Indigenous Abies firma trees were surveyed on Mount Unzen, Japan to clarify the dieback mechanisms of A. firma trees caused by herbivorous insects. A curculionid, Parendaeus abietinus, was revealed to be a univoltine species that caused the fall of needles as a result of adult and larval feeding on A. firma needles, and a scolytid, Polygraphus proximus, was regarded as a bivoltine species that produces subcortical galleries. Different degrees of needle infestation by P. abietinus among census years suggested that the population density of P. abietinus was remarkably high from 1995 to 1998, thereafter decreasing to 2001. Abies firma trees suffering from serious needle fall were frequently attacked and colonized by P. proximus in 1998 and 1999, and died soon after colonization; however, following the decline in the population density of P. abietinus, the number of A. firma trees colonized by P. proximus significantly reduced from 1999 to 2001. The possible dieback process of A. firma is considered as follows: (1) Around 1995, P. abietinus increased its population density and caused serious needle falling of A. firma; (2) in 1998 and 1999, P. proximus successfully colonized the bark of defoliated trees and played an important role in the dieback of A. firma; (3) after 1998, the population density of P. abietinus decreased; (4) thereafter, P. proximus could not colonize the bark of A. firma that survived serious defoliation and actively produced oleoresin.

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A male-produced crude aggregation pheromone from Eysarcoris lewisi (Distant) (Heteroptera: Pentatomidae) was collected from volatiles emitted by adult males. It was purified with high performance liquid chromatography using a silica gel column, followed by preparative gas chromatography. The attraction activity of each fraction was field tested using water pan traps. The isolated compound of the aggregation pheromone component was analyzed by GC-MS and NMR, and the chemical structure was estimated to be (Z)-2-methyl-6-(4-methylenebicyclo[3.1.0]hex-1-yl)hept-2-en-1-ol.

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Commercial colonies of European bumble bee, Bombus terrestris, have been widely used for pollinating tomato plants in greenhouses in Japan; however, a wild colony of the species was found in Hokkaido in 1996, after which several studies were conducted to investigate the ecological impact of the naturalization of B. terrestris to Japanese ecosystems. To quantify the status of naturalization, it is necessary to establish a method for estimating the population size of the alien bees established in the field. Locating bumble bee colonies in the field is, however, very difficult because they usually nest underground. In this study, we estimated the number of colonies using genetic data from B. terrestris queens foraging near greenhouses in Biratori, Hokkaido, Japan, applying the Shared Loci Correspondence Analysis (SLCA). Sampled 39 queens were assigned to 30 different families, which indicates that most queens came from different colonies. Based on the findings, we discuss the importance of urgent measures to prohibit B. terrestris from escaping from greenhouses and becoming naturalized in Hokkaido.

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A total of 53 strains of onion thrips, Thrips tabaci Lindeman, were established, of which 14 strains were collected from persimmon fruit and 39 strains from a vetch weed, Vicia angustifolia, in commercial persimmon orchards in Wakayama Prefecture. Susceptibility to insecticides was evaluated by the Petri dish-spraying tower method. Pyrethroid-resistant strains and pyrethroid-susceptible strains of T. tabaci were found in persimmon fruit. The LC₅₀ values of cypermethrin and fluvarinate of the pyrethroid-resistant strain were >240 ppm and >800 ppm, respectively, with resistance ratio of >200; however, no significant differences were observed between the two strains in the susceptibility to the other five insecticides, including organophosphates and neonycotinoids. There was a wide range of variability in mortality from 1.1 to 100% caused by cypermethrin at 60 ppm (LC₅₀ values from 0.390 ppm to 177 ppm) in T. tabaci collected from vetch in 5 different persimmon orchards. Regional difference was detected in the proportion of the pyrethroid-resistant strain: up to 67% of the strains collected from Naga where some orchards had been heavily applied with pyrethroids, compared to 17% of the strains collected from Kawahara where organophosphates and neonycotinoids had been applied.

