School of Life Science, Shanxi University, Taiyuan, China.
PLoS One. 2013 Aug 7;8(8):e71600. doi: 10.1371/journal.pone.0071600. Print 2013.
To investigate the effect of the secondary metabolites of entomopathogenic fungus on the hemocyte immunity of host insect, the secondary metabolite complex (SMC) of Beauveriabrongniartii was used in three concentrations (5.5, 55, and 550 µg/mL), and the 4(th) instar larvae of the pine caterpillar Dendrolimustabulaeformis were employed as host insects. The larvae were inoculated with the SMC solutions by injection in bioassays. Apoptosis of the larval hemocytes was observed using fluorescence microscopy (FM), transmission electron microscopy (TEM), and flow cytometry (FCM). The FM results showed that in the treated groups, larval hemocytes exhibited symptoms of early apoptosis at 6 h post-treatment by radiating a non-uniform kelly fluorescence and exhibited symptoms of late apoptosis at 12 h post-treatment by radiating a non-uniform orange fluorescence. Under TEM, the following ultra-structural changes associated with apoptosis of the larval hemocytes were observed in the treated groups: the nuclei were hypertrophied, slight folds were on the nuclear envelope, the chromatin became concentrated, the mitochondrial cristae disappeared or were disorderly, most cells developed blebs, and fibrillar aggregation appeared and accumulated in the cytoplasm. Apoptosis of the larval hemocytes was detected by FCM at 6 h post-treatment; the percentage of early apoptotic cells in the SMC 5.5, 55, and 550 µg/mL treatment groups were 11.93%, 13.10%, and 18.42%, respectively. Late apoptosis first occurred at 12 h post-treatment; the highest rate of apoptosis was 36.54 ± 4.37% at 24 h post-treatment in the SMC 55 µg/mL treatment group. In general, the cellular apoptosis rate was positively correlated with the SMC concentration and the time post-treatment. These results indicate that secondary metabolites of B. brongniartii are able to attack the hemocytes of D. tabulaeformis larvae and induce cellular apoptosis, thereby providing new evidence that secondary metabolites of mycopathogens can act on host immune systems.
为了研究昆虫病原真菌次生代谢物对宿主昆虫血细胞免疫的影响，采用 3 种浓度（5.5、55 和 550μg/ml）的球孢白僵菌次生代谢物复合物（SMC），以马尾松毛虫 4 龄幼虫为宿主昆虫，通过注射法将 SMC 溶液接种到幼虫体内进行生物测定。利用荧光显微镜（FM）、透射电子显微镜（TEM）和流式细胞术（FCM）观察幼虫血细胞凋亡情况。FM 结果显示，处理组幼虫血细胞在处理后 6h 时呈现出早期凋亡的辐射状非均匀凯莉荧光，在处理后 12h 时呈现出晚期凋亡的辐射状非均匀橙色荧光；TEM 下观察到处理组幼虫血细胞凋亡的超微结构变化：细胞核肥大，核膜有轻微褶皱，染色质浓缩，线粒体嵴消失或排列紊乱，大部分细胞形成小泡，细胞质中出现纤维状聚集物；FCM 在处理后 6h 检测到幼虫血细胞凋亡，SMC 5.5、55 和 550μg/ml 处理组的早期凋亡细胞百分比分别为 11.93%、13.10%和 18.42%，晚期凋亡在处理后 12h 首次发生，在 SMC 55μg/ml 处理组中，24h 后凋亡率最高，为 36.54±4.37%。总体而言，细胞凋亡率与 SMC 浓度和处理后时间呈正相关。这些结果表明，白僵菌的次生代谢物能够攻击马尾松毛虫幼虫的血细胞并诱导细胞凋亡，为病原真菌次生代谢物能够作用于宿主免疫系统提供了新的证据。
