The mating sequence has been studied in the white-spotted longicorn beetle, Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae). Both females and males are attracted to wounded host plant Citrus unshiu, and adults of both sexes, and both female and male elytra contained the sesquiterpenes that were identical with those in C. unshiu. Moreover, at close range orientation by males, some visual cues derived from females acted synergistically with olfactory factors. And, female contact sex pheromone was isolated from female elytra and revealed to consist of eight hydrocarbons, four ketones, and three lactones. The mating sequence of A. malasiaca was roughly considered to occur as follows. Emerged adults from trunks of the host plants must search for their mates over large areas. Highly mobile males may be attracted from long distances by the signal of sesquiterpenes released from wounded host plants which indicate the presence of other individuals and serve for intraspecific communication in A. malasiaca. At close range orientation, the sesquiterpenes also act as attractant for both females and males, and then visual cue guided them to their mates or rivals. After a male orients to another individual, he identifies it as a mate or a rival. Once a male recognizes a female, mating occurs.

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This paper introduces a new method for the mass-rearing of a typical riverine insect, Cheumatopsyche brevilineata. This caddisfly could be easily reared continuously under laboratory conditions in rotary-flowing water. The rearing system supported the full life cycle year-round, and the culture scale supplied sufficient larvae for bioassays every week. The hatchability of egg masses dipped at F₃ and F₉ but was otherwise stable at ≥70%. The sensitivity of first-instar larvae to fenitrothion was stable throughout successive generations of a fenitrothion-sensitive strain, and increased gradually in a resistant strain.

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In aphidophagous ladybird beetle, larvae reflexively exude hemolymph, i.e. reflex bleeding, when disturbed. In the present study, to evaluate the effectiveness of hemolymph as a defense against intraguild predation and its bleeding cost, laboratory experiments were conducted using two species, Coccinella septempunctata brucki and Harmonia axyridis. In the first experiment, to evaluate the effect of H. axyridis hemolymph as a means of avoiding intraguild predation by C. septempunctata brucki, the incidence of cannibalism of C. septempunctata brucki eggs by fourth instars was determined in relation to the presence of H. axyridis hemolymph. In this experiment, 80% eggs of C. septempunctata brucki were cannibalized when no H. axyridis hemolymph was present; however, only 20% of eggs were cannibalized when H. axyridis hemolymph was present. In the second experiment, average adult body weight of H. axyridis at its emergence differed after individuals had experienced no versus daily reflex bleeding during the fourth instar. The body weight of adult H. axyridis averaged 32.8±1.3 mg (n=11) and 35.3±0.9 mg (n=19) for males and females, respectively, when they did not reflex bleed during the fourth instar; however, their body weight tended to be lower when they had reflex bled daily during the fourth instar.

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The maize orange leafhopper, Cicadulina bipunctata, is a serious pest of second crops of forage maize in Kyushu, Japan, because it induces maize wallaby ear symptom (MWES), characterized by stunted growth and severe swelling of leaves. We investigated the seasonal occurrence, host plants, and distribution of C. bipunctata in Kyushu from 2004 to 2007. Population densities of C. bipunctata rapidly increased from late July, and generally peaked between September and October. Cicadulina bipunctata fed mainly on Eleusine indica, Digitaria ciliaris, and Setaria viridis from summer to autumn, and overwintered as adults mostly on Sasa spp., followed by wheat and Italian ryegrass. In early spring, the first generation seemed to grow largely on wheat. These results suggest that earlier planting of the second crop of forage maize and management of Sasa spp. in early winter are likely to be effective strategies for reducing the infestation of forage maize by C. bipunctata. Field surveys in 2005 and 2007 revealed that C. bipunctata was distributed at 34 of 54 sites, including Fukuoka, Saga, Kumamoto, Miyazaki, and Kagoshima prefectures. Because such a wide distribution has not been reported in previous studies, C. bipunctata is considered to have expanded its range in recent years.

