Chinese Journal of Applied Entomology 2015, 52(6): 1466 1473. DOI: 10.7679/j.issn.2095 1353.2015.177 摘 * 绿僵菌素对四种昆虫细胞的毒性 何广位 1, 2** 胡维娜 1, 2** 胡琼波 1,2*** 1. 510642 2. 510642 要 目的 Destruxins Bombyx mori Bm12 Ostrinia furnacalis Ofh S2 Sf9 方法 MTT A B DA DB DABM 4 IC 50 结果 24 h < 25 μg /ml Ofh Bm12 S2 Sf9 DA DB DABM DA DB DABM Ofh IC 50 219.19 112.29 34.86 μg/ml Bm12 S2 Sf9 IC 50 250 μg/ml DA DB Ofh Sf9 Bm12 S2 6.25 μg/ml Bm12 Ofh S2 Sf9 结论 Ofh S2 4 关键词 Toxicity of destruxins to four insect cell lines HE Guang-Wei 1, 2** HU Wei-Na 1, 2** 1, 2*** HU Qiong-Bo (1. The Key Laboratory of Natural Pesticide and Chemical Biology, South China Agricultural University, Guagzhou 510642, China; 2. Key Laboratory of Integrated Pests Management of Southern China, Ministry of Agriculture, Guagzhou 510642, China) Abstract [Objectives] Destruxins are insecticidal secondary compounds isolated from Metarhizium anisopliae. In this study, the toxicity of different destruxins was tested on four insect cell lines, i.e., Bombyx mori Bm12, Ostrinia furnacalis hemocytes Ofh, Drosophila melanogaster S2 and Spodoptera frugiperda Sf9. [Methods] MTT and microscopy analysis were employed to evaluated the effects of destruxins A and B (DA and DB), and a 1/1 (m/m) mixture of these compounds (DABM), on the four insect cell lines. Relative toxicity was assessed by comparing IC 50 s values and morphological changes in the cell lines. [Results] The results indicate that Ofh cells were more susceptible to lower dosages (< 25 μg/ml) of DA and DB, with IC 50 values of 219.19, 112.29 and 34.86 μg/ml for DA, DB and DABM, respectively, compared to IC 50 values > 250 μg/ml in the other cell types. Furthermore, DA and DB had a synergistic effect on Ofh cells, but an antagonistic effect on Sf9 cells, and an additive effect on Bm12 and S2 cells. Microscopy revealed morphological changes in the different cell lines, even at dosages as low as 6.25 μg/ ml. Moreover, these morphological changes became more apparent at higher dosages. Ruptured cells, strumae, aggregation, expanded cells, vacuoles and reduced cell numbers were found in Bm12 cells. In Ofh cells, cells * Supported projects 863 2011AA10A204-2 ** First author E-mail heguangwei1976@qq.com; 591877328@qq.com *** Corresponding author E-mail hqbscau@scau.edu.cn Received 2014-01-04 Accepted 2014-05-08
