Leaves of the privet tree, Ligustrum obtusifolium, contain a large amount of oleuropein, a phenolic secoiridoid glycoside, which is stably kept in a compartment separate from activating enzymes. When the leaf tissue is destroyed by herbivores, enzymes localized in organelles start to activate oleuropein into a very strong protein denaturant that has protein-crosslinking and lysine-decreasing activities. These activities are stronger than ever reported from plant systems and have adverse effects against herbivores by decreasing the nutritive value of dietary protein completely. We report here that strong oleuropein-specific ␤-glucosidase in organelles activates oleuropein by converting the secoiridoid glucoside moiety of oleuropein into a glutaraldehyde-like structure, which is also an ␣,␤-unsaturated aldehyde. Oleuropein activated by ␤-glucosidase had very strong proteindenaturing, protein-crosslinking, and lysine-alkylating activities that are very similar to, but stronger than, those of glutaraldehyde. Aucubin, another iridoid glycoside, had similar activities after ␤-glucosidase treatment. We also detected polyphenol oxidase activity in organelles that activate the dihydroxyphenolic moiety to have protein-crosslinking activities. These data suggest that the privet tree has developed an effective defense mechanism with oleuropein, a unique multivalent alkylator ideal as a protein-crosslinker. Our results that iridoid glycosides are precursors of alkylators may elucidate the chemical bases that underlie various bioactivities and ecological roles of iridoid glycosides.

The white grub Dasylepida ishigakiensis has a 2‐year life cycle and spends approximately 9 months as a nonfeeding larva, pupa and adult on a subtropical island. Evidence is presented indicating that this beetle has two diapauses that appear to synchronize this long life cycle with the seasons. Larvae exposed to 20, 22.5, 25 and 27.5 °C late in the third (last) stadium pupate rapidly except for some individuals kept at the highest temperature. The latter pupate upon transfer to 22.5 °C, indicating that larval diapause is maintained at high temperature but terminates upon transfer to a lower temperature. Pupal development is directly temperature‐dependent in the range 20–30 °C. Adults develop reproductive organs (i.e. the ovary in females and the seminal vesicles and accessory glands in males) rapidly at 15 and 20 °C, whereas those kept at 25 °C take a long time to do so. Ovarian development is completely suppressed at 30 °C but initiated upon transfer to 20 °C. In the laboratory, males with well‐developed reproductive organs mate even with sexually immature females, whereas females with undeveloped ovaries show no sexual behaviour. Although the two diapauses of this species are thermally regulated (i.e. a characteristic commonly expressed by insects in summer diapause), adults of this beetle emerge from pupae late in the autumn and remain in the soil for 2 months. Adult diapause effectively serves to synchronize the time of sexual maturation with the coldest month of the year.

Chemical components that attract males in the laboratory were extracted from the female elytra of the white-spotted longicorn beetle, Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae), and found to be comprised of several sesquiterpene hydrocarbons. Males located females that had fed on the bark of the host plant Citrus unshiu more frequently than those fed on an artificial diet in the laboratory. Male attraction was also confirmed in the corresponding sesquiterpene fractions of the host plant. The sesquiterpene fractions of female elytra and C. unshiu leaves showed quite similar GC profiles. In the field, the beetles were found on C. unshiu trees baited with intact females or males as frequently as those baited with the leaf extracts containing the sesquiterpenes at different purification levels. The beetles were found on those baited trees significantly more frequently than on unbaited control trees. Males, as well as females attracted both sexes, and the male elytra also contained the sesquiterpenes that were identical with those in female and C. unshiu. This indicated that active components in the elytra are acquired from C. unshiu by feeding, contact and/or adsorption. These sesquiterpenes may serve for intraspecific communication in A. malasiaca. The major sesquiterpenes from the C. unshiu leaves were isolated and elucidated by NMR analyses of four sesquiterpene hydrocarbons, b-elemene, b-caryophyllene, a-humulene, and a-farnesene.

We conducted a series of experiments with the white-spotted longicorn beetle Anoplophora malasiaca (Thomson), and its host plant, Citrus unshiu, to examine the origin of the sesquiterpene hydrocarbons (SHCs; including b-elemene, b-caryophyllene, a-humulene, afarnesene, and several unidentified compounds) that are contained in the elytra of the beetles and act as an attractant. In the laboratory, mechanically wounded citrus branches, as well as those fed upon by A. malasiaca, attracted males more frequently than intact branches. Solid phase microextraction (SPME) and subsequent analyses by gas chromatography (GC) analyses detected measurable SHCs from the air around both mechanically wounded and beetle-infested branches, as well as trace amounts from intact branches. The SHCs were also detected for a certain time from beetles that had fed on the citrus branches, but the amounts decreased rapidly after they were removed from the host. This decrease generally corresponded with a reduction of attractiveness of the beetles in a behavioural assay. Isolated females acquired the SHCs after exposure to, but not upon contact with, other females that had fed on C. unshiu branches. We hypothesize that the citrus SHCs are adsorbed in, retained on, and released from the wax layer of the beetle elytra. Since these compounds are released from branches when beetles feed, they may indirectly signal the presence of beetles to others in the field. The high response rate to SHCs by males is likely representing mate searching behavior. The SHCs act as kairomones with a releaser effect in the communication system of A. malasiaca.

