Perception Mechanism Of The Navel Orangeworm, Amyelois Transitella (Walker) To Plant Volatile Chemicals

The navel orangeworm, Amyelois transitella (Walker), is an important pest on pistachio, almonds, and walnuts in California, USA. It bores into and feeds the nuts both in the orchard and in the process of producing and store, frequently causing serious damage. It is difficult to control because of the bore-feeding characteristic. Now, the population control method is only by orchard cleaning after harvest and insecticide spraying when the insect population is high. Therefore, it is important to study the semiochemical detection mechanism, to identify the relationship between the insect and its hosts, to explore insect chemical ecological method for successful A. transitella population management.Here, two general odorant binding proteins from A. transitella, AtraGOBP1 and AtraGOBP2, were expressed and purified, and the interactions between these two GOBPs and plant volatile chemicals were also studied. At the same time, the electrical response of A. transitella antennae elicited by plant volatile chemicals was examined with electroantennography, and the perception mechanism was also discussed. Based on these researches, the effect of traps made with plant volatile chemicals on oviposition behavior was tested in both room and field to use these chemicals to monitor and control this pest. The main results are shown as follows:1. AtraGOBP1 and AtraGOBP2 protein were expressed and purified successfully in the transformed bacteria, Escherichia coli, which contains the project protein DNA (AtraGOBPl or AtraGOBP2 gene) and incubated in LB medium. The bacteria was harvested by centrifuging at 4500×g, followed by three times of freeze and thaw performance to cause lesion in the bacterial membrane. The protein was extracted with Tris-HCl buffer which followed by high speed centrifuge to remove the cell debris. High purity protein was got after the proteins solution passing through FPLC equipped with DEAE, Q-Seph, MONO Q, and gel filtration columns.2. The results from LC-ESI-MS analysis show that AtraGOBPl gave a molecular mass of 16,897 Da consistent with the calculated molecular mass of 16,903 Da given the formation of three disulfide bridges (expected 16,897 Da). Deconvolution of the mass spectrum from AtraGOBP2 gave a molecular mass of 16,161 Da in close agreement with the calculated molecular mass of 16,166 Da considering the formation of three disulfide linkages (expected, 16,160 Da). Far-ultraviolet circular dichroism spectra from both proteins resembled that of the pheromone-binding protein AtraPBP1, with maximum at ca.193 nm and two minima at 209 and 223 nm. Therefore, both AtraGOBP1 and AtraGOBP2 areα-helical-rich proteins, which is a common feature of moth OBPs. And they experienced a pH-dependence structure conformation change from pH 5.5 to pH 7.3. With cold binding assay method, it was found that both AtraGOBP1 and AtraGOBP2 were able to bind with decanal and nonanal at pH 7, while no obvious binding was detected at pH 5 or in control. For the rest plant volatile chemical, linalool,1-phenylethanol, PAPE and IBAPE, it is difficult to draw conclusion because the high background levels at pH 5 and the control. Competitive fluorescence binding with NPN as a reporter was performed to examine the binding ability of these chemical with these to GOBPs. The results showed that decanal and nonanal displaced NPN at pH 7, in contrast no displacement was found at pH 5 or in the control. None of the rest tested ligands displaced NPN, which means these two GOBPs were unable to bind with these chemicals.4. EAG recordings from live moths showed that both male and female antennae responded to these plant volatile chemicals in a dose-dependent manner. Experiments were also conducted to determine the effect of age on EAG response of female and female to plant volatile chemicals. The result showed that 0 to 4 days old male and female were sensitive to the tested plant volatile chemicals, and there were an obvious sensitivity decrease on day-5 in contrast with previous stages. Combined the information from interaction of the tested ligands and the GOBPs, we got the conclusion that there should be more odorant binding proteins were involved in the semiochemicals transport from environment to odor receptors.5. With the trap made with plant volatile chemical on filter papers with soaked paper towel to keep humidity, the effect of these chemicals on A. transitella oviposition behavior was examined at three different doses (10μl×10μg/μl,50μl×10μg/μl,100μl×10μg/μl). Then 15 males and 10 females were pun inside the nylon cage (60×60×60 cm) to examine the oviposition behavior. The result showed that both linalool and 1-phenylethanol attracted the female to lay egg on the trap. No eggs were found on the trap of other plant volatile chemicals. The bioassay in the field showed that:eggs of A. transitella were found on the trap made with linalool and 1-phenylethanol at dose of 50μl×10μg/μl,100μl×10μg/μl. However, no eggs were found on the trap with dose 10μl×10μg/μl. At the same condition, no eggs were found on the trap of other plant volatile chemicals at all the tested doses.