| Small brown planthopper(SBPH), Laodelphax striatellus, belongs to Hemiptera Delphacidae, which is one of the important pests of rice in Asia. SBPH damages the rice not only by directly sucking the plant juices but also by transferring a variety of plant virus rice, caused a serious loss of rice production. The eco-friendly pest control measures should be developed to control this notorious pest. Bacillus thuringiensis(Bt) is a gram positive bacteria, can produce a series of highly specific insecticidal crystal proteins(ICPs), however, little has been reported about the ICPs with high insecticidal activity on the planthoppers, aphids and other Hemiptera insect pests. ST-1, ST-2 proteins were isolated and purified from Bacillus thuringiensis strain IPPBI0TSUC1012, which showed a high insecticidal activity to SBPH nymphs. In this study, pathological changes of midgut epithelial cells of SBPH were observed after feeding with ST-1, ST-2 proteins. Investigation of localization of ST-1, ST-2 proteins in the midgut epithelial cells of SBPH and preliminarily studies of the receptors of ST-1, ST-2 proteins provide a theoretical basis for studying the toxicological mechanism of the ST-1, ST-2 proteins. These studies should be great helpful for the screening of effective insecticidal protein of hemipterous insects and enlarging the insecticides spectrums of insecticidal Bt proteins. The main results are as follows:1. The 3rd instar nymph of the SBPH were fed with artificial diets containing ST-1, ST-2 proteins(5μg/ml) and Cry1 Ac protein(5μg/ml). The pathological changes in the midgut were observed by transmission electron microscope(TEM). The results showed that the microvilli of SBPH nymphs midgut epithelial cells were degraded, along with cytoplasmic within shrinkage and vacuoles, inner cristae of mitochondria swelled, endoplasmic reticulum was broken into small vesicles, nuclear membrane swelled, chromatin condensation. The damage became serious with the increase of feeding time. In contrast, the microvilli and perimicrovillar matrix of SBPH were healthy, but epithelial cells appeared different degree of damage when feeding with artificial diets contained Cry1 Ac protein. SBPH fed on artificial diet had no obvious pathological changes.2. The distribution of ST-1, ST-2 proteins and the location of ST-1, ST-2 proteins receptors in the midgut of the 3rd instar SBPH nymphs feeding on an artificial diets with ST-1, ST-2 proteins(5 μg/ml) were studied by using Immunohistochemical method with a polyclonal antibody against ST-1 and ST-2 proteins. Western blot results showed that the preparation of polyclonal antibody dilution ratio can reach 20000 times, and the specificity is high. The results of immunohistochemistry showed that the ST-1 and ST-2 proteins mainly distributed in SBPH midgut epithelial cells on the surface of BBMV and perimicrovillar matrix. The whole tissue immune result showed that, ST-1, ST-2 proteins were distributed in the midgut epithelial cells of SBPH and mainly concentrated on the surface of the cell membrane. ST-1 and ST-2 proteins is scattered in the muscle layers of the midgut, suggesting that ST-1, ST-2 proteins may exist non specific binding in the midgut muscles.3. In order to explore the possible receptor types of ST-1, ST-2 proteins in the midgut of SBPH on BBMV, ST-1, ST-2 proteins were incubated and combined with BBMV before and after the BBMV treated with methyl-β-cyclodextrin(MbCD) and phosphatidylinositol-specific phospholipase C(PI-PLC). The results showed that the binding ability of ST1 and ST-2 proteins with BBMV did not decreased significantly after treated with MbCD and PI-PLC, suggesting that the receptors of ST-1 and ST-2 proteins are not the cholesterol and GPI anchoring proteins in SBPH. The insecticidal mechanism of the proteins ST-1 and ST-2 may differ from the pore-forming and an unknown mechanism should exist, which is worthy of further investigations. |
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