Plants can respond to infestation by herbivores with the emission of specific herbivore-induced plant volatiles. Many carnivorous arthropods that feed on herbivorous prey use these volatiles to locate their prey. Despite the growing amount of research papers on the interactions in tritrophic systems, it has remained unclear how carnivorous arthropods use herbivore-induced plant volatiles in prey-location. We investigated three important aspects of information use by the predatory mite Phytoseiulus persimilis, a specialised natural enemy of herbivorous spider mites. First, we showed that the foraging efficiency of predatory mites was not hampered by the presence of volatiles induced by non-prey caterpillars on brussels sprouts plants. Second, we revealed an important role for the volatile compound methyl salicylate. Predatory mites appear to use the presence of this compound, rather than its relative contribution to a volatile blend, to discriminate between two volatile blends. Third, we demonstrated that the role of methyl salicylate in the foraging behaviour of P. persimilis depends on previous experiences of the predators with this compound. Our research improves the understanding of the selection pressures that act on the foraging responses of carnivorous arthropods, and consequently on the selection pressures on volatile emission by plants in response to herbivory.

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Rapid progress has been made in research on insect pheromones and other semiochemicals in Korea during the last two decades. Sex pheromone compositions have been identified in several major orchard and other horticultural moth pest species and some other species in Korea, such as Helicoverpa assulta, Grapholita molesta, Carposina sasakii, Adoxophyes orana, Lyonetia prunifoliella, Phyllonorycter ringoniella, Dichocrocis punctiferalis, Ostrinia furnacalis and Peridroma saucia. Some of these identified sex pheromones have already been successfully incorporated into IPM systems, and are now being used as direct and indirect pest control means in Korea. Korean populations of many of these species showed significant differences in pheromone compositions from those reported in the neighboring countries of Japan and China, suggesting that such interpopulational pheromone polymorphism is more common across the insect world than previously thought. Sex pheromone compositions were also identified in some aphid species in Korea such as Aphis spiraecola and Tuberocephalus momonis, and it was soon found that the aphid sex pheromone components were highly attractive to their predator lacewings. The presence of kairomones responsible for attracting egg parasitoids was shown in a pheromone component, hot pepper odor and male scale factor for H. assulta, and in male accessory glands for Lymantria dispar. Studies on the regulation of pheromone biosynthesis and release were carried out simultaneously in some species in Korea, resulting in the characterization of PBAN (pheromone biosynthesis activating neuropeptide) in H. assulta and Adoxophyes sp. A semiochemical research system has been firmly established in Korea through these studies. In some areas, successful monitoring and mating disruption technology have been effectively transferred from researchers to the end users in Korea, which will soon be expanded to wider areas. Continuous attempts to identify and use semiochemicals as well as wider applications of semiochemicals in pest control are expected in Korea in the coming years. Since Korean research on semiochemicals does not cover vast areas due to limited resources, focused research and practical application will be needed for selected target insect species.

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The rice water weevil, Lissorhoptrus oryzophilus, was first detected in Japan in 1976 in a paddy field, in Tokoname, near Nagoya. This weevil is a recent invader and is assumed to have entered with hay from California. Ten years later, the weevil succeeded in spreading throughout all Japanese paddy fields and also expanded to Korean, Taiwan and east China. The rice water weevil now is regarded as one of the common insect pests of rice in these areas. Investigations on taxonomy, biology, population dynamics, damage and economic importance, estimation of population density and forecasting, and control are reviewed.

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We investigated the attraction of Riptortus clavatus to different component compositions of its aggregation pheromone (mixture of three components) in the field by the use of sticky traps. In two-component tests, each of (E)-2-hexenyl(E)-2-hexenoate (EE) and (E)-2-hexenyl(Z)-3-hexenoate (EZ) mixed with myristyl isobutyrate (MI) showed equally synergistic effects in attracting R. clavatus relative to MI alone. There was no correlation between the degree of attractiveness and the dosages of EE or EZ added to MI. No significant difference was observed in the numbers of insects caught between traps baited with the three-component blend (MI+EE+EZ) and traps with two-component blends (MI+EE or MI+EZ). In addition, two-component blends (MI : EE or MI : EZ=2 : 1) were almost as attractive as the natural blend (MI : EE : EZ=1 : 5 : 1), and even in the absence of the main component, EE. R. clavatus responded to a wide range of the pheromone component blends without a particular optimum blending ratio. This might characterize the aggregation pheromone of this bug.

