Phylogeographical evidence for historical long-distance dispersal in the flightless stick insect Ramulus mikado.

Exploring how organisms overcome geographical barriers to dispersal is a fundamental question in biology. Passive long-distance dispersal events, although infrequent and unpredictable, have a considerable impact on species range expansions. Despite limited active dispersal capabilities, many stick insect species have vast geographical ranges, indicating that passive long-distance dispersal is vital for their distribution. A potential mode of passive dispersal in stick insects is via the egg stage within avian digestive tracts, as suggested by experimental evidence. However, detecting such events under natural conditions is challenging due to their rarity. Therefore, to indirectly assess the potential of historical avian-mediated dispersal, we examined the population genetic structure of the flightless stick insect Ramulus mikado across Japan, based on a multifaceted molecular approach [cytochrome oxidase subunit I (COI) haplotypes, nuclear simple sequence repeat markers and genome-wide single nucleotide polymorphisms]. Subsequently, we identified unique phylogeographic patterns, including the discovery of identical COI genotypes spanning considerable distances, which substantiates the notion of passive long-distance genotypic dispersal. Overall, all the molecular data revealed the low and mostly non-significant genetic differentiation among populations, with identical or very similar genotypes across distant populations. We propose that long-distance dispersal facilitated by birds is the plausible explanation for the unique phylogeographic pattern observed in this flightless stick insect.

Hybridisation between host races broadens the host range of offspring in Eotetranychus asiaticus (Acari: Tetranychidae).

Host adaptation in herbivorous arthropods is one of the first steps to sympatric speciation, and spider mites (Acari: Tetranychidae) are useful model organisms for studying this phenomenon. Many researchers have studied the process of host adaptation via artificial selection experiments. Recent analyses suggest that hybridisation has diversified host ranges, although empirical evidence is scarce. We explored the host exploitation of two strains of Eotetranychus asiaticus established from Ternstroemia gymnanthera (Pentaphylacaceae) and Japanese cinnamon, Cinnamomum yabunikkei (Lauraceae), and evaluated the effect of hybridisation on offspring host use. Transplant experiments showed that females oviposited and immature mites developed only on their native hosts, suggesting specialisation to the secondary metabolites of each host plant. However, F1 hybrids from reciprocal crosses developed on both host plants (survival rate: 92-100%) with normal female-biased sex ratios. Furthermore, all backcrosses to the parental strains yielded B1 offspring that were also viable on both host plants with normal sex ratios (69-87% and 39-92% females on T. gymnanthera and C. yabunikkei, respectively). B1 survival rates in interstrain crosses were varied (11-63%) and lower than those in intrastrain crosses (88-93%). We could not detect any reproductive barriers in these experiments, and host preference may be the sole factor determining pre-mating isolation. The survival rates and sex ratios we observed suggest cytochromosome interactions. In conclusion, hybridisation, which results in heterozygotes and recombination, is an underexplored way to provide spider mites with a novel host plant.

Development of microsatellite markers for the geographically parthenogenetic stick insect Phraortes elongatus (Insecta: Phasmatodea).

Many plant and animal species exhibit geographic parthenogenesis, wherein unisexual (= parthenogenetic) lineages are more common in their marginal habitats such as high latitude or altitudes than their closely related bisexual counterparts. The Japanese stick insect, Phraortes elongatus (Thunberg) (Insecta: Phasmatodea), is known as a geographically parthenogenetic species due to the existence of both bisexual and unisexual populations. Here, we developed microsatellite markers to infer the genetic variation among populations of P. elongatus. Totally, 13 primer pairs were developed for the species, and they were tested on 47 samples collected from both a bisexual population and a unisexual population. All 13 loci were polymorphic in the bisexual population, whereas no loci were polymorphic in the unisexual population. The loss of variation in the unisexual population implies automixis with terminal fusion or gamete duplication as the mode of parthenogenesis. The markers developed in this study will be helpful for further comprehensive analysis of the genetic diversity and gene flow between bisexual and parthenogenetic lineages of P. elongatus.

Photoperiodic control of reproductive arrest in the oak-inhabiting spider mite Schizotetranychus brevisetosus (Acari: Tetranychidae).

