Abstract: Objective　To investigate the effect of the fungus strain Penicillium aurantiocandidum Z12 with a molluscicidal activity on the immune function of lymphocytes in Oncomelania hupensis, so as to provide insights into elucidating the mechanism of its molluscicidal actions. Methods　The fermentation broth of the P. aurantiocandidum Z12 strain at a concentration of 1% was prepared. A total of 390 O. hupensis snails without Schistosoma japonicum infections were acclimated in dechlorinated water in the laboratory for 3 days. For the molluscicidal assay, 180 snails were randomly assigned into six groups, of 10 O. hupensis snails in each group. O. hupensis snails in three experimental groups were exposed to 10 mL fermentation broth of the P. aurantiocandidum Z12 strain for 24, 48, 72 hours, and snails in three control groups were immersed in 10 mL dechlorinated water for the same time points. Each test was repeated in triplicate. All snails were transferred to dechlorinated water for recovery for one hour following immersion, and snail activity was assessed. Inactive snails were identified for survival with the shell tapping method, and snail mortality was estimated to assess the molluscicidal activity of the P. aurantiocandidum Z12 strain against snails. In addition, 210 snails were randomly divided into seven groups, of 10 snails in each group. Snails in six experimental groups were immersed in the fermentation broth of the P. aurantiocandidum Z12 strain for 2, 4, 8, 16, 24, 48 hours, and snails in six control groups were immersed in the same volume of dechlorinated water for one hour. Each test was repeated in triplicate. All snails were transferred to dechlorinated water for recovery for one hour following immersion. Then, snails were crushed, and the head⁃foot soft tissues were collected, washed in phosphate⁃buffered saline (PBS), pieced, filtered, and centrifuged to yield concentrated lymphocyte solutions. Cells were mounted on glass slides, fixed in methanol and stained with 10% Giemsa solutions for 30 minutes, and the counts and proportions of small acidophilic hyalinocytes, large acidophilic hyalinocytes, basophilic hyalinocytes, small basophilic granulocytes, and large basophilic granulocytes were recorded under a microscope. The major axis length and nuclear major axis length of lymphocytes were measured with the software BEION 4.20. Results　The mortality rates of O. hupensis snails were 20.0% (6/30), 40.0% (12/30) and 63.3% (19/30) following immersion in 1% fermentation broth of the P. aurantiocandidum Z12 strain for 24, 48, 72 hours, respectively ([χ2] = 11.657, P < 0.01), and were 6.7% (2/30), 16.7% (5/30) and 30.0% (9/30) following immersion in dechlorinated water for 24, 48, 72 hours, respectively. There was no significant difference in the snail mortality between the experimental and control groups at 24 hours ([χ2] = 2.308, P = 0.129); however, significant differences were observed at 48 hours ([χ2] = 4.022, P = 0.045) and 72 hours ([χ2] = 6.696, P = 0.010). Following immersion in 1% fermentation broth of the P. aurantiocandidum Z12 strain for 8 to 48 hours, the proportions of small acidophilic hyalinocytes (S = -21.000, Z = -3.160, P < 0.05) and large acidophilic hyalinocytes (S = -4.000, Z = -1.980, P < 0.05) appeared a tendency towards a remarkable decline in O. hupensis snails, and the proportion of small basophilic granulocytes gradually increased (S = 3.000, Z = 2.120, P = 0.034). During the whole exposure period, the proportions of large basophilic granulocytes (S = 2.000, Z = 0.150, P = 0.880) and basophilic hyalinocytes (S = 9.000, Z = 1.500, P = 0.134) showed no apparent linear trend in O. hupensis snails, and no significant changes were seen in the major axis length (F = 1.530, P = 0.170) or the nuclear major axis length of small acidophilic hyalinocytes (F = 1.395, P = 0.232), or in the major axis length (F = 0.543, P = 0.774) or the nuclear major axis length of large acidophilic hyalinocytes (F = 0.611, P = 0.721). Similarly, the major axis length and nuclear major axis length of basophilic hyalinocytes (F = 2.490 and 0.508, both P values > 0.05), small basophilic granulocytes (F = 1.851 and 1.167, both P values > 0.05), and large basophilic granulocytes (F = 0.195 and 0.609, both P values > 0.05) fluctuated over time during the exposure period; however, no significant differences were seen at different time points. Conclusion　The fermentation broth of the P. aurantiocandidum Z12 strain may pose an immunosuppressive effect on acidophilic hyalinocytes in O. hupensis snails.

