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

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