Populations of Schizotetranychus brevisetosus Ehara (Acari: Tetranychidae), which live on the evergreen oak (Quercus glauca), survive the coldest months as either adult females or winter eggs. Adult females comprise the majority of the population in early November and oviposit from late November to early March. Most winter eggs hatch by late March, and adults of the next generation emerge in April. This species is considered an egg-diapausing species, but the environmental cues that regulate female reproductive arrest and resumption are mostly unknown. We investigated the photoperiodic responses of autumn reproductive arrest in 10 populations collected from different elevations in Shikoku, Japan. All populations showed long-day responses to critical daylength (CDL) around 12.2 h light (12.2L) at 20 °C, though there was no linear relationship between CDL and altitude. This result explains the steep decline in the proportion of summer eggs in November. Nonreproductive females developed under 10L at 20 °C commenced oviposition 14.3-20.6 days after transferring to 15L. This long pre-oviposition period explains the reduction in eggs before winter reproduction and suggests shallow adult diapause. Eggs thus obtained hatched in 12.9-15.3 days, similarly to summer eggs. Therefore, egg diapause in S. brevisetosus is much shallower than in species on deciduous hosts, which lay their winter eggs in early autumn to hatch at leaf flush in spring. The reproductive arrest and short hatching period may be an adaptation allowing egg-laying in midwinter, when predation pressure is low.

Production of winter eggs in Schizotetranychus brevisetosus (Acari: Tetranychidae) inhabiting evergreen Japanese blue oak.

The overwintering pattern of parasitic herbivorous arthropods is closely related to host phenology, because defoliation imposes strong selection pressures on various developmental stages. This relationship has been well studied in populations of spider mites (Acari: Tetranychidae) on deciduous hosts, but is little studied in populations on evergreen hosts, probably because their leaves are always available. However, spring defoliation may also influence the life cycle. We studied the overwintering pattern of Schizotetranychus brevisetosus (Acari: Tetranychidae), a specialist on evergreen oak, Quercus glauca, in Kochi, Japan. Only adult females and their eggs (winter eggs) survived the coldest months. We also observed a conspicuous seasonal change in egg colour and size: December winter eggs were 1.7× larger than September summer eggs, suggesting the winter eggs are diapausing. Adult females produced summer eggs until November and winter eggs from late November until they disappeared in March. The winter eggs hatched in early March. The immature stages developed in mid- to late March, when 39% of old leaves fell, some of which carried hundreds of immature individuals. Therefore, even in evergreen hosts, defoliation places potentially strong selection pressure on the immature stages. Despite this, S. brevisetosus appears not to change its egg hatching season to avoid the defoliation season, so it may have evolved a behavioural adaptation to escape falling leaves. The existence of several species with similar lifestyles suggests the importance of host phenology and predation pressure for evolution of the overwintering pattern.

Variation in nesting behavior of eight species of spider mites, Stigmaeopsis having sociality.

Nesting behavior is considered to be an important element of social living in animals. The spider mites belonging to the genus Stigmaeopsis spend their lives within nests produced from silk threads. Several of these species show cooperative sociality, while the others are subsocial. In order to identify the origins of this social behavior, comparisons of nest sizes, nesting behaviors (making nests continuously or separately), and their associated traits (fecal deposition patterns) were made for eight cogeneric Stigmaeopsis species showing various levels of social development. All of these species inhabit bamboo plants (Poaceae). We initially addressed the proximate factor of nest size variation. The variation in nest size of the eight species corresponded well with the variation in dorsal seta sc1 length, suggesting that nest size variation among species may have a genetic basis. The time spent within a nest (nest duration) increased with nest size on the respective host plants. Nest arrangement patterns varied among species showing different sized nests: Large nest builders continuously extended their nests, while middle and small nest-building species built new separate nests, which resulted in different social interaction times among species, and is thought to be closely related to social development. Fecal deposition behaviors also varied among Stigmaeopsis species, suggesting diversity in anti-predatory adaptations. Finally, we discuss how the variation in sociality observed within this genus is likely the result of nest size variation that initially evolved as anti-predator strategies.

Lethal male combats in Schizotetranychus brevisetosus (Acari: Tetranychidae) on blue Japanese oak (Quercus glauca).