6 : 1467 became expanded but appeared to return to normal with aggregation. Vacuoles and a few expanded, ruptured and aggregated cells were found in the S2 cell line, and membrane shrinkage, broken cells and a decrease in cell numbers were recorded in the Sf9 cell line. [Conclusion] The Ofh cell line was the most sensitive to destruxins, and the non-host insect Drosophila melanogaster S2 cell line the least sensitive. The results suggest that although relatively large doses of destruxins are required to kill insect cells, smaller dosages are sufficient to cause changes in cell morphology. Key words destruxins, insect cells, toxicity Destruxins Metarhizium anisopliae Pedras et al. 2002 2004 39 A B DA DB Liu and Tzeng 2012 Galleria mellonella Vilcinskas et al. 1997a 1997b Aspergillus niger 0.005 μg/ml Vey et al. 2002 Drosophila melanogaster Escherichia coli IMD Pal et al. 2007 Fan et al. 2013 Spodoptera litura SL-1 Chen et al. 2014 A B 4 1 材料与方法 1.1 细胞与培养 Bombyx mori (Linnae) Bm12 TNM-FH Hyclone Ostrinia furnacalis Guenee Ofh EX-CELL 420 Sigma Drosophila melanogaster (Meigen) S2 Schneider Sigma Spodoptera frugiperda (J.E.Smmith) Sf9 SFX Hyclone 4 10% Gibco 27 Bm12 S2 Sf9 2~4 d Ofh 3~4 d 1.2 绿僵菌素与处理 A B DA DB Hu et al. 2006 DA DB m/m DABM 1 mg 100 μl DMSO Sigma 10 000 μg/ml 1-80
1468 Chinese Journal of Applied Entomology 52 4 000 2 000 1 000 500 250 125 62.5 μg/ml 1 1.3 MTT 法检测绿僵菌素对 4 种细胞的毒性 MTT MTT Formazan 臢 OD OD % =1 OD OD / OD OD 100 96 Corning Incorporated 90 μl 24 h 1 10 5 /ml 10 μl 6.25 12.5 25 50 100 200 400 μg/ml 1% DMSO 4 27 24 h 50 μl 1 MTT 27 4 h 1 000 r/min 150 ml DMSO imark 490 nm (OD) SPSS IBM IC 50 1.4 形态学观察 24 h 2 结果与讨论 2.1 绿僵菌素对 4 种细胞的毒性 MTT 24 h Bm12 Ofh S2 Sf9 1 Bm12 Ofh Sf9 S2 DA DB 6.25~25.00 μg/ml 3 35%~48% S2 20% 1 4 DA DB DABM Ofh DABM 12.5 μg/ml Ofh 45% Bm12 Sf9 S2 13% 35% 15% 400 μg/ml Bm12 Sf9 S2 55% 46% 56% IC 50 IC 50 IC 50 1 DA DB Bm12 Sf9 Fig. 1 图 1 绿僵菌素处理 24 h 后 4 种昆虫细胞的死亡率 Mortalities of four cell lines after 24 h treated with destruxins DA: A DB: B DABM: A B 1/1 DA: Destruxin A; DB: Destruxin B; DABM: The 1/1 mixture of destruxin A and destruxin B.
6 : 1469 Cell line Treatment Table 1 表 1 绿僵菌素对 4 种细胞的毒性回归方程和 IC 50 值 IC-p equations and IC 50 values of destruxins against four cell lines IC-p equation Chi-Square test (P) IC 50 95% mg/l IC 50 (95% confident interval) Bm12 DA y=0.001 0.319x 0.656 260.09 (175.83-488.25) - CTC DB y=0.001 0.209x 0.999 281.04 (150.70-3 202.64) - DABM y=0.003 1.002x 0.542 326.79 (277.92-399.84) 82.67 Ofh DA y=0.000 0.095x 0.999 219.19 - - DB y=0.001 0.083x 0.607 112.29 - - DABM y=0.002 0.061x 0.631 34.86-425.96 S2 DA y=0.003 0.894x 0.526 350.58 (289.52-451.47) - DB y=0.003 0.833x 0.968 295.10 (247.42-367.36) - DABM y=0.003 0.946x 0.036 303.92 (223.65-488.65) 105.44 Sf9 DA y=0.002 0.385x 0.951 250.46 (181.83-392.19) - DB y=0.001 0.263x 0.985 260.10 (161.70-634.18) - DABM y=0.001 0.390x 0.998 508.29-50.21 DA: A DB: B DABM: A B 1/1 DA: Destruxin A; DB: Destruxin B; DABM: The 1/1 mixture of destruxin A and destruxin B. IC 50 Ofh S2 DB DA IC 50 4 Ofh DA DB DABM Ofh IC 50 219.19 μg/ml 112.29 μg/ml 3 IC 50 250 μg/ml 1 DA DB 4 DABM Ofh CTC 425.96 DA DB S2 CTC 50.21 DA DB CTC 80~120 1 2.2 绿僵菌素对 4 种细胞形态的影响 Bm12 Ofh S2 Sf9 6.25~400 μg/ml DA DB DABM Bm12 24 h 2 DA DB DABM Ofh Plastohemocytes Oenocytoide 3 S2 4 Sf9 5 3 讨论 4 Ofh IC 50 DA DB Ofh IC 50 219.19 112.29 μg/ml DABM IC 50 34.86 μg/ml 6.25 μg/ml