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Experiments were conducted to investigate whether there is a trade-off between hunting ability and starvation tolerance in spiderlings of a wandering spider, Pardosa pseudoannulata Boes. et Str. The width of the carapace (the cover over the cephalothorax) was used as an index of hunting ability, as a larger carapace was confirmed as being advantageous for capturing prey. Abdomen width was used as an index of starvation tolerance because individuals with larger abdomens survived longer under fasting condition. Effects of carapace width and abdomen width on starvation tolerance were examined using multiple regression analysis. The carapace width was found to have a negative effect but the abdomen width had a positive effect on starvation tolerance. Moreover, the ratio of abdomen width to carapace width was significantly different among clutches. These results suggested that there is a trade-off between carapace width and abdomen width, that is, between hunting ability and starvation tolerance, and also suggested that the trade-off might be an allocation strategy specific to each clutch.

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A termite egg-mimicking fungus was discovered in nests of the Japanese subterranean termite Reticulitermes speratus in 1997 and was reported for the first time in 2000. This fungus has a mutualistic relationship with termites. When termite workers recognize eggs laid by queens, they gather the eggs to tend them. The corticioid fungus Fibularhizoctonia sp. forms sclerotia that morphologically and chemically mimic termite eggs. By mimicking eggs, the fungus is protected and may be transported by termites to a competitor-free habitat. In turn, the sclerotia enhance egg survival, probably because the antifungal and antibacterial compounds produced by the fungus protect the eggs from putative pathogens. This novel termite-fungus interaction has previously been reported only in R. speratus in Japan. In this study, I conducted wide-range sampling in Japan and the United States to investigate the distribution of egg-mimicking fungi in five Reticulitermes spp. Here I show that R. flavipes and R. virginicus in the United States, as well as R. speratus, harbor the egg-mimicking fungus. No egg-mimicking fungi were found in R. okinawanus in Okinawa and R. hesperus in California. A BLAST homology search for rDNA sequences categorized the egg-mimicking fungi isolated from R. flavipes and R. virginicus as a species of Fibularhizoctonia.

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White-spotted longicorn beetle, Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae), females were observed to use both olfactory and visual cues for orientation to males in the laboratory. When a freshly killed male was fixed at ca. 10 cm ahead and 5 cm to the right or left from a starting point on a 75° slope panel, 36% of the females oriented themselves toward the male before direct contact. Similar behavioral responses to a glass-rod model treated with male extract were observed. When a black or white glassrod treated with the extract was presented, the orientation toward the black rod was significantly greater than that to the white one. Few females oriented toward rods without extract. This indicates that the olfactory cue is essential for female orientation, and that a visual cue enhances the effect of the olfactory cue.

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To evaluate the efficiency of herbivore exclusion by the ant, Lasius japonicus Santschi on the vetch, Vicia angustifolia L. with extrafloral nectaries, we analyzed ant foraging behavior and ant aggressiveness against herbivores and discuss the mechanism of herbivore exclusion by ants in relation to the number of ants visiting a plant. In a field census, the number of alfalfa weevil Hypera postica larvae, which is the main herbivore on V. angustifolia, decreased with the number of L. japonicus workers. The encounter rate between workers of L. japonicus and herbivores increased as more workers visited the plant. The aggressiveness of an individual worker against herbivores also increased as more workers visited the plant. Consequently, there was a negative correlation between the number of workers on a plant and the residence time of an herbivore on a plant. Therefore, we expect that the efficiency of herbivore exclusion by ants increases rapidly when the number of ants on a plant attains a certain level.

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A new technique was developed for the efficient rearing of phytoseiid mites. The developed artificial rearing arena consists of a 24-well tissue culture plate and pieces of woolen yarn. The rearing efficiencies of four species of phytoseiid mites belonging to different genera, Phytoseius nipponicus, Amblyseius eharai, Typhlodromus vulgaris and Typhlodromips tsugawai, were compared in regard to the newly developed arena and several types of conventional artificial arenas. P. nipponicus, A. eharai, and T. vulgaris increased significantly in number on the new artificial arenas. T. tsugawai also multiplied well on the new artificial arena, although no significant differences were observed in the number of mites between the new artificial arena and a conventional artificial arena made of a plastic board containing pieces of woolen yarn. These results confirmed the usefulness of the new artificial arena, and lead to the expectation that this technique could be applied for rearing many other species of phytoseiid mites.

