Objective: To investigate the physiological characteristics of Bacillus thuringiensis subspecies israelensis (Bti) with double mutations of cwlE and sigK genes and to assess the activity of Bti against larvae of Culex pipiens pallens under different external factors, so as to provide the theoretical evidence for the use of engineered bacteria of Bti for effective mosquito control.
Methods: B. thuringiensis wild-type strain Bt-59 and Bt-59 strain with cwlE mutation [Bt-59 (ΔcwlE)] were cultured in nutrient broth media for 24 hours, and Bt-59 strains with sigK mutation [Bt-59 (ΔsigK)] and double mutations of cwlE and sigK [Bt-59 (ΔcwlE-sigK)] were cultured in nutrient broth media for 48 hours. Then, 5 μL of culture media were transferred to glass sides, and cell morphology and mother cell lysis were observed under an optical microscope. The optical densities of Bti strain culture media were measured at different time points of culture, and the growth curves of Bt-59, Bt-59 (ΔcwlE), Bt-59 (ΔsigK), and Bt-59 (ΔcwlE-sigK) strains were plotted. The differences in carbon source metabolism of four Bti strains were analyzed using the Biolog microplate culture method, and the metabolic activity of these strains was estimated with average well color development (AWCD). The fermentation media of these four Bti strains were diluted into final concentrations of 2.000, 1.000, 0.500, 0.250, and 0.125 μL/L, and the median lethal concentrations (LC50 values) of these four strains against the third instar larvae of Cx. pipiens pallens were determined. In addition, the fermentation media of Bti strains were processed as follows: pH adjusted to 5, 7 and 9; treated at 30, 40 ℃ and 50 ℃ for 12 hours; and exposed to irradiation with ultraviolet lights for 0 hour and 6 hours. Then, 20 third instar larvae of Cx. pipiens pallens were exposed to the above processed fermentation media at a final concentration of 1 μL/L in 200 mL of water at 26 ℃ for 24 hours, and the mosquito mortality was estimated to evaluate the effects of pH, temperature and ultraviolet irradiation on the larvicidal activity of four Bti strains.
Results: The growth curves of the Bt-59 strain and its mutants shared a similar changing trend, and both experienced a stable phase 6 hours post-culture. Both spores and crystal proteins were found in Bt-59 and Bt-59 (ΔcwlE) cells, and only crystal proteins were found in Bt-59 (ΔsigK) and Bt-59 (ΔcwlE-sigK) cells. No lysis was found in the cell wall of the Bt-59 (ΔcwlE-sigK) strain, and the crystal protein was embedded in the mother cell. Biolog microplate culture assay showed that the AWCD values of four Bti strains showed a similar changing trend over time, and 33 carbon sources were found to be metabolized by all of the four strains, including dextrin, D-maltose and D-trehalose. The LC50 values of the fermentation media of Bt-59, Bt-59 (ΔcwlE), Bt-59 (ΔsigK), and Bt-59 (ΔcwlE-sigK) strains were 0.60, 0.51, 0.70 μL/L and 0.72 μL/L against Cx. pipiens pallens, respectively. The adjusted mortality of larval Cx. pipiens pallens reduced by 76.60%, 76.00%, 66.67%, and 0 following exposure to the fermentation media of Bt-59, Bt-59 (ΔcwlE), Bt-59 (ΔsigK), and Bt-59 (ΔcwlE-sigK) strains at a pH of 5 relative to at a pH of 7, and the adjusted mortality reduced by 49.02%, 51.06%, 36.36%, and 4.44% following 6-hour exposure to ultraviolet irradiation relative to 0-hour exposure, while the adjusted mortality was 68.33% to 83.33% following treatment with the fermentation media of four Bti strains at different temperatures.
Conclusions: Bt-59 (ΔcwlE-sigK) strains do not generate spores, and the absence of cwlE and sigK does not affect the growth, carbon source metabolism, and larvicidal activity of Bti strains against larval Cx. pipiens pallens. Cell wall embedding of Bt-59 (ΔcwlE-sigK) strains may protect larvicidal crystal proteins of Bti strains from external environmental factors, including ultraviolet irradiation, and pH alteration.