Male spider mites (Acari: Tetranychidae) guard preimaginal quiescent females to reserve paternity. In a few species of Stigmaeopsis that make densely woven silky nests on the surface of host plants, mature males attack and even kill rivals to gain their rights to inseminate young females in the nest. Here we report that the adult males of Schizotetranychus brevisetosus (Acari: Tetranychidae), whose web nest construction behavior evolved independently from Stigmaeopsis spp., also exhibit highly aggressive behavior towards one another. Combat mortality was measured in an experimental design where two males in the treatment group were forced to live in a nest made by adult females on a leaf arena, while one male in the control group was allowed to live in a nest on another arena. We found that the 5-day mortality of males in the treatment group (56 and 37 % in two replicates) was significantly higher than in the control group (9 and 12 %) and that lethal combats frequently arose in the treatment group. However, we found no consistent trend for the length of the first legs, which is known to affect the outcome of battle in Tetranychus urticae and Stigmaeopsis miscanthi. This might be due to a relatively small number of measurable specimens, and the effects of body size should be explored in future studies.

Reproductive isolation between Stigmaeopsis celarius and its sibling species sympatrically inhabiting bamboo (Pleioblastus spp.) plants.

Stigmaeopsis celarius Banks (hereafter Sc) is a spider mite living and feeding on the leaves of various bamboo species such as Moso bamboo [Phyllostachys edulis (=P. pubescens)] and Pleioblastus spp. (Poaceae). A previous phylogenetic study revealed a cryptic, phylogenetic sister species to Sc (hereafter Ss). Although its life type appears to be similar to that of Sc, individuals of Ss make much smaller nests compared with Sc, and the nests have been found mostly on Nezasa bamboo (Pleioblastus argenteostriatus). To investigate whether Sc and Ss are reproductively isolated, we explored their populations in southwestern Japan, and crossed them to examine mating behaviors and fertilization success. Field surveys revealed that the nests of these two species occur on the same leaves and, thus, the individuals of these species may make frequent contact. Reciprocal crosses suggested that the two species are reproductively isolated. Though Sc males have tried to mate with Ss females, copulation seldom occurred because of their long opisthosoma (hind body), which prevented the insertion of the aedeagus into the genitalia of Ss females. In contrast, most Ss males ignored Sc females, and eggs were not fertilized even in the few cases where copulation appeared to occur. These results suggest that strong selection pressure is imposed on body length to prevent interspecific hybridization in the contact area of these species.

Effectiveness of cervical cancer screening based on a mathematical screening model using data from the Hiroshima Prefecture Cancer Registry.

Here we assessed the effectiveness of cervical cancer screening using data from the Hiroshima Prefecture Cancer Registry regarding patient age at the start of screening and differences in screening intervals. A screening model was created to calculate the health status in relation to prognosis following cervical cancer screening and its influence on life expectancy. Epidemiological data on the mortality rate of cervical cancer by age groups and mortality rates from the Hiroshima Prefecture Cancer Registry were used for the model projections. Our results showed that life expectancy when screening rate was 100% compared with 0% was extended by approximately 1 month. Furthermore, when the incidence of cervical cancer was 0% compared with the screening rate was 100%, life expectancy was extended by a maximum of 3 months. Moreover, among individuals affected by cervical cancer, a difference of 13 years in life expectancy was calculated between screened and unscreened groups.

Relationship between body colour, feeding, and reproductive arrest under short-day development in Tetranychus pueraricola (Acari: Tetranychidae).

In Tetranychus spider mites (Acari: Tetranychidae), diapausing females have a conspicuous orange body colour, which is used as an indicator of diapause induction in many laboratory studies. However, to which extent body colour reflects reproductive activity is scarcely investigated. In this study, we investigated the relationship between body colour, reproductive arrest, and food intake in the inbred strain of T. pueraricola individually reared at 20 °C with a 10:14 h light: dark photoperiod. Our results showed that (1) body colour is a good indicator of reproductive arrest 11 days after adult emergence but does not completely reflect reproductive status at an earlier age; (2) even orange females intermittently feed, and the arrest of feeding comes after the change in body colour; and (3) reproducing females have a higher risk of death than non-reproducing females. These results suggest that measurement of diapause incidence by body colour alone may miss the variation in reproductive status in early adult life.

Host suitability of house fly, Musca domestica (Diptera: Muscidae), pupae killed by high or low temperature treatment for a parastoid, Spalangia endius (Hymenoptera: Pteromalidae).