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A tortoise beetle, Cassida nebulosa feeds on chenopodiaceous weeds, Chenopodium album and C. album var. centrorubrum as its main hosts. The beetle is known as a pest of another chenopodiaceous plant, the sugar beet. However, as a chenopodiaceous vegetable, spinach is rarely harmed by C. nebulosa. Newly emerged adults that had no feeding experience developed a positive feeding response to spinach as well as to C. album var. centrorubrum within 24 h. On the other hand, adults reared on C. album var. centrorubrum did not accept spinach, and their feeding response to spinach was still weak even after rearing on spinach for 4 d. When C. nebulosa were reared on leaves of C. album var. centrorubrum treated with the methanol extracts of spinach, they accepted spinach as well as C. album var. centrorubrum. In contrast, when C. nebulosa were reared on leaves of spinach treated with the methanol extracts of C. album var. centrorubrum, consumption of spinach by the adults was less than that by the adults reared on leaves treated with solvent only. These results indicate that C. nebulosa has the potential to harm spinach due to the habituation to feeding deterrents in spinach. However, the harm to spinach by C. nebulosa could be suppressed because feeding experience on C. album var. centrorubrum can prevent C. nebulosa from habituating to spinach.

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The effect of previous rice plant infestation of the white-backed planthopper, Sogatella furcifera, on the incidence of rice blast disease caused by the fungal pathogen, Magnaporthe grisea, was investigated. The fungal pathogen M. grisea was less likely to cause symptoms of leaf blast on rice plants that had been previously infested with S. furcifera than on uninfested plants. This indicates that resistance to M. grisea was induced in rice plants by infestation of S. furcifera. The number of blast lesions per plant decreased with increasing numbers of infesting S. furcifera from 0 to 10 pairs. The active period of induced resistance was maintained for a relatively long time being greater than 15 d. The difference in the incidence of leaf blast between S. furcifera infested and uninfested plants in the booting stage became smaller than that in the tillering stage, although there was a still significant difference in blast incidence between both infested and uninfested plants in booting stage. There was no significant difference in the incidence of leaf blast between damaged plants treated with needling and untreated control plants. This suggests that the simple mechanical effect to the plants caused by stylet insertion (penetration) and subsequent probing with the feeding behavior of S. furcifera was not related to the observed phenomenon. In S. furcifera infested plants, the expression of two genes regarding β-1,3-glucanase, Gns4 and Gns5, was confirmed by RT-PCR analysis. These results show that infestation of S. furcifera apparently induced physiological changes including gene expression that were related to pathogen resistance to M. grisea in rice plants.

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A new assay method was developed for easily and precisely determining whether beetles were knocked down (define knockdown) by phosphine fumigation for the purpose of evaluating the phosphine resistance of the cigarette beetle. The knockdown time (KT) was investigated for six strains of beetles exposed to phosphine. The susceptibilities of the six strains were investigated by checking adult mortalities from phosphine exposure. One strain showed high resistance and almost all beetles survived even at 1,000 ppm. The KT₅₀ values of the high resistant strain exposed to 100, 200 and 300 ppm phosphine were 32,800, 5,000 and 3,650 min, respectively. The KT₅₀ values of susceptible and low resistant strains, with LC₅₀ values lower than 50 ppm, were smaller than 250 min even at 100 ppm. The KT₅₀ values of the middle resistant strain, with an LC₅₀ value of 140 ppm, were 920, 340 and 290 min at 100, 200 and 300 ppm, respectively. The time difference between the low resistant beetles knocked down last and the highly resistant beetles knocked down first was 86, 18 and 8 h at 100, 200 and 300 ppm, respectively. The probit regression line for the middle resistant strain lay between that of the susceptible strain and that of the highly resistant strain, although it was nearer to that of the susceptible strain.

