Analysis of the molecular mechanism underlying the maintenance of endosymbiotic system in aphid

内共生系统是通过寄主和共生细菌Buchnera之间的互惠关系来维持的，Buchnera是一种特殊的寄主细胞，称为细菌细胞。在本研究中，我们展示了Buchnera与蚜虫共生的动态，得到了以下结果：1)细菌细胞中的Buchnera密度随寄主的生命周期而波动，这种波动依赖于年龄和形态。在有翼翼状突起的最后蜕皮前后，当间接飞行肌肉发育时，Buchnera密度急剧下降，而在无翼翼状突起的早期幼虫阶段，Buchnera密度下降。2)对最终蜕皮后的细菌细胞蛋白进行的蛋白质组学分析表明，与翼状突起的细菌细胞相比，翼状突起的细菌细胞中的羧基肽酶-卵黄生成样蛋白(CPVL)的表达增强。用抗CPVL抗血清进行免疫印迹分析表明，在发育过程中，棉铃虫CPVL在翼部细菌细胞中被激活，并作为一种溶酶体酶降解Buchner.3)LysoTracker组织化学分析表明，在最终蜕皮后，翼部细菌细胞中的溶酶体系统被急剧激活。此外，电子显微镜观察发现许多降解的Buchnera含有不规则间隔的电子致密区域，并被膨胀的共生体膜所包围。这些发现表明，寄主溶酶体系统参与了对共生系统中Buchnera密度的年龄和形态的调节，降解的Buchnera可能作为营养物质提供给寄主。我们认为，本研究的结果将有助于阐明蚜虫内共生系统维持的分子机制。

Endosymbiotic system in the aphid is maintained through a mutualistic association between the host and a symbiotic bacterium, Buchnera, which is harbored in particular host cell called bacteriocyte. In the present study, we demonstrated the dynamics of Buchnera-aphid symbiosis, and following results were obtained.1) Buchnera density in bacteriocytes fluctuated according to the life cycle of the host in an age- and morph-dependent manner. Buchnera density decreased drastically just around the final ecdysis in the winged morph alatae, when the indirect flight muscles develop, whereas it decreased at the early larviposition stage in the wingless morph apterae.2) Proteomic analysis of the bacteriocyte p roteins just after the final ecdysis revealed the enhanced expression of carboxypeptidase-vitellogenic like (CPVL), in the alatae bacteriocytes compared to the apterae bacteriocytes. Immunoblotting analysis using anti-CPVL antiserum revealed that Aphid-CPVL is activated in the alatae bacteriocytes accompanying with the processing, and functions as a lysosomal enzyme to degrade the Buchnera.3) Histochemical analysis using LysoTracker indicated that the lysosomal system is drastically activated in the alatae bacteriocytes just after the final ecdysis. Further, electron microscope observations revealed many degrading Buchnera that contained irregularly spaced electron-dense areas and were enclosed by a distended symbiosome membrane in the alatae bacteriocytes.These findings suggest that the host lysosomal system is involved in age-and morph-dependent regulation of Buchnera density in the aphid symbiotic system and the degraded Buchnera may be supplied as nutrients to the host. We considered that the result obtained in the present study would contribute in elucidating the molecular mechanism underlying the maintenance of endosymbiotic system in aphid.