed no obvious effect on purifiedtrypsin-like, the dependence of concentration were not observed.All of the results showed that the strong activation of trypsin-like in the midgut of M.separata may be one of the factors that periplocoside P and T cause toxicity to insects.