［摘要］ 目的观察cwlE和sigK双突变苏云金杆菌以色列亚种 (Bacillus thuringiensis subspecies israelensis, Bti) 生理特性及其在不同外界因素影响下对淡色库蚊幼虫的杀灭活性, 为应用持效Bti工程菌杀蚊提供实验依据。方法在营养肉汤培养基中, 将苏云金杆菌野生株Bt-59和cwlE 突变株［Bt-59 (ΔcwlE) 株］培养24 h、将sigK 突变株［Bt-59 (ΔsigK) 株］及cwlE和sigK 双突变株［Bt-59 (ΔcwlE-sigK) 株］培养48 h后, 各取5 μL培养液分别置于载玻片上, 光学显微镜下观察细胞形态及母细胞裂解情况。通过测定不同培养时间菌株培养液的光密度, 绘制Bt-59、Bt-59 (ΔcwlE) 、Bt-59 (ΔsigK) 和Bt-59 (ΔcwlE-sigK) 株生长曲线。利用Biolog微平板培养技术分析4种菌株碳源代谢差异, 计算平均颜色变化率 (average well color development, AWCD) 以评价其代谢活性。将以上4种菌株发酵液经梯度稀释至终浓度分别为2.000、1.000、0.500、0.250、0.125 μL/L, 以3龄淡色库蚊幼虫作为供试蚊虫, 测定菌株的24 h致死中浓度 (median lethal concentration, LC50) 。分别取菌株发酵液进行如下处理：调节pH值至5、7和9, 置于30、40、50 ℃下处理12 h, 经紫外灯照射0、6 h, 随后分别取终浓度为1 μL/L的上述菌株发酵液至200 mL水中, 加入20只3龄淡色库蚊幼虫, 并置于26 ℃恒温培养箱中, 24 h后计算蚊虫死亡率以评价pH值、温度以及紫外线照射对上述4种菌株杀灭淡色库蚊幼虫效果的影响。结果Bt-59及其突变株生长曲线变化趋势基本一致, 均在培养6 h 后进入稳定期。Bt-59 和Bt-59 (ΔcwlE) 株细胞内均含有芽胞和晶体蛋白; Bt-59 (ΔsigK) 和Bt-59 (ΔcwlE-sigK) 株不产芽胞, 细胞内只有晶体蛋白, 其中Bt-59 (ΔcwlE-sigK) 株细胞壁不发生裂解, 形成的晶体蛋白包埋在母细胞中。4种菌株的AWCD值随时间变化趋势基本一致, 均能代谢糊精、D-麦芽糖、D-海藻糖等33种碳源。Bt-59、Bt-59 (ΔcwlE) 、Bt-59 (ΔsigK) 和Bt-59 (ΔcwlE-sigK) 株发酵液对淡色库蚊幼虫的24 h LC50值分别为0.60、0.51、0.70 μL/L和0.72 μL/L。与pH = 7条件下相比, 在pH = 5条件下, 经Bt-59、Bt-59 (ΔcwlE) 、Bt-59 (ΔsigK) 和Bt-59 (ΔcwlE-sigK) 株发酵液处理后的淡色库蚊幼虫校正死亡率分别降低了76.60%、76.00%、66.67%、0; 与紫外线照射0 h相比, 经紫外线照射6 h后, 经Bt-59、Bt-59 (ΔcwlE) 、Bt-59 (ΔsigK) 和Bt-59 (ΔcwlE-sigK) 株发酵液处理后淡色库蚊幼虫校正死亡率分别降低了49.02%、51.06%、36.36% 和4.44%; 在不同温度下, 经4 种菌株发酵液处理后的淡色库蚊幼虫校正死亡率在68.33% ~ 83.33%。结论Bt-59 (ΔcwlE-sigK) 株不产芽胞, 且cwlE和sigK缺失不影响Bti生长、碳源代谢和杀灭淡色库蚊幼虫效果。Bt-59 (ΔcwlE-sigK) 株细胞壁包埋可保护Bti杀虫晶体蛋白免受紫外线照射、pH值改变等外界环境因素的影响。.
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