| . Plant secondary metabolites result from the interaction between plants and environment (life and non-life) during the long period of evolution in plants. Currently, the discovery of new insecticidal compounds from plant secondary metabolites and subsequently using them as the lead-compounds for further modification has been one of the important ways for the research and development of new pesticides. In this dissertation, fraxinellone, isolated from the root bark of Diatamnus dasycarpus Turcz., Family Rutaceae, was firstly studied its insecticidal activities. Then, the action mechanism of fraxinellone against Mythimna separata was studied by combination of the poisoned symptoms, histopathology research (light micrograph and transmission electron microscopy), and biochemistry methods. The results were as follows:1. Fraxinellone showed the delayed insecticidal activity. The highest mortality of the treated insect larvae was usually reached after 2-3 days. The poisoned insects exhibited typical stomach toxicity symptom, i.e. vomit and diarrhea, and lost much water to death.2. Fraxinellone possessed high antifeeding activity. The antifeeding rates of fraxinellone at 5.0, 10.0, and 20.0 mg/mL against 5th instar larvae of M. separata were all more than 60% in 2 h. After treatment of 24 h, the medium antifeedant concentration (AFC50) values against 5th instar larvae of M. separata, 2nd instar larvae of Plutella xylostella and 2nd instar larvae of Agrotis ypsilon were 11.47,7.78 and 13.40 mg/mL, respectively. After treatment of 24 h, the antifeeding rates against 2nd instar larvae of Pieris rapae, 4th instar larvae of Helicoverpa armigera, and 3rd instar larvae of A. ypsilon at 20.0 mg/mL were 78.00%, 73.20%, and 27.83%, respectively. After treatment of 48 h, the antifeeding rate against 3rd instar larvae of P. rapae was 80.80%. Besides that, it also showed antifeeding activity against 5th instar larvae of A. ypsilon and final instar larvae of M. separata.3. Through typical stomach toxicity methods, fraxinellone showed strong stomach-poison activity against 5 Lepidoptera insects, e.g., M. separata, P. rapae, P. xylostella, H. armigera, and A. ypsilon. At 20.0 mg/mL, fraxinellone displayed the strongest insecticidal activity against M. separata among of all the tested insects. But fraxinellone showed weak touch-poison effects against the tested insects. The medium lethal concentration (LC50) values of fraxinellone against 2nd instar larvae of P. xylostella, 3rd instar larvae of M. separata, and 4th instar larvae of Culux pipiens pallens were 7.10, 15.95, and 6.38×10-2 mg/mL, respectively. The medium lethal dosage (LD50) value of fraxinellone against 5th instar larvae of M. separata was 135.70μg/g.4. Fraxinellone had certain effect on the development of larvae of M. separata. After treated by 5.0,10.0 and 20.0 mg/mL, the development duration from 5th to 6th instar larvae of M. separata was prolonged to 1, 2 and 3 days, respectively, and three corrected mortalities were all large than 50%. At 10.0 and 20.0 mg/mL, the growth inhibition rates against M. separata were all large than 30%. The pupation rate and the eclosion rate of M. separata decreased along with the increasing concentration. Moreover, fraxinellone could result in abnormal pupae.5. Fraxinellone showed strong inhibition activity against egg hatching of M. separata. After treated by 5.0, 10.0, and 20.0 mg/mL, the egg hatching durations were lagged 3-4 days, and only 50% egg hatched. Furthermore, only a few newly emerged larvae developed to 2nd instar larvae, and the growth inhibition rates were 73.94%, 86.52%, and 85.43%, respectively.6. It is positive relationship between the insecticidal activity of fraxinellone and temperature, i.e., the high temperature (32 oC) led to higher insecticidal activity than the low ones (12 oC and 22 oC).7. Through dissection, the alimentary tract of M. separata had no obvious damage. But the midguts of insects, which lost their water heavily, were out of flexible, and got fragile. Under light micrograph observation, after treated by fraxinellone at 20.0 mg/mL, the transverse section of M. separata midgut exhibited irregular shape, part of the epithelial cells were discontinuous, the cavity of globet cell turned larger, the nucleus of columnar cell changed swollen, the peritrophic membrane almost could not be seen, and the midgut contents were distributed in confusion in the gut lumen.8. Through transmission electron microscopy (TEM), the organelles in M. separata midgut were found to be largely affected after treated by fraxinellone at 20.0 mg/mL. The ultrastructure of no symptom larvae of M. separata almost the same as that of control, but only the lysosome body emerged in midgut cell. When the treated M. separata lost water, the microvilli and the chromatin were well distributed, but the mitochondria swollen irregularly, their cristae fractured and dissolved, and the RER dilated. Along with time, some treated insects lost water heavily. Under subcellular morphological observation, the cytoplasma had fewer electrons dense, the chromatin of nuclei became concentrated, and a lot of secondary lysosome emerged in cytoplasma. The cristae of some mitochondria disappeared, and got vacuolated, but the two-layer membrane remained intact, and the RER still dilated. The globet cell wall invaginated, and the inner organelle disordered, even some of the epithelium cell contents flowed into the midgut lumen. Fraxinellone could let microvilli rupture or incline, even partially fall off.9. By compared with the midgut digestive enzyme activities of the control M. separata, fraxinellone inhibited theα-amylase and lipase activities of 5th and 6th instar larvae of M. separata. On the contrary, fraxinellone increased the level of protease activity of the 5th and 6th instar larvae. That is, protease perhaps played a key role in the catabolism process of fraxinellone in the midgut. Through further studies on specific proteases in the 5th instar larvae of M. separata, it showed the active alkaline trypsin-like enzyme (BAPNA as the substrate) and the chymotrypsin-like enzyme (BTEE as the substrate) activities were declined along with time after treated by fraxinellone, but the activity of weak alkaline trypsin-like enzyme (TAME as substrate) was increased. To the 6th instar larvae of M. separata, the active and weak alkaline trypsin-like enzyme activities were both firstly decreased, and then were increased. But the chymotrypsin-like enzyme activity was inhibited, and its activity got lower along with time. On the contrary, to the 5th instar larvae of A. ypsilon, the activity of chymotrypsin-like enzyme was enhanced obviously, while the two trypsin-like enzyme activities were almost inhibited.10. Carboxylesterase (CarE) and glutathione S-transferase (GST) played an important role in the metabolism of fraxinellone in the midgut of M. separata. Fraxinellone could increase the activities of CarE and GST of 5th and 6th instar larvae of M. separata to some extent, but the activities of NADPH-P450 reductase and O-demethylase were reduced.
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