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A hypersensitive bioassay of nucleopolyhedrovirus infecting a silkworm, Bombyx mori, (BmNPV) has been developed. Peroral administration of a chitin synthesis inhibitor, Polyoxin AL wettable powder (Polyoxin AL), to a silkworm resulted in a decrease of the median lethal concentration (LC₅₀) of BmNPV. The LC₅₀ in the silkworm ingesting Polyoxin AL decreased as the silkworm developed from the 2nd to the 4th stadium. LC₅₀ in a 2nd stadium silkworm ingesting Polyoxin AL was lower than that in a silkworm refrigerated at 4°C. Silkworms at the 2nd stadium that had ingested Polyoxin AL were used to detect BmNPV in the dust from 15 sericultural farmhouses. Dust samples were collected at a leaf storage space, a rearing room and a mounting room just before the beginning of silkworm rearing after the formalinisation of the rearing environment. The dust was collected again at the same places immediately after harvesting the cocoons. In addition to the BmNPV detection in the dust, occurrence of innerside-stained cocoons was examined at the same 15 farmhouses where the dust was collected. The following features of BmNPV contamination are recognized. In some farmhouses, BmNPV was not inactivated even after treatment with formalin. BmNPV accumulated in only one cycle of the silkworm rearing of about 3 weeks in almost all the farmhouses. Of all three places in which the dust was assayed, the mounting room tended to be most heavily contaminated by BmNPV. More innerside-stained cocoons occurred in a farmhouse where more silkworms died of BmNPV in an assay of the dust collected before the beginning of the silkworm rearing. On the other hand, these features of BmNPV contamination were not detected using silkworm larvae that had not ingested Polyoxin AL. This new bioassay for BmNPV utilizing Polyoxin AL-treated larvae is an effective tool to assess the cleanliness of the sericultural environment.

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Effect of leaf age on the flight response of a parasitic wasp, Cotesia kariyai, to a plant-herbivore complex was studied in a wind tunnel. Both young and old leaves infested with host larvae (common armyworms: Mythimna separata) attracted more wasps than their respective uninfested leaves. Preference of the wasps to uninfested young leaves vs. uninfested old leaves was not significantly different in choice experiments. However, when young infested leaves were compared against old infested leaves in choice experiments, the wasps showed a significant preference for the former. In the single stand experiments, % landing response to young infested leaves was not significantly different from the old infested leaves. The fact that attractiveness of infested corn leaves to wasps differs according to leaf age suggests differential allocation of induced indirect defense in the corn plant.

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The leaf beetle, Gastrophysa atrocyanea Motschulsky (Coleoptera: Chrysomelidae) has been regarded as a bioagent suitable for the extermination of certain weeds, especially Rumex obtusifolius. We examined conditions for artificial mass rearing of this insect. An artificial diet based on the host-plant leaf powder and supplemented with feeding stimulants, was developed for laboratory rearing of the leaf beetles. In particular, the hatchability of eggs laid by the fourth generation and female longevity were equivalent to those for fresh host leaves. Consequently, the fifth generation was achieved by rearing on artificial diet.

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We previously demonstrated that a novel 252-kDa protein (P252) isolated from brush border membranes (BBM) of Bombyx mori, hybrid Shurei×Shogetsu, specifically bound Cry1Aa, Cry1Ab and Cry1Ac toxins with a K_d of 29, 179 and 20 nM, respectively. P252 was found in a Triton X-100–soluble fraction of BBM from first, third and fifth instar larvae, suggesting that it may be an important element of midgut epithelial cell membranes. P252 was not partitioned into a Triton X-100–insoluble BBM fraction and nearly identical partitioning of P252 was observed using detergents CHAPS and Igepal. These results suggested that P252 localized in non-raft regions of BBM. Immunofluorescence analysis using anti-P252 antiserum demonstrated the existence of P252 in BBMV. Cy3-labeled Cry1Aa, Cry1Ab and Cry1Ac were shown to bind to BBMV, and the addition of anti-P252 antiserum reduced the number of BBMV showing Cy3 fluorescence by 30%. This clearly suggested an important role for P252 in Cry1A binding to BBMV. CD spectra of a mixture of purified P252 with either Cry1Aa, Cry1Ab or Cry1Ac were compared with those of respective free Cry1A toxins and only one of the mixtures (Cry1Aa/P252) was shown to be significantly changed compared to that of native Cry1Aa.

