Study On Molecular Mechanisms Of Mating Disruption Of Sitotroga Cerealella By Active Substances From Garlic Essential Oil

The Angoumois grain moth, Sitotroga cerealella Olivier(Lepidoptera: Gelechiidae), is a serious stored-grain pest worldwide, especially on stored wheat and rice. In some areas in China, more than 40% of the total losses in stored grains were caused by this pest alone. At present, chemical controls are still the most effective tools for stored-product insect management systems. However, the preference of larvae to feed inside grains, which provides the moth pest additional protection from direct contact with insecticides, makes it difficult to warrant full success by chemical treatments. Moreover, increasing resistance to some chemical insecticides, as well as the presence of insecticidal residues in grains after chemical treatments, highlights the need for alternatives to conventional chemical insecticides in order to control the most significant stored-grain pest.Our previous studies demonstrated that garlic essential oil and its two major components, diallyl disulfide(DADS) and diallyl trisulfide(DATS), can cause reduction in the number of eggs deposited by S. cerealella females. However, the mechanism for the reduction in egg number caused by garlic substances remains unclear. Another researcher in our team found that these garlic active substances could cause reduction in mating rate and frequency of the female moth, indicating that these substances possess great potential in regulating moth?s mating behavior, resulting in ovipositional inhibition. Considering the molecular mechanisms by which moths sense and recognize environmental odors(the binding of odors with odorant-binding proteins(OBPs) is the first step of odorant reception), it is possible that OBPs play an important role in the mating disruption of S. cerealella by garlic active substances. Therefore, the aim of the present study is to determine the targets of mating disruption of S. cerealella by active substances of garlic essential oil and explore its molecular mechanisms. The results are shown as follows: 1. Effects of garlic active substances on the olfactory behavior of adult S. cerealellaA Y-tube olfactometer bioassay was used to test the effects of the garlic active substances on the olfactory behavior of S. cerealella adults. These results showed that, at a dose of 10 μL, both male and female moths displayed no difference in choice behavior between DATS and clean air, indicating no directional bias in the system. Additionally, synthetic sex pheromone of female moth(7Z, 11E-hexadecadien-1-ol acetate, HDA) had a strong attractive effect on males(RI =- 0.49; P < 0.001), and had high behavioral repellent or deterrent activity against females(RI = 0.64; P < 0.000001). When mixed with DATS, HDA showed no significantly attractive effect on males(RI =-0.10; P > 0.05) and a much weaker repellent efficacy on females(RI = 0.31; P < 0.05) compared with the treatment of HDA alone. In addition, behavioral responses to DADS were similar to what was observed for DATS. These results suggested that garlic substances interrupted the moths? responses to the sex pheromone. 2. Ultrastructural observations on antennal sensilla of S. cerealellaThe ultrasturcture of the antennal sensilla of S. cerealella were investigated by scanning electron microscopy and transmission electron microscopy. There were seven types of sensilla on both sex antennae, including sensilla trichodea, sensilla basiconica, sensilla coeloconica, sensilla styloconica, sensilla auricillica, sensilla squamous and B?hm bristles, of which the sensilla trichodea were significantly more abundant on male antennae than those of females. Based on the literature, the possible functions of the antennal sensilla in S. cerealella were discussed according to their morphology, fine structures, distribution and numbers. These results can help us to understand the olfactory mechanism of chemical communication between males and females, and may be useful to lay the foundation for future behavioral studies. 3. Cloning and expression patterns of ScerOBPsThree novel OBP genes, ScerGOBP1(GenBank: KF513152), ScerGOBP2(GenBank: KF513150) and ScerPBP(GenBank: KF513151), were cloned from S. cerealella antennae by homology cloning and RACE strategy. The results of sequence analysis and quantitative real-time PCR showed that all three ScerOBPs shared the characteristics of classic OBP family, namely, they were predicted to be of small size, low pI and hydrophilic, predicted to be secreted due to presence of signal peptide sequences, predicted to encode six cysteine residues at conserved positions, and expressed specifically in antennae. ScerPBP expression was malebiased(P < 0.05), but ScerGOBP1 and ScerGOBP2 were similar between sexes(P > 0.05). In addition, ScerPBP expression was significantly higher than ScerGOBP1 and ScerGOBP2 in male antennae. These results will lay the foundation for the following study on the structure and function of ScerOBPs. 4. Expression, purification and binding characteristics of ScerOBPsRecombinant protein of ScerGOBP1, ScerGOBP2 and ScerPBP were produced using a prokaryotic expression system and purified using affinity chromatography column(GSTrapTM 4B). The binding properties of two garlic substances(i.e. DADS and DATS), and S. cerealella sex pheromone(HDA) with ScerOBPs were investigated by fluorescence competitive binding assay. The results indicated that DATS had similar or even higher binding affinity to ScerPBP than HDA, but was bound by ScerGOBP1 and ScerGOBP2 with no or poor affinity. And, binding affinities to DADS were similar to what was observed for DATS. These results suggested that high affinity of ScerPBP with these garlic substances provides possibilities for pheromone-reception interference by these compounds, which will disturb courtship behavior. 5. Homology modeling and molecular docking of ScerPBPThe 3D structure of ScerPBP was constructed using homology modeling, and the binding modes between the ligands and ScerPBP were searched by molecular docking. 3D structure showed that ScerPBP consists of 6 α-helixes, which form a binding pocket. In addition, molecular docking indicated that both DADS/DATS and HDA could be successfully docked into the active site of Scer PBP and shared the same hydrophobic binding cavity, further suggesting that garlic active substances probably acted as inhibitors of ScerPBP, and competed with HDA for ScerPBP binding.In a word, all the research work will help us to know the molecular mechanism on the mating disruption of S. cerealella by active substances from garlic essential oil, that is, garlic substances compete with the sex pheromone of S. cerealella for ScerPBP binding, which reduce the binding opportunity of ScerPBP in male antennae with the sex pheromone released by females, and thus disrupting the moths? mating behavior and ultimately resulting in ovipositional inhibition. These results will reveal the molecular mechanism on the moth how to sense and recognize environmental odors and produce corresponding behavioral responses, and also will help to find a new target and a new idea for controlling these pests.