The objective of this study was to establish a high quality progeny production system for the house fly parasitoid, Spalangia endius (Hymenoptera: Pteromalidae), by stockpiling hosts. We performed two host killing methods before host storage: (i) heat-killed by 30 min exposure to 50°C or (ii) freeze-killed by 10 min exposure to -80°C. The average number of parasitoids that emerged from nonstored house fly pupae after heat- or freeze-killing was not significantly different from live pupae. When house fly pupae stored at -20°C after heat-killing were supplied to S. endius, progeny production was significantly less than live pupae. Moreover, productivity became very low when house fly pupae refrigerated at 3°C after heat- or freeze-killing were supplied to S. endius. On the other hand, when house fly pupae stored at -80°C for 1 year after heat-killing were supplied to S. endius, the average number of parasitoids that emerged was not significantly different from live pupae. The average number of parasitoids that emerged from freeze-killed hosts kept for more than 8 weeks at -80°C was significantly fewer than live pupae. Thus, this study clarified that a higher-quality host can be maintained not only by simply storing at -80°C but also by adding heat treatment before storage.

Coelomic fluid: a noninvasive source of DNA in earthworms.

To investigate whether coelomic fluid secreted by earthworms can be a noninvasive source of DNA, we amplified and sequenced DNA extracted from the coelomic fluid and muscle tissue of eight worms. The sequences obtained using DNA extracted from both sources were identical. All cytochrome c oxidase subunit I (COI) mitochondrial DNA sequences, including those retrieved from GenBank, formed a monophyletic group of Metaphire sieboldi. The results indicate that we successfully extracted total DNA from coelomic fluid secreted by earthworm.

Analysis of genetic variation and phylogeny of the predatory bug, Pilophorus typicus, in Japan using mitochondrial gene sequences.

Pilophorus typicus (Distant) (Heteroptera: Miridae) is a predatory bug occurring in East, Southeast, and South Asia. Because the active stages of P. typicus prey on various agricultural pest insects and mites, this species is a candidate insect as an indigenous natural enemy for use in biological control programs. However, the mass releasing of introduced natural enemies into agricultural fields may incur the risk of affecting the genetic integrity of species through hybridization with a local population. To clarify the genetic characteristics of the Japanese populations of P. typicus two portions of the mitochondrial DNA, the cytochrome oxidase subunit I (COI) (534 bp) and the cytochrome B (cytB) (217 bp) genes, were sequenced for 64 individuals collected from 55 localities in a wide range of Japan. Totals of 18 and 10 haplotypes were identified for the COI and cytB sequences, respectively (25 haplotypes over regions). Phylogenetic analysis using the maximum likelihood method revealed the existence of two genetically distinct groups in P. typicus in Japan. These groups were distributed in different geographic ranges: one occurred mainly from the Pacific coastal areas of the Kii Peninsula, the Shikoku Island, and the Ryukyu Islands; whereas the other occurred from the northern Kyushu district to the Kanto and Hokuriku districts of mainland Japan. However, both haplotypes were found in a single locality of the southern coast of the Shikoku Island. COI phylogeny incorporating other Pilophorus species revealed that these groups were only recently differentiated. Therefore, use of a certain population of P. typicus across its distribution range should be done with caution because genetic hybridization may occur.

Overwintering survival and postdiapause fecundity in a population of the Kanzawa spider mite Tetranychus kanzawai (Acari: Tetranychidae) on Orixa japonica (Rutaceae).

In several adult-diapausing insects and mites, long duration of diapause decreases egg production after diapause termination. However, such nonlethal effects are much less studied than overwintering survival. In this study, lethal and nonlethal effects of different periods of diapause were investigated in an adult-diapausing spider mite, Tetranychus kanzawai Kishida (Acari: Tetranychidae), through field experiments. Diapausing females produced in the laboratory were kept in their natural habitat from October or November to the next April, and survival rate and postdiapause fecundity were compared between the months. The survival rate was lower, but not significantly, in the October treatment. Postdiapause fecundity was also not significantly different between the months. These results suggest that the effects of diapause on survival and reproduction are quite small after October, when diapausing mites are increasing in their natural habitat.