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The relative effects of intra and interspecific interactions on the larval survival of two species of aphidophagous ladybirds, Coccinella septempunctata brucki and Harmonia axyridis, were assessed at two different prey abundances. In single species rearing, the number of larvae of both species decreased during the latter developmental stages, mainly due to cannibalism. When prey was abundant, the survival of both species improved; however, in mixed species rearing, the mortality of C. septempunctata brucki due to cannibalism or intraguild predation was higher than in single species rearing, whereas the mortality was lower in H. axyridis. Consequently, the survival of C. septempunctata brucki decreased while that of H. axyridis improved. In addition, in the 18 intraguild predation events observed, H. axyridis was always the intraguild predator and C. septempunctata brucki was its prey. Thus, when these two species co-exist in the field, H. axyridis is likely to be an important intraguild predator of C. septempunctata brucki. In addition, when prey is scarce, it is likely that cannibalism and intraguild predation become more important factors in the intra and interspecific relationships of these two species.

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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.

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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 μg 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 μg/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.

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Laboratory and field experiments were conducted to investigate the bioefficacy of sweet flag (Acorus calamus) against the pulse beetle Callosobruchus chinensis, which infests stored chick pea seeds. Dried powders of the leaf and rhizome as well as their extracts in methanol (ME) and petroleum ether (PEE) were evaluated as bio-insecticides against the beetle. Rhizome powder (5 mg/g seed) was found to be more efficacious, causing 100% mortality, than leaf powder (20 mg/g seed), showing 91.1% mortality. Rhizome powders also showed 100% ovicidal activity and thus completely inhibited F₁ emergence at a lower dose than that of leaf powders. The ME and PEE of leaves and rhizome showed 12 to 100% mortality. The percentage of egg laying decreased from 12.8 to 100 with increasing doses of powders and extracts. No F₁ adult emerged at 0.3–0.4 mg/g ME of leaves and rhizome and PEE of rhizome. In six-month field trials, all treatments at laboratory doses proved efficacious (75–98%) over organophosphate insecticide malathion (74.4%) in terms of feeding deterrence. Powder- and extract-treated chick pea seeds did not lose their viability and germinated at 100%, similar to control seeds. It was concluded that leaf and rhizome powders and their solvent extracts showed significant mortality, and inhibition of egg laying and F₁ emergence of C. chinensis at varying concentrations. Hence, the leaves and rhizome of A. calamus may be recommended as admixtures in the integrated management of beetle infestation of pulse seeds during storage.

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Age-specific survival rate (l_x) and fecundity (m_x) of Monochamus carolinensis (Olivier) adults were determined under field conditions using two caged populations, each composed of ten pairs of adults. The average longevity was 8.0 and 7.3 weeks for females and males, respectively. Oviposition marks made by females contained 0 to 4 eggs. The average number of eggs in an oviposition mark was 0.73 over a females' life span. The average potential (∑m_x) and realized (∑l_xm_x) fecundities were estimated to be 330.6 and 249.7 eggs, respectively. A comparison of 16 populations of four Monochamus species reported so far including this study showed positive correlations between body size, longevity, and mean fecundity for adult females, suggesting that the body size is an important trait when evaluating Monochamus species as vectors of the pinewood nematode.

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The peritrophic membrane (PM) is a non-cellular tissue involved in the protection of midgut epithelium from mechanical damage and insults from pathogens. This study was carried out to determine the involvement of PM in mosquitoes after infection with dengue virus. Aedes aegypti (Diptera: Culicidae) mosquitoes were fed sucrose and human blood with and without dengue virus type 2 (D2-16681), and collected at 0.5, 1, 6, and 12 h, respectively. Specimens were prepared for examination under light and electron microscopy. The results showed that PM was produced only in the blood-fed mosquitoes. The infected blood meal induced the mosquitoes to produce PM in their midgut earlier and thicker than in mosquitoes with blood alone. The initial evidence of PM occurred at 1 h post-blood meal (PBM) as a matrix-like structure. By 6 h PBM, PM had become a layer, which persisted at 12 h. Among mosquitoes fed with blood alone, this structure was found only from 6 and 12 h PBM. Dengue virus type 2 induced different modifications of mosquito PM construction and structures, confirmed under an electron microscope.