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Seasonal abundance and within-plant distribution of Acyrthosiphon spp. and their natural enemies were investigated on alfalfa in Obihiro, Hokkaido, Japan in 2001. Densities of A. pisum and A. kondoi peaked in early June. Coccinellid and syrphid predators appeared to respond to aphid numbers and were synchronized with aphid populations. Two species of braconid (aphidiine) parasitoids (Aphidius ervi and Praon barbatum) were collected. Parasitism rates for each aphid species were variable through the season, but the estimated number of parasitized aphids reached a peak at the same time as overall aphid densities. After the first harvesting of alfalfa (14 June) aphid densities became very low, and coccinellid and syrphid predators were not found. In contrast to the coccinellid and syrphid predators, the heteropteran predators, Nabis stenoferus and Orius spp. became common during summer. Aphids, immature parasitoids (live parasitized aphids and mummies), Coccinella septempunctata and syrphids occurred more on the upper parts of alfalfa stems than on the lower parts, but this trend was not found for Hippodamia tredecimpunctata. The temporal and spatial distributions of members of the aphidphagous guild are likely to influence natural enemy–aphid relationships and intraguild interactions.

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A photo-stable juvenoid, pyriproxyfen, affected every life stage of Plutella xylostella at critical points in their development. Egg hatch was reduced by about 90% when eggs were treated at 0–1 d. Third instar larvae treated with the compound failed to pupate. At a dose of 100 ppm, supernumerary larvae were produced and most of the dead larvae showed abnormalities associated with the chimera form. Pupae formed at this higher dose were arrested in their development, and all died. Final instar larvae 7 d after treatment pupated successfully. However, adult eclosion was reduced to about 50% even at a low dose of 4 ppm. About 20% of treated pupae failed to eclose, but the rest were unaffected and eclosed normally. Following treatment of adults, egg production was reduced to 10 per female and these failed to hatch. Furthermore, adults placed in contact with pyriproxyfen in an acetone : canola oil formulation on aluminium foil produced eggs that failed to hatch, especially when eggs were at an early stage of development within the female at the time of treatment. Results indicate that a device treated with an appropriate formulation of pyriproxyfen to contaminate both sexes of P. xylostella adults and their eggs would be superior to methods of targeting larvae in control operations.

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To evaluate the effect of pollen released from transgenic insecticidal corn on non-target lepidopteran insects, corn pollen deposition density on the leaves of sunflower and black nightshade was measured near a cornfield. At 12 d from the start of anthesis, the highest cumulative pollen density on leaves was approximately 160 grains per cm² at 1 m from the edge of the cornfield, falling to 20 grains at 5 m and less than 10 grains at 10 m. The pollen density calculated using a mathematical model in a previous study evidently had overestimated values. To evaluate precisely the effect of corn pollen expressing Bacillus thuringiensis (Bt) endotoxin (Cry1Ab) on the survival of lepidopteran larvae, we improved the bioassay methods using the pale grass blue, Pseudozizeeria maha, the leaf disc of the wood sorrel, Oxalis corniculata, and transgenic Bt corn (Event-176). When the surface of the leaf was pretreated with a small amount of 80% acetone solution, the preselected pollen dose was successfully applied onto the leaf disc. Larval survival of P. maha was significantly affected at pollen density of more than 20 grains per cm² on the leaf disc. It is unlikely that pollens from Bt corn expressing Cry1Ab have wide-scaled deleterious effects on non-target P. maha near cornfields, because of low pollen deposition dose on the leaves.

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Three cucurbitaceous feeding leaf beetle species, Aulacophora indica, A. lewisii, and A. nigripennis, were investigated with regard to the relationship between feeding preference by the beetles and cucurbitacins B, E, I, and E-glucoside contents in the leaves of 18 cucurbitaceous plant species. HPLC analysis and TLC analysis of A. lewisii revealed that the cucurbitacin B and E-glucoside, which are strong feeding stimulants for A. indica and A. lewisii, were abundant in Lagenaria siceralia, Sicyos angulatus, and Cucumis melo leaves. In a feeding preference test using fresh leaves, A. indica preferred various cucurbitaceous plant species and methanol extracts regardless of the contents of the four cucurbitacins. A. lewisii mostly fed on Luffa cylindrica leaves in which the four cucurbitacins were not detected. A. lewisii was also stimulated to feed by the methanol extracts of Lagenaria siceraria and L. cylindrical, while A. nigripennis, which is only slightly stimulated to feed by the four cucurbitacins, fed primarily on Trichosanthes kirilowii var. japonica leaves. Among 18 cucurbitaceous plants, only Diplocyclos palmatus leaves were completely rejected by the three beetle species, and these beetle species did not even feed on D. palmatus leaves treated with the cucurbitacins. These results indicate that some leaf constituents as well as the four cucurbitacins play an important role in host plant selection by the three cucurbitaceous feeding beetle species.