1470 应用昆虫学报 Chinese Journal of Applied Entomology Fig. 2 52 卷 图 2 绿僵菌素处理 Bm12 细胞 24 h 后的显微图片 Profiles of Bm12 cells after 24 h treated with destruxins CK. 1% DMSO 对照 A/B/AB1~7. DA DB 和 DABM 分别以 6.25 12.5 25 50 100 200 和 400 μg/ml 处理细胞 ag 细胞聚集 db 碎片 ex 膨胀 str 瘤状突起 va 空泡 比例尺=100 μm 下图同 CK: 1% DMSO control; A/B/AB1 7: The treatments of DA, DB and DABM at the concentrations of 6.25, 12.5, 25, 50, 100, 200, 400 μg/ml. ag: Aggregation; db: Debris; ex: Expand; str: Strumae; va: Vacuole. Scale bar =100 μm. The same below. Fig. 3 图 3 绿僵菌素处理 Ofh 细胞 24 h 后的显微图片 Profiles of Ofh cells after 24 h treated with destruxins pl 浆血细胞 oe 类绛色细胞 比例尺=100 μm pl: Plastohemocytes; oe: Oenocytoide. Scale bar =100 μm.
6期 何广位等: 绿僵菌素对四种昆虫细胞的毒性 Fig. 4 图 4 绿僵菌素处理 S2 细胞 24 h 后的显微图片 Profiles of S2 cells after 24 h treated with destruxins br 细胞破碎 比例尺=50 μm br: Broken. Scale bar= 50 μm. Fig. 5 图 5 绿僵菌素处理 Sf9 细胞 24 h 后的显微图片 Profiles of Ofh cells after 24 h treated with destruxins sh 细胞收缩 比例尺=100 μm. sh: Shrinkage. Scale bar =100 μm. 1471
S2 IC 50 300 μg/ml S2 Bm12 Sf9 Bm12 Sf9 A B S2 4 6.25 μg/ml Vey et al. 2002 Fan et al. 2013 S2 A B S2 4 参考文献 (References) Chen XR, Hu QB, Yu XQ, Ren SX, 2014. Effects of destruxins on free calcium and hydrogen ions in insect hemocytes. Insect Science, Doi 10.1111/1744-7917.12028. Fan JQ, Chen XR, Hu QB, 2013. Effect of destruxins A on hemocytes morphology of Bombyx mori. Journal of Integrative Agriculture, 12(6): 1042 1048. Hu QB, Ren SX, 2004. Review of destruxins of Metarhizium anisopliae Sorokin. Chinese Journal of Biological Control, 20(4): 234 242. [,, 2004.., 20(4): 234 242.] Hu QB, Ren SX, Wu JH, Chang JM, Musa DP, 2006. Investigation of destruxin A and B from 80 Metarhizium starins in China, and the optimization of cultural conditions for the strain MaQ10. Toxicon, 48(5): 491 498. Liu BL, Tzeng YM, 2012. Development and applications of destruxins: a review. Biotechnology Advances, 30: 1242 1254. Pal S, St Leger RJ, Wu LP, 2007. Fungal peptide destruxin a plays a specific role in suppressing the innate immune response in Drosophila melanogaster. Journal of Biological Chemistry, 282: 8969 8977. Pedras MSC, Zaharia LI, Ward DE, 2002. The destruxins: synthesis, biosynthesis, biotransformation, and biological activity.
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