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Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis (Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins (Cry2A, Cry1C, Cry1Aa, and Cry1Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid (EDTA), PMSF, tosyl phenylalanine chloromethyl ketone (TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). EDTA could significantly inhibit the degradation of Cry2A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1C and Cry1Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.

Higher boll worm survival rates were detected after high temperature presented during square period in Bt cotton. The objective of this study was to investigate the effects of high temperature level on the Bt efficacy of two different types of Bt cotton cultivars at squaring stage. During the 2011 to 2013 cotton growth seasons, high temperature treatments ranged from 34 to 44°C in climate chambers, and field experiments under high temperature weather with various temperature levels were conducted to investigate the effects of the high temperature level on square Bt protein concentration and nitrogen metabolism. The climate chamber experiments showed that the square insecticidal protein contents reduced after 24 h elevated temperature treatments for both cultivars, whereas significant declines of the square insecticidal protein contents were detected at temperature >38°C, and only slightly numerical reductions were observed when temperature below 38°C. Similar high temperature responses were also observed at the two field experimental sites in 2013. Correspondingly, high temperature below 38°C seems have little effect on the square amino acid concentrations, soluble protein contents, glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) activities as well as protease and peptidase activities; however, when the temperature was above 38°C, reduced soluble protein contents, enhanced amino acid concentrations, decreased GPT and GOT activities, bolstered protease and peptidase activities in square were detected. In general, the higher the temperature is (>38°C), the larger the changes for the above compound contents and key enzymes activities of the square protein cycle. The findings indicated that the unstable insect resistance of the square was related to high temperature level during square stage.

Expression of insecticidal protein for transgenic Bacillus thuringiensis (Bt) cotton is unstable and related to nitrogen metabolism. The objective of this study was to investigate the relationship between leaf carbon nitrogen ratio (C/N) and insecticidal efficacy of two Bt cotton cultivars. C/N ratio and Bt protein content were both measured at peak square period and peak boll period respectively under 5-7 d high temperature and different nitrogen fertilizer rates on the Yangzhou University Farm and the Ludong Cotton Farm, China. All plants were grown in field. The results showed that the C/N ratio enhanced slightly and the Bt protein content remained stable at peak square period, but significant increases for the C/N ratio and decreases markedly for the leaf Bt protein concentration were detected at the peak boll period. The similar patterns at the two growth periods were found for the leaf C/N ratio and Bt protein content by different N fertilizer treatments. When nitrogen rate was from 0 to 600 kg ha−1, the C/N ratio was reduced by 0.017 and 0.006 for Sikang 1 and Sikang 3 at peak square period, compared to the 1.350 to 1.143 reduction for Sikang 1 and Sikang 3 at peak boll period, respectively. Correspondingly, the leaf Bt protein contents were bolstered by 2.6-11.8 and 26.9-36.9% at the two different growth periods, respectively. The results suggested that enhanced C/N ratio by high temperature and nitrogen application may result in the reduction of insectiocidal efficacy in Bt cotton, especially in peak boll period.