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Asian citrus greening disease (CGD) is caused by Candidatus Liberibacter asiaticus, transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama. An effective method for controlling the pathogen is currently unavailable; therefore, controlling psyllids is a more practical strategy for minimizing damage from CGD. However, ecological/ethological field studies on this insect are limited due to a lack of effective marking methods. This study examined the effectiveness of a marking method for psyllids using fluorescent powder (FZ-2803). Equal numbers of marked and unmarked psyllids were released at a distance of 2 m from a light. The collection of marked psyllids near the light was significantly lower than that of unmarked psyllids until 4 h after marking. After 5 h, no significant difference was observed. No significant differences in mortality were detected between the two groups during a 40-day period after marking. The persistence of a mark on the psyllid body was confirmed visually in the laboratory for 40 days. One hundred marked psyllids were released in an orange jasmine field (18 m×18 m). The marked insects were detected visually, and the proportion of marked insects was 30% after 20 days and 20% after 40 days. These results indicate that the marking method using fluorescent powder does not affect survival, and it can be used for ca. 5 weeks in the field.

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Mass trapping with a synthetic sex pheromone lure to control the sugarcane click beetle Melanotus okinawensis Ohira (Coleoptera: Elateridae) was conducted in an agricultural area (81.4 ha) on Ikei Island (158.3 ha), Okinawa, Japan, with 970–1,000 traps (10.6 traps/ha) from 2000 to 2005. The total number of beetles captured by 725 traps was 22,701 in 2000, which decreased to 27.6% in 2005. Mean trap catch by 50 monitoring traps showed a similar decrease (to 26.1%) during the six-year trapping. Mean catches by 10 monitoring traps in an untreated area (Miyagi Island: 107 ha) did not decrease. Trends of population density in the area estimated by release-recapture experiments indicated a population decrease of about 90% from 2000 to 2005. Furthermore, the number of wild adults discovered on sugarcane and collected by hand in the treated area also decreased every year, by up to 86.2% by 2005. The population in the untreated area did not decrease consistently. The mating rates of females collected weekly in 2001 and 2002 were 73.9 and 85.7% in the treated area, which was slightly lower than in untreated areas (92.7 and 96.7%). We defined the male removal rate as the proportion of reduced survival duration. We estimated the male removal rate using two methods. The estimate obtained from the number of recaptured individuals was 0.345 while the estimate obtained from the difference in sex ratios between the treated and untreated areas was 0.319. These values appeared too small to explain the great reduction in population density in the treated area. Nevertheless, these results indicated that mass trapping with a high density of sex pheromone traps may effectively reduce an isolated population of M. okinawensis.

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Radio binding assay and dot-virus protein binding assay were carried out to study the difference in binding of the brush border membrane fraction (BBMF) in Aedes albopictus (Skuse) and Culex quinquefasciatus (Say) (Diptera: Culicidae). The BBMF of Ae. albopictus displayed higher saturation and relative binding characteristics, while the BBMF of Cx. quinquefasciatus showed low saturation and non-relativity. The virus overlay protein-binding assay was then adopted to detect virus-binding protein in the midgut of the two mosquitoes. We detected one 35-kDa virus binding protein in the midgut of Ae. albopictus, whereas none was found in the midgut of Cx. quinquefasciatus. The relationship between dengue-2 virus binding to the midgut of mosquitoes, and the mosquito mesenteron infection barrier against dengue 2 virus are discussed.

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Petroleum ether extracts of 63 varieties of plant species, including 37 varieties of leaves, 14 varieties of seeds, 10 varieties of flowers and 2 varieties of fruits were tested for larvicidal activity against 3rd instar larvae of important vector mosquitoes i.e. Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti (Diptera: Culicidae). A 24 h bioassay showed that six plant extracts, Acacia nilotica, Argemone mexicana (leaves and seeds), Citrullus colocynthis, Jatropha curcas and Withania somnifera were toxic, with an LC₅₀ value of less than 100 ppm against all three vector mosquitoes tested.

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Various morphological and chemical features of male white grub beetle Dasylepida ishigakiensis were compared among four island populations of Miyako, Irabu, Ishigaki, and Iriomote Islands in Okinawa Prefecture, Japan. Males of the Miyako and Irabu populations were significantly brighter in color and larger in size than those of the Ishigaki and Iriomote populations, although these differences were not significant within the two population groups. This was also true for male cuticular hydrocarbon compositions. The compositions were almost identical between Ishigaki and Iriomote and between Miyako and Irabu, but differed between the former and latter two populations. The overall findings suggest that the populations on Miyako and Irabu are different from those on Ishigaki and Iriomote.

