Abstract: Objective　To assess the stability of the molluscicidal activity of the fermentation broth supernatant of Bacillus indicus F19, so as to provide insights into development of novel molluscicides derived from this isolate. Methods　Healthy Oncomelania hupensis snails free from Schistosoma japonicum infections and acclimated in the laboratory for 7 days were selected. The fermentation broth supernatant of B. indicus F19 was prepared. For each treatment condition, four experimental groups and one control group were established, with 10 mL of the fermentation broth supernatant each group. O. hupensis snails were exposed to different pH values (3, 6, 9 and 12), temperatures (40, 60, 80 ℃ and 100 ℃), and ultraviolet (UV) exposure durations (1, 2, 3 h and 4 h). Then, the supernatant was diluted with dechlorinated water to concentrations of 50%, 10%, and 5% (v/v), and 10 mL of each dilution solution was added to three separate tubes, with an equal volume of dechlorinated water as a blank control. Twenty O. hupensis snails were transferred to each tube, immersed for 48 h and 72 h, and collected for viability assessment using the shell⁃tapping method. The mortality of O. hupensis snails was calculated and compared among dilution groups and the blank control group to evaluate the molluscicidal activity of the fermentation broth supernatant of B. indicus F19.  Results　There was a significant difference in the mortality of O. hupensis snails immersed in 5% fermentation broth supernatants of B. indicus F19 for 48 h at various pH levels ([χ2] = 11.556, P < 0.05), and the mortality reduced to 13.33% (8/60) in the experimental groups at a pH value of 12, which was significantly lower than in the blank control group [33.33% (20/60)] ([χ2] = 6.708, P < 0.05). There was a significant difference in the mortality of O. hupensis snails immersed in 10% fermentation broth supernatants of B. indicus F19 for 72 h at various pH levels ([χ2] = 31.691, P < 0.001), and the mortality reduced to 71.67% (43/60) and 65.00% (39/60) in the experimental group at pH values of 9 and 12, which was significantly lower than in the blank control group [100.00% (60/60)] ([χ2] = 8.571 and 12.110, both P values < 0.01). The mortality of O. hupensis snails both reduced to 23.33% (14/60) following immersion in 5% fermentation broth supernatants of B. indicus F19 heated in a water bath at 80 ℃ and 100 ℃ for 48 h, which was lower than that of unheated O. hupensis snails [33.33% (20/60)] and snails heated in a water bath at 40 ℃[35.00% (21/60)] and 60 ℃ [33.33% (20/60)], and there was no significant difference in the mortality of O. hupensis snails among groups ([χ2] = 3.898, P > 0.05). The mortality of O. hupensis snails was 68.33% (41/60), 66.67% (40/60), 65.00% (39/60), and 68.33% (41/60) following immersion in fermentation broth supernatants heated in a water bath at 40, 60, 80 ℃ and 100 ℃ for 72 h, which was all lower than that of snails in the control group [71.70% (43/60)]; however, there was no significant difference seen in the snail mortality among groups ([χ2] = 0.674, P > 0.05). The mortality of O. hupensis snails was 73.33% (44/60), 71.67% (43/60), 71.67% (43/60), and 75.00% (45/60) following 48 h immersion in 10% fermentation broth supernatants of B. indicus F19 irradiated by UV for 1, 2, 3 h and 4 h, 33.33% (20/60), 31.67% (19/60), 35.00% (21/60), and 33.33% (20/60) following 48 h immersion in 10% fermentation broth supernatants of B. indicus F19 irradiated by UV for 1, 2, 3 h and 4 h, and 71.67% (43/60) and 33.33% (20/60) following 48 h immersion in 10% fermentation broth supernatants of B. indicus F19 without UV irradiation, and no significant differences were found among groups ([χ2] = 0.229 and 0.150, both P values > 0.05). In addition, the mortality of O. hupensis snails was 76.67% (46/60), 71.67% (43/60), 70.00% (42/60), and 76.67% (46/60) following 72 h immersion in 5% fermentation broth supernatants of B. indicus F19 irradiated by UV for 1, 2, 3 h and 4 h, and 71.67% (43/60) in the control group, and there was no significant difference in the snail mortality among groups ([χ2] = 1.193, P > 0.05). Conclusion　 The molluscicidal action of the fermentation broth supernatants of B. indicus F19 is tolerant to high temperature and UV irradiation; however, the activity is attenuated under alkaline conditions.

