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.

The feeding behavior in nectar-feeding insects is triggered by a sugar-receptor response in contact chemosensilla. The contact chemosensilla are distributed not only on tarsi and the outside of the proboscis but also on the inside of the food canal in Lepidoptera. Although the chemosensilla inside the food canal are assumed to detect sweet taste during the passage of nectar through the food canal, their electrophysiological function has received little attention. In the nectar-feeding Asian swallowtail butterfly, Papilio xuthus (Lepidoptera: Papilionidae), we found 15-to 30-μm-long sensilla neatly lined up along the inside galea wall, which forms the food canal in the proboscis. The receptor neurons of these sensilla responded to sucrose. We hypothesized that starch and sucrose compete with each other for a taste receptor site on the sensilla. When we added starch and sucrose to the food-canal sensilla, the electrophysiological responses of food-canal sensilla were inhibited in parallel with the food-sucking behavior of the butterflies. These results suggest that the food-canal sensilla are involved in the behavioral control of nectar-sucking in this butterfly species.

Foretarsal ventral surface observation of Japanese Papilio butterflies showed that the shapes of fifth foretarsi and numbers and localization of contact chemosensilla and spines in these areas are closely related to both phylogeny and behavior in these species. My results basically supported the classification that Japanese Papilio divides into five subgenera -- Papilio (P. machaon), Princeps (P. xuthus and P. demoleus), Achillides (P. maackii and P. bianor), Menelaides (P. helenus, P. polytes, P. protenor and P. macilentus) and Iliades (P. memnon). Moreover, female foretarsal morphology also corresponded to the physical features of their preferring host plant leaves. The specificity of female P. machaon, female P. macilentus and female P. maackii seemed to also relate to the geographical diversities of these species and their hostplant, and their co-evolution.

Papilio maackii females prefer a rutaceous plant, Phellodendron amurense, for oviposition, whereas another semi-sympatric Rutaceae feeder, Papilio protenor, never exploits this plant as a host in nature. However, the larvae of both species perform well on this plant in the laboratory. Phellamurin, a flavonoid present in the organic fraction from P. amurense inhibits egg laying by P. protenor. We examined whether phellamurin is involved in the differential acceptance of P. amurense by the two butterflies. The ovipositing females of P. maackii readily accepted P. amurense and a methanolic extract of the foliage, while P. protenor rejected them entirely. However, the aqueous fraction derived from the extract elicited significant oviposition responses of similar levels from the two species. Phellamurin did not induce oviposition behavior in P. protenor females. In contrast, P. maackii was stimulated to oviposit by phellamurin at concentrations exceeding 0.2%. The response was dose-dependent and reached ca. 70% at 2% phellamurin, which is approximately equivalent to its natural abundance in young leaves of P. amurense. Since the aqueous fraction was very stimulatory to both species, the combined effect of phellamurin and the aqueous fraction on oviposition was tested. The addition of phellamurin to the aqueous fraction enhanced the ovipositional activity of P. maackii, but dramatically suppressed the oviposition response of P. protenor even at 0.1% concentration. These results, taken together with those obtained from electrophysiological recordings with foretarsal chemosensilla, indicate that phellamurin acts as an oviposition stimulant for P. maackii, and as a potent deterrent for P. protenor. The results suggest that host range expansion or host shifts may be made by ovipositing females that overcome phytochemical barriers.

Many butterflies acquire nutrients from non-nectar sources such as puddles. To better understand how male Papilio butterflies identify suitable sites for puddling, we used behavioral and electrophysiological methods to examine the responses of Japanese Papilio butterflies to Na+, K+, Ca2+, and Mg2+. Based on behavioral analyses, these butterflies preferred a 10-mM Na+ solution to K+, Ca2+, and Mg2+ solutions of the same concentration and among a tested range of 1 mM to 1 M NaCl. We also measured the ion concentrations of solutions sampled from puddling sites in the field. Na+ concentrations of the samples were up to 6 mM, slightly lower than that preferred by butterflies in the behavioral experiments. Butterflies that sipped the 10 mM Na+ solution from the experimental trays did not continue to puddle on the ground. Additionally, butterflies puddled at sites where the concentrations of K+, Ca2+, and/or Mg2+ were higher than that of Na+. This suggests that K+, Ca2+, and Mg2+ do not interfere with the detection of Na+ by the Papilio butterfly. Using an electrophysiological method, tip recordings, receptor neurons in contact chemosensilla inside the proboscis evoked regularly firing impulses to 1, 10, and 100 mM NaCl solutions but not to CaCl2 or MgCl2. The dose–response patterns to the NaCl solutions were different among the neurons, which were classified into three types. These results showed that Japanese Papilio butterflies puddle using Na+ detected by the contact chemosensilla in the proboscis, which measure its concentration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00114-012-0976-3) contains supplementary material, which is available to authorized users.