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We investigated the prevalence of infestation by the bumblebee tracheal mite, Locustacarus buchneri (Acari: Podapolipidae), in four commercial colonies of Bombus terrestris (Hymenoptera: Apidae). All the foundresses, 91.8±5.67% (mean±SE) of worker bees, 73.9±6.85% of males, and 77.4±21.5% of new queens had the mite in their abdominal air sacs. The mean number of eggs, adult females, and mobile mites infesting a single bumblebee worker were 76.9±76.8 (range: 4–301, n=40), 3.78±3.45 (1–11, 40), and 3.45±2.98 (1–11, 20), respectively. From these values, we calculated the number of eggs per adult mite female to be 23.9±21.7 (range: 4–126, n=40). As a single worker bee had about 76 mites in her abdominal air sacs and approximately 90% of the bees in a colony of about 160 bees were infested by the mite, an infested B. terrestris colony has the potential to produce more than 10,000 L. buchneri individuals during colony development.

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The larvicidal activities of three sesquiterpenes, alantolactone, isoalantolactone and dihydroisoalantolactone, isolated from the roots of Inula helenium (Compositae) against 3rd and 4th instars of Aedes albopictus (Diptera: Culicidae) and Paratanytarsus grimmii (Diptera: Chironomidae), were examined. The two sesquiterpenes, alantolactone and isoalantolactone, showed LC₅₀ values of 2.7 μg/ml and 11.9 μg/ml for A. albopictus, and 5.1 μg/ml and 4.1 μg/ml for P. grimmii within 48 h, respectively. Alantolactone was significantly more toxic than isoalantolactone against A. albopictus; however, dihydroisoalantolactone did not entirely show lethal effects against the larvae of both species at a concentration of 1,000 μg/ml.

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The effects of associative learning of plant chemicals on host-searching behavior in Ascogaster reticulata Watanabe, an egg-larval parasitoid of the smaller tea tortrix, Adoxophyes honmai Yasuda, were investigated. Learned responses of conditioned and non-conditioned plant species were examined by a two-choice assay. Before the two-choice assays, females were given a rewarding oviposition experience or unrewarding experience on a leaf disc or leaf extract of either tea, camellia, sasanqua, bayberry, chinquapin, rose, Japanese cedar, fern pine, mulberry and corn. Following the experience, females were tested in a choice test between tea leaf and an alternative plant leaf. The results of the two-choice assays with leaf square discs were similar to tests using leaf extracts. Females that were conditioned with tea leaf showed a significant preference for tea leaf over the alternative plant species, except for leaves of the genus Camellia (camellia and sasanqua). Wasps conditioned with the other nine plant leaves only showed a significant preference for bayberry, mulberry, fern pine and corn. Females that had rewarding and unrewarding experiences were able to distinguish between tea and camellia. These results suggest that A. reticulata females are able to distinguish different plant species except for closely related species and some plant species; however, combinations with rewarding and unrewarding experiences enabled females to discriminate closely related species.

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In aphids, an identification system based on adult morphology is difficult to apply to larvae or adults of a different morph because of their small size and extensive polymorphism. In this work, identification of aphids using random amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific primers was studied. A total of 20 RAPD primers were screened on eleven members of Aphidinae, i.e., Acyrthosiphon pisum, Aphis craccivora, Aphis gossypii, Aphis rumicis, Cavariella salicicola, Indomegoura indica, Lipaphis erysimi, Myzus formosanus, Myzus hemerocallis, Myzus persicae, and Toxoptera odinae. DNA fragments potentially useful as species-specific markers were selected for six aphid species. A primers set, A02ApF/A02ApR, based on the nucleotide sequence of a specific 462 bp fragment obtained from Ac. pisum by RAPD-PCR using primer A02, amplified this fragment only from Ac. pisum genomic DNA, but not from that of other species. The specificity of the primers for M. persicae was further confirmed using individuals from several field populations. It is concluded that RAPD PCR-SCAR (sequence characterized amplified region) DNA assay is a useful method for the detection of species-specific DNA fragments in mixed populations, and that pertinent data could facilitate development of a realistic quarantine system for pea aphids.