Key words: Oncomelania hupensis, Schistosomiasis, Penicillium aurantiocandidum, Immune system, Lymphocyte, Granulocyte, Hyalinocyte 

摘要： 目的　探讨灭螺真菌橘灰青霉菌Z12对钉螺淋巴细胞免疫功能的影响，为阐明其灭螺机制提供依据。方法　制备1%橘灰青霉菌Z12菌株发酵液。选取血吸虫感染阴性的钉螺共390只，于实验室去氯水中适应性饲养3 d。取180只钉螺，随机分为6组，每组10只；3个实验组钉螺分别置于10 mL橘灰青霉菌Z12菌株发酵液中浸泡24、48、72 h；3个对照组分别置于等量去氯水中浸泡24、48、72 h，各组均设3个平行处理单元；浸泡结束后，各组钉螺均转入去氯水中复苏1 h，观察钉螺活动性，对无活动的钉螺采用敲击法判定死活，计算钉螺死亡率，以评价橘灰青霉菌Z12杀螺效果。另取210只钉螺，随机分为7组，每组10只；6个实验组分别置于10 ml橘灰青霉菌Z12菌株发酵液中浸泡2、4、8、16、24、48 h，对照组置于等量去氯水中浸泡1 h，各组分别设3个平行处理单元；处理结束后，各实验组钉螺均复苏1 h；随后将各组钉螺破壳取头足部软体，经磷酸盐缓冲液洗涤、夹碎、过滤及离心后获得淋巴细胞浓缩液，制片并采用10% Giemsa染液染色30 min后，于显微镜下计数嗜酸性小透明细胞、嗜酸性大透明细胞、嗜碱性透明细胞、嗜碱性小颗粒细胞、嗜碱性大颗粒细胞5类淋巴细胞数量并计算构成比；此外，采用BEION V4.20医学图像软件测量各组钉螺各类淋巴细胞长径及核长径。结果　经1%橘灰青霉菌Z12发酵液浸泡24、48 h和72 h后，各实验组钉螺死亡率分别为20.0%（6/30）、40.0%（12/30）和63.3%（19/30），差异有统计学意义（[χ2]  = 11.657，P < 0.01）；经去氯水浸泡24、48 h和72 h后，各对照组钉螺死亡率分别为6.7%（2/30）、16.7% （5/30）和30.0%（9/30）。浸泡24 h后实验组和对照组钉螺死亡率差异无统计学意义（[χ2]  = 2.308，P = 0.129）；浸泡48 （[χ2]  = 4.022，P = 0.045）、72 h（[χ2]  = 6.696，P = 0.010）后，两组钉螺死亡率差异有统计学意义。经1%橘灰青霉菌Z12发酵液浸泡8 ~ 48 h时（染毒中后期），钉螺嗜酸性小透明细胞和嗜酸性大透明细胞构成比呈显著下降趋势（S = -21.000、       -4.000，Z = −3.160、-1.980；P 均< 0.05），嗜碱性小颗粒细胞占比逐渐上升（S = 3.000，Z = 2.120， P = 0.034）。在染毒全期，嗜碱性大颗粒细胞（S = 2.000，Z = 0.150，P = 0.880）、嗜碱性透明细胞（S = 9.000，Z = 1.500，P = 0.134）占比无明显线性变化趋势，嗜酸性小透明细胞长径（F = 1.530，P = 0.170）、核长径（F = 1.395，P = 0.232）与嗜酸性大透明细胞长径（F = 0.543，P = 0.774）、核长径（F = 0.611，P = 0.721）均无显著变化，嗜碱性透明细胞（F = 2.490、0.508，P 均 > 0.05）、嗜碱性小颗粒细胞（F = 1.851、1.167，P 均> 0.05）、嗜碱性大颗粒细胞（F = 0.195、0.609，P均 > 0.05）长径及细胞核长径均呈波动变化，但不同时间差异亦均无统计学意义。结论　橘灰青霉菌Z12发酵液对钉螺嗜酸性透明细胞可能具有免疫抑制作用。

关键词: 钉螺, 血吸虫病, 橘灰青霉菌, 免疫系统, 淋巴细胞, 颗粒细胞, 透明细胞