Key words: Oncomelania hupensis, Schistosomiasis, Bacillus indicus F19, Molluscicidal activity, Stability 

摘要： 目的　评价印度芽孢杆菌F19发酵上清液灭螺活性的稳定性，为后续开发该菌株相关灭螺药物提供参考。方法　选取血吸虫感染阴性的健康活体钉螺作为实验对象，于实验室饲养7 d。制备印度芽孢杆菌F19发酵上清液，每种处理条件均设置4个实验组和1个对照组，每组各取10 mL发酵上清液。将发酵液调节至不同酸碱度（pH值为3、6、9、12）、加热至不同温度（40、60、80、100 ℃）以及经紫外光照射不同时长（1、2、3、4 h）后，分别采用去氯水配置成稀释体积比为50%、10%及5%的溶液。各取10 mL上述稀释液分别加入3支试管中，设置等体积去氯水作为空白对照组，每管加入20只钉螺，采用浸杀法浸泡钉螺48、72 h后取出，用敲击法判断钉螺死活，计算并比较各组稀释液及空白对照组钉螺死亡率以评估灭螺活性。结果　将稀释体积比为5%的发酵上清液调节至不同酸碱度，浸杀钉螺48 h后，各组钉螺死亡率差异有统计学意义（[χ2] = 11.556，P < 0.05）；上清液pH值为12时钉螺死亡率降至13.33%（8/60），显著低于未经酸碱调节处理的对照组［33.33%（20/60）］（[χ2] = 6.708，P < 0.05）；将稀释体积比为10%的发酵上清液调节至不同酸碱度，浸杀钉螺72 h后，各组钉螺死亡率差异亦有统计学意义（[χ2] = 31.691，P < 0.001）；上清液pH值分别为9、12时钉螺死亡率分别为71.67%（43/60）和65.00%（39/60），均低于对照组钉螺死亡率［100.00%（60/60）］（[χ2] = 8.571、12.110，P均< 0.01）。稀释体积比为5%的发酵上清液经80 ℃和100 ℃水浴加热，浸杀钉螺48 h后，钉螺死亡率均降至23.33%（14/60），略低于未经高温处理的对照组［33.33%（20/60）］和40 ℃［35.00%（21/60）］、60 ℃［33.33%（20/60）］水浴加热处理组，但各组差异无统计学意义（[χ2] = 3.898，P > 0.05）；经40、60、80、100 ℃加热处理的上清液浸杀72 h后，钉螺死亡率分别为68.33%（41/60）、66.67%（40/60）、65.00%（39/60）、68.33%（41/60），对照组为71.70%（43/60），各组差异亦无统计学意义（[χ2] = 0.674，P > 0.05）。稀释体积比为10%、5%的发酵上清液经紫外光照射1、2、3、4 h，浸杀钉螺48 h后，各处理组钉螺死亡率分别为73.33%（44/60）、71.67%（43/60）、71.67%（43/60）、75.00%（45/60）和33.33%（20/60）、31.67%（19/60）、35.00%（21/60）、33.33%（20/60），未经紫外光照射的对照组钉螺死亡率分别为71.67%（43/60）和33.33%（20/60），各组死亡率差异均无统计学意义（[χ2] = 0.229、0.150，P均> 0.05）。稀释体积比为5%的发酵上清液经紫外光照射1、2、3、4 h，浸杀钉螺72 h后，各处理组钉螺死亡率分别为76.67%（46/60）、71.67%（43/60）、70.00%（42/60）、76.67%（46/60），对照组钉螺死亡率为71.67%（43/60），各组差异亦无统计学意义（[χ2] = 1.193，P > 0.05）。结论　印度芽孢杆菌F19发酵液的灭螺活性具有耐高温和耐紫外光的特性，但在碱性条件下其灭螺活性减弱。

关键词: 钉螺, 血吸虫病, 印度芽孢杆菌F19, 灭螺活性, 稳定性