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The pathogenicity of Japanese strains of the entomopathogenic fungi, Paecilomyces fumosoroseus, Beauveria bassiana and Aschersonia aleyrodis, against the nymphs of the silverleaf whitefly, Bemisia argentifolii, was compared with that of strains from foreign commercial products, including P. fumosoroseus, B. bassiana and Verticillium lecanii. With a single dose of 6×10⁸ conidia/ml, the highest mortality was observed for the Japanese strain P. fumosoroseus PF3110, although it did not cause significantly different mortality than the strains from foreign commercial products. The LC₅₀ values of the native strain were determined on various days after inoculation and the LT₅₀ values were determined at different doses. The native strain, P. fumosoroseus PF3110, has potential as a microbial control agent against this whitefly.

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The present paper describes a novel Y-track olfactometer for the analyses of olfactory responses of insects which are easily disturbed and then feign to be dead. The olfactometer has been demonstrated to be useful for the analyses of olfactory responses of the vine weevil, Otiorhynchus sulcatus F.

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We compared the effects of intrinsic quality and seasonal abundance of two host plants, Gynostemma pentaphyllum and Trichosanthes cucumeroides (Cucurbitaceae), on host use by a phytophagous lady beetle, Epilachna admirabilis. Although, only G. pentaphyllum exists as an available host in April, T. cucumeroides sprouts from late May and its leaf abundance surpasses that of G. pentaphyllum in August, the egg-laying period for E. admirabilis. In the laboratory, T. cucumeroides supported higher growth than did G. pentaphyllum and larvae grew faster as the dietary proportion of T. cucumeroides increased. We expected females to switch to T. cucumeroides for feeding and egg deposition. However, most adults remained and laid eggs on G. pentaphyllum. There is a poor correlation between larval performance and adult egg-laying site selection. On the other hand, first and second instar larvae showed a greater preference for T. cucumeroides in a host preference test. Additionally, 70–80% of first instar larvae were found on G. pentaphyllum in the field, and the number of larvae on T. cucumeroides increased with developmental stage. We suggest that larvae often switch food sources from G. pentaphyllum to T. cucumeroides depending on the intrinsic quality and food availability of these two host plants.

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The larval ectoparasitoid of Pseudaletia separata, Euplectrus separatae oviposits exclusively on the dorsolateral surface from the 3rd thoracic to the 2nd abdominal segment of the host. Larval parasitoids are well known to be exposed to risks from aggressive defense behavior when they attempt to parasitize a host. E. separatae female sting the host to inject a venom prior to oviposition. At this time the female wasp is exposed to a serious risk from aggressive host behavior. However, the female avoids the risk by moving to a specific segment of the host. The question arose as to how the female wasp recognizes the specific segments of the host. The female wasp mounted the host larva from the posterior dorsal part and moved forward on the host. The female used chemical cues to recognize the posterior part of the host body. Diethyl ether extracts from the last abdominal segments of the host were more attractive to females than extracts from the head. Motionless behavior in the host after the female wasp mounted did not induce the forward movement of the female wasp. Once the female was on the host she used crawling of the host body (peristaltic movement) as a stimulus to move forward on the host. Another key stimulus to cause the parasitoid to cease movement forward on the specific segments of the host was examined. When a host head was attached to the 3rd abdominal segment, the female wasp shifted the oviposition site adequately behind the head on the host body, suggesting that the head is used as a visual cue to pinpoint a suitable oviposition site.

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To clarify tissues susceptible to chill injuries in the pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), 5 d old non-diapausing pupae (ND5) were subjected to a non-freezing temperature of −20°C, with or without prior acclimation at 5°C. Cold acclimation of pupae for 8 d or longer substantially decreased the mortality after exposure to −20°C for 5 d. Analysis of the morphology of dead pupae suggested that they died at two discrete developmental stages: after formation of the adult morphology (type 1), and at a stage indistinguishable from ND5 in appearance (type 2). The two types of death were differentially induced by changing the period of −20°C treatment. A majority of pupae exhibited type 1 death after a 2 d treatment, whereas type 2 death was predominant after a 12 d treatment. Regarding the type 1 death, pharate adults appeared to have died because they failed to emerge from the puparial case. Possibly, the neuromuscular system was impaired by the short term chilling at −20°C. On the other hand, the Malpighian tubules were seriously damaged by long term chilling at −20°C, and this is thought to be the cause of type 2 death.

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