Synergistic Defensive Function of Raphides and Protease through the Needle Effect

Raphides, needle-shaped calcium oxalate crystals in tissues of many plants, have been thought to play defensive roles against herbivores without detailed bioassays for their defensive roles and modes of function using purified raphides. In order to examine the defensive roles and modes of function of raphides in a clear experimental system, we performed bioassays giving the larvae of the Eri silkmoth, Samia ricini (Saturniidae), leaves of their host plant, the castor oil plant, Ricinus communis (Euphorbiaceae), painted with the raphides purified from kiwifruits, Actinidia deliciosa (Actinidiaceae), in presence or absence of cysteine protease, which often coincide with raphides in plant tissues. Raphides alone or cysteine protease alone showed only weak defensive activities around experimental concentrations. However, when raphides and cysteine protease coexisted, they synergistically showed very strong growth-reducing activities, and the mortality of caterpillars was very high. In contrast, amorphous calcium oxalate did not show synergism with cysteine protease on defensive activities, indicating that the needle-shape of raphides is essential for the synergism. The present study provides the first clear experimental evidence for the synergism between raphides and other defensive factors. Further, the study suggests that "the needle effect", which intensify the bioactivities of other bioactive factors by making holes to the barriers (cell membrane, cuticle, epithelium, the nuclear membrane, etc.) and facilitate the bioactive factors to go through them and reach the targets, is important in the defensive activities of raphides, and possibly in the allergy caused by raphides, and in the carcinogenic activities of other needle-shaped components including asbestos and plant derived silica needles.