Effect of host plants on diapause induction in immature and adult Tetranychus kanzawai (Acari: Tetranychidae).

I determined the difference in sensitivity of immature and adult mites to host plants with regard to diapause induction. I used immature and adult polyphagous spider mites, Tetranychus kanzawai Kishida (Acari: Tetranychidae), and two host plants, Orixa japonica Thunb. (Rutaceae) and Phaseolus vulgaris L. (Fabaceae). The diapause incidence was investigated in two adult mite-host and two immature mite-host combinations of treatments. The incidence of diapause induction was much higher in mites developing on O. japonica than those on P. vulgaris, whichever host plants they fed on in adulthood. The host plants affected adult mites to a much lesser extent than the immature mites, though the mites feeding on O. japonica showed a consistently higher diapause incidence. These results suggest that host plants affect diapause induction to a greater extent in immature mites than in adult mites.

Dominant and recessive inheritance patterns of diapause in the two-spotted spider mite Tetranychus urticae.

In this study, we investigated the diapause incidence in 3 geographic strains of the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae). Under diapause-inducing conditions of 12:12 light:dark at 15 degrees C, the diapause incidence was nearly 100% in a strain from northern Japan (Sapporo), whereas it was nearly 0% in 2 strains from southern Japan (Itoman and Takanabe). Reciprocal crosses clearly showed that the nondiapause phenotype is inherited in a completely dominant manner, and no maternal effect was detected. Backcrosses to the Itoman and Takanabe strains suggested that dominant nondiapause alleles control the nondiapause phenotype. To clarify the genetic basis of nondiapause in the northern population, we also established a nondiapausing variant ("selected nondiapause" abbreviated as snd) from the Sapporo strain. Crossing experiments revealed that a single recessive allele is responsible for the nondiapause phenotype. Thus, both dominant and recessive inheritance patterns of diapause were detected in the T. urticae populations studied here.

Suppression of ovarian development and vitellogenin gene expression in the adult diapause of the two-spotted spider mite Tetranychus urticae.

Tetranychus urticae (Acari: Tetranychidae) possesses a sac-like ovary with characteristic oocytes that protrude from the ovarian surface. In nondiapause females, transparent oocytes became opaque with yolk deposition between days 0 and 1 in the adult stage at 20 degrees C. In diapause females, however, ovarian development ceased at a stage having transparent oocytes without yolk deposition; this stage corresponded to the day-0 stage of the nondiapause females. Four partial fragments of the vitellogenin (Vg) genes of T. urticae were isolated. This is the first report on the Vg genes of mites. The deduced amino acid sequences of these four Vg gene fragments contained the von Willebrand factor D domain and the GLCG motif, which were reported to be the common features of Vg sequences in insects and ticks. Northern blot analysis did not detect Vg mRNA in the diapause adult females of T. urticae. It is, therefore, suggested that diapause mites do not synthesize Vg mRNA and that vitellogenesis is regulated at the transcriptional level in diapause.

Development times under long- and short-day conditions in the Kanzawa spider mite Tetranychus kanzawai (Acari: Tetranychidae).

In a theoretical framework, the development time of arthropods is closely associated with population growth, so that strong selection pressure may be imposed on development patterns. In this context, selection towards shorter development times may act on female mites in the season when population size is growing. In contrast, such selective pressure may be weaker just before hibernation, because the stage adjustment may be more important than population growth in such seasons. Thus, selection to shorten development times may act more strongly in long daylength conditions, so that different developmental responses among the seasons (reaction norm) may evolve. I predicted that the development time is shorter under the long-day conditions of population-growing seasons than under the short-day conditions of hibernating seasons, and that genetic variation at the long-day is smaller than that at the short-day. In order to test this prediction, the egg-to-adult development time of a population of Tetranychus kanzawai (Acari: Tetranychidae) was investigated under short (9L:15D) and long (15L:9D) daylength conditions at 18 degrees C. In addition, genetic variation in female development times under these conditions was estimated by using half-sib design. The development time of females at short day was 1 day longer than at long day under the experimental conditions. Male development times follow the pattern of females with <1 day delay. However, heritabilities under these two treatments were quite low and neither was significant. Thus, only the first prediction was supported by the results. In addition, maternal and/or non-additive effects in the female development time were highly significant under both daylength conditions examined.