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Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) B- and Q-biotypes are exotic insects in Japan and are consequently regarded as insect pests in agricultural fields. Using B- and Q-biotypes, we investigated the hatching rate, survival rate during the larval stage and the total development period from egg to adult. Whiteflies were allowed to lay eggs on several host plants: cabbage, Brassica oleracea var. capitata; cucumber, Cucumis sativus L.; tomato, Solanum lycopersicum L.; eggplant, Solanum melongena L.; bell pepper, Capsicum annuum cv. grossum; and five cultivars of kidney bean, Phaseolus vulgaris (cvs. Kentucky101, Nagauzura, Morocco, Satsukimidori and Honkintoki), and the effects on the above traits were evaluated. Clear differences between the two biotypes were detected. A particularly important difference was that the B-biotype successfully developed to adults on only one of five cultivars of kidney bean, whereas the Q-biotype was able to utilize all the test plants as hosts regardless of the cultivar. This finding suggests that the determination of host plants in B. tabaci is greatly influenced by not only plant species but also by cultivar. In addition, there is a high possibility that the Q-biotype has the ability to adapt to a wider range of plant species and cultivars compared with the B-biotype.

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Torymus sinensis and Torymus beneficus (Hymenoptera: Torymidae) are, respectively, introduced and indigenous parasitoid wasps that attack the invasive chestnut gall wasp, Dryocosmus kuriphilus (Hymenoptera: Cynipidae) in Japan. Torymus beneficus has two emergence types in spring, here designated early-spring and late-spring. It is very difficult to distinguish these Torymus species and emergence types of T. beneficus by their morphological and ecological characteristics. We designed four allele-specific primers for the internal transcribed spacer 1 region of nuclear ribosomal DNA, and developed three kinds of allele-specific PCR. These methods enabled us to distinguish the species, emergence type, and F₁s. This is the first report of the use of molecular markers to distinguish T. sinensis, the late-spring strain of T. beneficus, and their F₁s.

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Oligonucleotide-probed fluorescent in situ hybridization (FISH) targeting 16S rRNA is a powerful technique for detecting and characterizing bacterial cells in environmental samples without cultivation; however, general application of the technique to insect endosymbionts has been hindered by the strong autofluorescence frequently observed in insect tissues. Here we describe a protocol that markedly reduces autofluorescence of insect tissues by hydrogen peroxide (H₂O₂) treatment, whereby 16S rRNA of bacterial endosymbionts is kept in a FISH-detectable condition. Among various histological fixatives, Carnoy's solution was superior in that whole insects were successfully fixed and autofluorescence of insect tissues was suppressed in comparison with the widely used formaldehyde-based fixatives. Treatment with both alcoholic 6% H₂O₂ solution and aqueous 6% H₂O₂ solution markedly reduced autofluorescence of the fixed insect tissues, wherein the former kept 16S rRNA of bacterial endosymbiont in a FISH-detectable condition while the latter failed to do so. The protocol was applicable to endosymbionts of diverse insects such as aphids, lice and bat flies. The protocol was applicable not only to fresh insect samples but also to archival insect samples preserved in acetone for several years. We propose a general and robust protocol for quenching autofluorescence of insect tissues for FISH detection of bacterial endosymbionts, which is potentially applicable to endosymbionts of a wider range of organisms with considerable autofluorescence.

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The pathogenicity of six strains of the entomopathogenic bacteria Photorhabdus and Xenorhabdus, symbiotically associated with the entomopathogenic nematodes Heterorhabditis and Steinernema, respectively, to the mushroom mite, Luciaphorus perniciosus was studied. Bacterial cultures of the Thai strain Xenorhabdus nematophila (X1) and the imported strain Photorhabdus luminescens (P1), containing 1×10⁸ cells/ml, showed the highest virulence to the female mite, resulting in 85 and 83% mortality within 3 days after application, respectively. The fecundity of the surviving L. perniciosus was also significantly reduced by the X. nematophila (X1) strain. These results demonstrate the potential of X. nematophila (X1) to control the mushroom mite.