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The grain aphid Sitobion miscanthi (Takahashi) is a major pest of cereal crops in China. Sexual reproduction is rarely found in this species, and the reproductive strategy is expected to be reflected in the population genetic structure. Using five microsatellites, we analyzed four populations of S. miscanthi sampled during the 2005–2006 wheat season (from October to May) from a Beijing suburb. Substantial allelic diversity was found, with an average 9.8 alleles per locus, but there were only 45 five-locus genotypes detected among the 143 aphid samples. Of the six commonest five-locus genotypes, four were found in all the seasonal populations, and must have reproduced by continuous parthenogenesis through the seasons (including a winter) because of the big differences between their observed and expected frequencies. All populations showed extremely high heterozygosity and heterozygosity excesses, and significantly deviated from Hardy-Weinberg equilibrium even in data sets including one individual per genotype. Those genetic features occur because S. miscanthi is almost anholocyclic. The population collected immediately after the winter significantly differentiated from the other three populations collected earlier or later (F_ST=0.059–0.064), while differentiations among the other three populations were not significant (F_ST=−0.016–−0.004). The probable causes of this seasonal variation were discussed.

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Syngrapha ottolenguii (Noctuidae: Plusiinae) is a typical Japanese alpine moth with adults appearing from mid to late summer. Field and laboratory studies show that this species has a univoltine cycle in 1 year, although one generation can be completed in 60 days at 16L-8D and 20°C. We found two strategies—short-day-induced prolongation of 2nd and 3rd instar larval stages for winter, and long-day-induced arrestment of reproductive maturation in adults for summer to enable the univoltine life history without obligatory diapause in the severe alpine climate. As a result, oviposition is delayed into autumn, and the late-hatching, cold-tolerant larvae overwinter under deep snow. Larvae before and after overwintering develop very slowly due to their unique behavior of hiding from the sun's radiant heat.

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The effects of different stimulation methods on colony development of Bombus terrestris queens were examined to increase rearing efficiency and reduce production costs. A total of 350 hibernated queens obtained from a commercial company were used. Each queen was placed separately in the starting box and four starting methods were tested: only the queen (q), one queen stimulated with one B. terrestris worker (qtw), one honeybee worker (qhw), and one B. terrestris pupa (qtp). There were significant differences among the treatments in terms of egg laying, colony production and saleable production ratio, the colony initiation time, the time of the emergence of the first worker, the number of egg cells in the first brood. Starting methods had no significant effect on the other colony characteristics. The highest egg laying ratio, colony production ratio and saleable colony production ratio were found in the qtw group (82.8, 53.3, 39.0%) followed by qtp (72.8, 42.8, 28.5%), qhw (57.1, 28.6, 16.2%) and q (35.7, 17.1, 14.3%) groups, respectively. The queens with qtw treatment developed more egg cells (4.39±0.21) in the first brood and started egg laying (11.75±0.83 days) earlier than in the other treatments. The results showed that adding a B. terrestris worker is the best starting method for the mass rearing of B. terrestris. Qtp treatment was found to be the second most successful starting method.

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The late male-killing phenomenon was reported in the oriental tea tortrix, Homona magnanima, but its gross pathology and histopathology have not been elucidated. The present study investigated pathological changes in larvae of a late male-killing strain of H. magnanima (LMK). Most male LMK larvae died during the last instar or pupal stage, and dead male larvae showed characteristic signs and symptoms of male killing. The body color of male LMK larvae started to change 4 d after molting into the fifth instar and became increasingly opaque white. Male LMK larvae weighed significantly less than normal-strain (NSR) males. Midgut epithelial cells of male LMK larvae developed normally and microvilli were observed on the luminal side 3 d after the final molt. Regenerative cells of male LMK larvae were observed on the basement membrane but these cells did not develop to pupal midgut cells. From 7 d after the final molt, midgut cells of male LMK larvae were discharged into the lumen as granules and regenerative cells elongated from the basement membrane. All midgut cells of male LMK larvae dropped into the lumen just before death and no cells were observed on the basement membrane. The fat body of male LMK larvae contained large fat granules in the cytoplasm but became a tumor-like cell mass that finally fused with epidermal cells. These changes were specific to LMK males; no differences in the developmental pattern or morphology were observed between female LMK and NSR larvae. From these results, it is suggested that the degradation of midgut epithelial cells may be the main reason for late male killing.