Precopulatory behavior of the white grub beetle, Dasylepida ishigakiensis, was observed under laboratory conditions to determine the environmental factors controlling mating behavior, which is synchronized among individuals at dusk in the field. When light intensity was gradually decreased, both females and males sequentially started moving their antennae, legs and abdomen, then walking, and females rhythmically extruded their abdomen while males opened the elytra to fly. Pheromone release by females was suggested in a wind tunnel assay in which males oriented themselves to both intact and crushed bodies of females, but not of males. When illumination was suddenly turned off, precopulatory behavior of females and males was similarly induced but the frequencies of calling and flying behaviors were significantly reduced as compared with those observed when light intensity was gradually reduced. This result may suggest that gradual darkening is an important factor for their synchronized appearance from the soil and subsequent mating behaviors, including pheromone release in females and orientation flight in males. These behaviors were significantly suppressed at low temperatures below 18°C. This result supports our previous conclusion that temperature is the primary factor controlling the emergence and mating activity of sexually mature beetles in the field.

The white grub beetle, Dasylepida ishigakiensis (Niijima et Kinoshita), is a serious pest of sugarcane in the Miyako Islands of Okinawa, Japan. Two evident electroantennographic (EAG) responses were observed by solid-phase microextraction sampling from female secretion and subsequent gas chromatography-electroantennographic detection (GC-EAD) analyses. These were identified as 2-propanol and 2-butanol by GC-MS analyses. GC-EAD on a chiral column showed two evident EAG signals corresponding to 2-butanol enantiomers, but the ratio and amounts could not be determined because of the small amounts. (R)-2-Butanol elicited the strongest EAG response, followed by (S)-2-butanol and 2-propanol, which showed 1/10 and 1/100 of the response of the first compound, respectively. In the field, only (R)-2-butanol attracted feral males when it was baited as 1 ml water solution of 1-100 mg of this compound, (S)-2-butanol, racemic mixture or 2-propanol on cotton ball lures (ca. 1.5 cm diameter) in vane-traps. When 2-propanol was blended with 10 mg/ml of (R)-2-butanol solution on cotton ball lures, male catches decreased as the amount of 2-propanol increased. From these results it was concluded that (R)-2-butanol was a component of the sex attractant pheromone of D. ishigakiensis while (S)-enantiomer and 2-propanol showed inhibitory effects when they were blended with the first compound.

Two active fractions were found during the isolation of contact sex pheromone of female elytra of the white‐spotted longicorn beetle, Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae), in addition to fraction of hydrocarbons that had previously been identified. One fraction was essential to evoke a series of precopulatory behaviors of males toward a glass dummy when coated together with the hydrocarbon blend. The other fraction enhanced this activity when added to the mixture. From the latter synergistic fraction, we isolated five novel compounds and identified them as 10‐heptacosanone, (Z)‐18‐heptacosen‐10‐one, (18Z,21Z)‐heptacosa‐18,21‐dien‐10‐one, (18Z,21Z,24Z)‐heptacosa‐18,21,24‐trien‐10‐one, and 12‐heptacosanone by GC‐MS and NMR analyses. A blend of four of these synthetic ketones, without 12‐heptacosanone, in the ratio and concentration found in female elytra extract (250 : 400 : 1000 : 180 ng FE−1) showed greater synergistic effect than the natural fraction containing the ketones. This effect was canceled out by further addition of 12‐heptacosanone (100 ng FE−1), which was still comparable to the effect of the natural ketone fraction.

Olfactory and visual cues were shown to mediate short-distance orientation in Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae). In a laboratory test, more than 80% (n = 42) of males walked straight upward when presented with an untreated surface with a 75 ° slope. When a freshly killed female was fixed at a short distance (10 cm ahead and 5 cm to left/right) from the starting point, 50% of males (n = 30) were oriented toward the female before direct contact. Similar behavioural responses were observed when female extract was directly applied to the slope or to a glass rod model fixed on the slope. When black, white, and transparent coloured rods with the extract were presented, the orientation response was significantly greater for black than to white and transparent rods, to which only a negligible response was observed.