Abstract: Objective　To evaluate the storage stability of metabolites from actinomycetes Streptomyces nigrogriseolus XD 2⁃7 and the mollcuscicidal activity against Oncomelania hupensis in the laboratory, and to preliminarily explore the mechanisms of the molluscicidal activity. Methods　The fermentation supernatant of S. nigrogriseolus XD 2⁃7 was prepared and stored at -20, 4 ℃ and 28 ℃ without light for 10 d; then, the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The fermentation supernatant was boiled in a 100 ℃ water bath for 30 min and recovered to room temperature, and then the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The pH values of the fermentation supernatant were adjusted to 4.0, 6.0 and 9.0 with concentrated hydrochloric acid and sodium hydroxide, and the fermentation supernatant was stilled at room temperature for 12 h, with its pH adjusted to 7.0; then, the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The fermentation product of S. nigrogriseolus XD 2⁃7was isolated and purified four times with macroporous resin, silica gel and octadecylsilane bonded silica gel. The final products were prepared into solutions at concentrations of 10.00, 5.00, 2.50, 1.25 mg/L and 0.63 mg/L, and the molluscicidal effect of the final productswas tested against O. hupensis following immersion for 72 h, while dechlorination water served as blank controls, and 0.10 mg/L niclosamide served as positive control. The adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels were measured in in O. hupensis soft tissues using high performance liquid chromatography (HPLC) following exposure to the final purified fermentation products of S. nigrogriseolus XD 2⁃7. Results　After the fermentation supernatant of S. nigrogriseolus XD 2⁃7 was placed at -20, 4 ℃ and 28 ℃ without light for 10 d, immersion in the stock solution and solutions at 10⁃ and 50⁃fold dilutions for 72 h resulted in a 100% (30/30) O. hupensis mortality. Following boiling at 100 ℃ for 30 min, immersion in the stock solution and solutions at 10⁃ and 50⁃fold dilutions for 72 h resulted in a 100.00% (30/30) O. hupensis mortality. Following storage at pH values of 4.0 and 6.0 for 12 h, immersion in the fermentation supernatant of S. nigrogriseolus XD 2⁃7 for 72 h resulted in a 100.00% (30/30) O. hupensis mortality, and following storage at a pH value of 9.0 for 12 h, immersion in the fermentation supernatant of S. nigrogriseolus XD 2⁃7 for 72 h resulted in a 33.33% (10/30) O. hupensis mortality ([χ2] = 30.000, P < 0.05). The minimum concentration of the final purified fermentation products of S. nigrogriseolus XD 2⁃7 was 1.25 mg/L for achieving a 100% (30/30) O. hupensis mortality. The ATP level was significantly lower in O. hupensis soft tissues exposed to 0.10 mg/L and 1.00 mg/L of the final purified fermentation products of S. nigrogriseolus XD 2⁃7 than in controls (F = 7.274，P < 0.05), while no significant difference was detected in the ADP level between the treatment group and controls (F = 2.485，P > 0.05). Conclusions　The active mollcuscicidal ingredients of the S. nigrogriseolus XD 2⁃7 metabolites are maintained stably at -20, 4 ℃ and 28 ℃ for 10 d, and are heat and acid resistant but not alkali resistant. The metabolites from S. nigrogriseolus XD 2⁃7 may cause energy metabolism disorders in O. hupensis, leading to O. hupensis death.

Key words: Oncomelania hupensis, Streptomyces nigrogriseolus, ?Secondary metabolite, Molluscicidal effect

摘要： 目的　评价放线菌黑色浅灰链霉菌（Streptomyces nigrogriseolus XD 2⁃7）代谢产物储存稳定性及其分离纯化产物实验室杀螺活性，并初步探讨其杀螺作用机制。方法　制备黑色浅灰链霉菌发酵上清液，将其于-20 ℃、4 ℃和28 ℃无光照条件下放置10 d后，测定其72 h浸杀灭螺效果；将发酵上清液置于100 ℃水浴中煮沸30 min后恢复至室温，测定其72 h浸杀灭螺效果；用浓盐酸和氢氧化钠调节发酵上清液pH值为4.0、6.0和9.0，室温下静置12 h，再调节发酵上清液pH至7.0，测定其72 h浸杀灭螺效果。用大孔树脂、硅胶和十八烷基硅烷键合硅胶依次对黑色浅灰链霉菌发酵产物进行4次分离纯化，将最终分离纯化产物配置成10.00、5.00、2.50、1.25 mg/L和0.63 mg/L浓度药液，测定其72 h浸杀灭螺效果。各实验设立空白对照组以脱氯水处理，阳性对照组以0.10 mg/L氯硝柳胺处理。采用高效液相色谱法测定经纯化代谢产物浸泡后钉螺软体组织中三磷酸腺苷（adenosine triphosphate, ATP）和二磷酸腺苷（adenosine diphosphate, ADP）含量。结果　黑色浅灰链霉菌发酵上清液在-20 ℃、4 ℃和28 ℃无光照条件下放置10 d后，原液及稀释10、50倍药液浸杀钉螺72 h，钉螺死亡率均为100.00%（30/30）；经100 ℃煮沸30 min后，原液及稀释10、50倍药液浸杀钉螺72 h，钉螺死亡率均为100.00%（30/30）。发酵上清液在pH值为4.0、6.0条件下存储12 h后浸杀钉螺72 h，钉螺死亡率均为100.00%（30/30）；在pH值为9.0条件下存储12 h后浸杀钉螺72 h，钉螺死亡率为33.33% (10/30，[χ2] = 30.000，P < 0.05)。发酵上清液最终分离纯化产物100.00%（30/30）杀死钉螺所需最低浓度为1.25 mg/L。以0.10 mg/L和1.00 mg/L浓度最终分离纯化产物处理钉螺后，钉螺软体组织中ATP水平较对照组显著降低（F = 7.274，P < 0.05），ADP水平与对照组差异无统计学意义（F = 2.485，P > 0.05）。结论　黑色浅灰链霉菌代谢产物中杀螺活性成分可在-20 ℃、4 ℃和28 ℃条件下稳定储存10 d，且耐热、耐酸而不耐碱，其杀螺机制可能是使钉螺能量代谢发生紊乱而死亡。

关键词: 湖北钉螺, 黑色浅灰链霉菌, 次级代谢产物, 杀螺效果