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In a previous study, we found two amino acid substitutions, Ser431Phe and Ala302Ser, in the Drosophila Ace paralogous acetylcholinesterase (AP-AChE) genes from pirimicarb-resistant Aphis gossypii. Ser431Phe is responsible for the development of pirimicarb resistance and Ala302Ser may play a role in insensitivity to organophosphorus insecticides. In this study, we developed the two types of molecular diagnostics for detecting these mutations. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method was developed to detect the Ser431Phe and Ala302Ser mutations. A PCR amplification of specific alleles (PASA) method, designated to detect Ser431Phe mutations allowed the three genotypes to be discriminated more rapidly and simply than by PCR-RFLP. Genotyping of samples collected from Japanese pear and Satsuma mandarin orchards in Japan was carried out using PCR-RFLP. We revealed that the genetic composition of the A. gossypii orchard population changes with time without exposure to insecticide and that molecular diagnostics are useful for monitoring the development of insecticide resistance. Although both mutations were frequently detected in field populations, pirimicarb-resistant aphids did not seem to always constitute a large proportion in field populations in Japan. We found several individuals which had Ser302 without Phe431. This finding denies the concept that this genotype is inviable from the viewpoint of enzymatic kinetics.

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The proteobacterium Buchnera aphidicola and its aphid hosts have a symbiotic relationship, in which B. aphidicola provides its hosts with essential amino acids. In the case of tryptophan, Buchnera carries genes for the biosynthesis of tryptophan on both its chromosomes (trpB and dnaN) and on plasmids (trpEG). To explore the evolution of B. aphidocola in Myzus persicae (Sulzer), we determined the partial sequences of trpB, dnaN and trpEG from B. aphidicola of four Chinese populations of M. persicae. These genes were found to have characteristics of the corresponding genes in B. aphidicola strains in other aphids. The trpB gene sequence in M. persicae was more closely related to that in Diuraphis noxia, and dnaN and trpEG genes were in the same clade as D. noxia. The phylogenies inferred from trpB, dnaN and trpEG were largely concordant, reflecting the systematics of the host aphids.

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Survival and growth efficiency of fourth-instar larvae of two species of aphidophagous ladybirds, Coccinella septempunctata brucki Mulsant and Harmonia axyridis Pallas were determined when fed aphid, Acyrthosiphon pisum Harris, or intraguild prey, another species of aphidophagous ladybird, Propylea japonica Thunberg. The percentage of fourth instars fed intraguild prey that completed their development was 42.9% for C. septempunctata brucki and 100% for H. axyridis, respectively. All fourth-instar larvae of both species fed aphids completed their development. The average growth efficiency (increase in body weight/weight of prey consumed) of fourth instars of H. axyridis was significantly lower than that of C. septempunctata brucki when fed aphids. In contrast, when fed intraguild prey, it was significantly higher than that of C. septempunctata brucki. Adaptive significance and determinants of prey specialization in aphidophagous ladybirds are discussed.

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Mechanisms of resistance to pirimicarb were surveyed in Iranian populations of the peach-potato aphid, Myzus persicae. Insecticide bioassays were carried out using topical application on three different populations collected from Karaj and Rasht in the Northern provinces. Results of the bioassays indicated that Rasht and Karaj-S populations were susceptible to pirimicarb. A population collected from the college greenhouse (Karaj-R) was resistant to pirimicarb. LD₅₀ (>100 ng/insect) of pirimicarb was not calculated for the Karaj-R population due to limited solubility of this insecticide. The activity and PAGE patterns of esterase in resistant (Karaj-R) and susceptible populations showed that one mechanism of resistance to pirimicarb was esterase-based resistance. Esterase activity of the Karaj-R population was 3.25-fold higher than the susceptible population (Karaj-S), and one of the two esterase bands with higher activity in the Karaj-R population showed strong susceptibility to pirimicarb. The acetylcholinesterase (AChE) of the Karaj-R population had lower affinity to artificial substrates, acetylthiocholine, propionylthiocholine and butyrylthiocholine, than Rasht and Karaj-S populations. The I₅₀ of pirimicarb for Karaj-R, Karaj-S and Rasht populations was 1.89×10⁻⁵ M, 1.37×10⁻⁸ M and 1.37×10⁻⁸ M, respectively. These results suggest that AChE of the Karaj-R population is insensitive to pirimicarb. Ratios of AChE insensitivity of the Karaj-R population to the Karaj-S population were 1,384.6, 11.8 and 2.7 for pirimicarb, monocrotophos and aldicarb, respectively; however, the ratios were 0.32, 0.15 and 0.10 for carbofuran, propoxure and MTMC, respectively, showing negatively correlated sensitivity to pirimicarb-insensitive AChE.

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