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To evaluate the pollination efficiency of a stingless bee, Melipona quadrifasciata, individuals originating from tropical and subtropical regions of South America were examined on tomatoes (cv. Momotaro 8) cultivated in a greenhouse in summer and autumn. We compared differences in the rates of foraged flowers, pollen weights foraged from anthers, rates of fruit set of tomatoes, seed number of tomato fruits and yields of tomato fruit weight between M. quadrifasciata and the bumblebee, Bombus terrestris. When the amount of tomato pollen was sufficient, there were no differences in pollination efficiency between the stingless bee and the bumblebee; however, the rates of foraged flowers, the rate of fruit set and the yields were significantly reduced in the stingless bee compared with the bumblebee when fertile tomato pollen decreased markedly during the hottest period of the summer. During this period, the rate of flowers foraged by bumblebees did not decrease, but the rates of fruit set, the seed number and the yields of tomato fruits decreased significantly in comparison with the other periods. It is known that tomato pollen production is inhibited at high temperatures; thus, we consider that the high temperatures in the greenhouse (average daily temperature higher than 28°C) caused the extreme decrease in pollen production. These results indicate that M. quadrifasciata can be used as a suitable pollinator of tomatoes if sufficient amounts of fertile pollen are provided.

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The courtship calling frequency of males and the time of mating during the day were compared between an Okinawa mass-reared strain and a Taiwan wild strain of the melon fly, Bactrocera cucurbitae (Coquillett). The mass-reared strain was maintained for about 150 generations under artificial rearing conditions and was concurrently used for the sterile insect technique (SIT) in Okinawa. The call frequency of males was significantly lower in the Okinawa mass-reared strain than in the Taiwan wild strain, and the mating time of mass-reared males was significantly later in the day than that of Taiwan wild-strain males. Moreover, the mating competitiveness of the Okinawa mass-reared strain was lower than the Taiwan wild strain in mate-choice tests. These results suggest that improvement in the mating competitiveness of mass-reared melon flies or the introduction of a new strain from a foreign country is urgently needed for the SIT program to eradicate the melon fly population in case of reinvasion in Japan.

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A cDNA of aquaporin (AQP, water channel) belonging to the major intrinsic protein (MIP) family was obtained from the digestive tract of the Formosan subterranean termite, Coptotermes formosanus. The cDNA encoded an AQP homolog designated as CfAQP with 249 amino acids. RT-PCR analysis revealed the expression of CfAQP in the digestive tract and other tissues, suggesting that CfAQP may play important roles not only in water recycling but also other essential water transport processes in the termite. Phylogenetic analysis of CfAQP with 71 insect MIP sequences from 17 species indicated that insect MIPs could be classified into 4 groups, designated as insect MIP Group 1 to Group 4, and that CfAQP is a member of Group 1, a sister group of vertebrate AQPs.

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The present study investigated the fitness consequence of pupation site of bagworms, Thyridopteryx ephemeraeformis, on arborvitae. The abundance and survival of pupae were higher on trees with a southern exposure than on those with a northern exposure. Neither sex-ratio nor female mating success was influenced by the exposure of trees. The ratio of females per male, survival of pupae, and mating success of females all increased from the bottom to the top of arborvitae. Our results suggest that bagworm larvae may enhance their fitness by pupating in locations most suitable for pupal survival and female mating success. Because adult female bagworms can not disperse, pupation by female larvae may further influence the performance of their larval progeny, either by favouring the long-range dispersal of ballooning larvae or by providing neonates with suitable foliage nearby their site of emergence.

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A cell line from the monarch butterfly Danaus plexippus designated BCIRL-DP-AM/JG was established from adult ovaries. The cell line consisted mainly of round cells and took a prolonged period of time in the growth medium ExCell 401 containing 10% fetal bovine serum and antibiotics before it could be subcultured on a regular basis. The cell line had a population doubling time of 32 h and was susceptible to four baculoviruses (MNPV) but was refractile to a fifth baculovirus, a single nucleopolyhedrovirus (HzSNPV). PxMNPV was the most infectious of the MNPV for BCIRL-DP-AM/JG cell line of D. plexippus producing a titer of 5.9×10⁷ TCID₅₀/ml. The non-occluded Hz-1 virus was also infectious for the D. plexippus cell line. The occlusion bodies produced by the BCIRL-DP-AM/JG cell line were infectious for 24 h old Trichoplusia ni larvae and gave LC₅₀ values equivalent to or better than the LC₅₀ for OB produced in the other susceptible cell lines by the MNPV under study. The identity of the monarch butterfly cell line was established by DAF-PCR.

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