野村 昌史

<title>Abstract</title><sec> <title>Objective</title> In insects, closely related species are often difficult or impossible to distinguish solely by morphological traits. Mitochondrial DNA (mtDNA) markers are often useful and reliable for distinguishing closely related species. However, useful mtDNA markers can be unavailable, particularly when such species pairs experienced hybrid introgression in the past. Although polymorphic nuclear DNA markers would be necessary to distinguish such species pairs, recombination, multiple copies, and slower mutation rates of the nuclear DNA compared with those of mtDNA often make it challenging. The objective of this study was to develop a multiplex polymerase chain reaction that can reliably amplify and distinguish the <italic>Tpi</italic> sequences of <italic>Eurema mandarina</italic> and <italic>Eurema hecabe</italic>. </sec><sec> <title>Results</title> We successfully analyzed the nucleotide sequences of the Z chromosome-linked triose phosphate isomerase (<italic>Tpi</italic>) gene to develop a multiplex polymerase chain reaction (PCR) that amplified ca. 120-bp products for <italic>E. mandarina</italic> and ca. 375-bp products for <italic>E. hecabe</italic>. We suggest that multiplex PCR using <italic>Tpi</italic> with appropriately designed primers can be used to accurately and reliably distinguish between other closely related Lepidoptera species. </sec>

<title>Abstract</title> Helicoverpa armigera (Hübner) is a notorious agricultural pest native to the Old World. Recently, its invasion into South and Central America has become a serious problem in the New World. The rapid detection of invasive pests is essential to eradicate them and prevent establishment. However, an extremely similar species, H. zea (Boddie) distributed in the New World makes identification difficult. Helicoverpa armigera and H. zea have only minor differences in male genitalia to separate them morphologically. Both species are attracted to the same pheromone lure, and it takes considerable time and effort to identify them from bulk samples obtained during trap monitoring. Although several molecular approaches based on PCR have been reported, these methods require expensive equipment and are unsuitable for onsite diagnostics. Here, we developed a rapid and convenient diagnostic method based on the loop-mediated isothermal amplification to distinguish H. armigera from related species: H. zea, H. assulta (Guenée), H. punctigera (Wallengren), and Chloridea virescens (Fabricius). The diagnostic method makes it possible to detect H. armigera within 90 min only using simple equipment. The method also worked with mixed DNA templates containing excess DNA from H. zea at the ratio of 1:999 (H. armigera:H. zea). This method can be an effective tool for onsite diagnostics during monitoring surveys for invasive H. armigera.

<title>Abstract</title>It is generally believed that when maternally inherited sex ratio distorters become predominant, either the host population goes extinct or nuclear suppressors evolve in the host. Here, we show an empirical case where all-female-producing <italic>Wolbachia</italic> is likely to be stably maintained at a high frequency. On an island population of the butterfly <italic>Eurema mandarina</italic>, a <italic>Wolbachia</italic> strain <italic>w</italic>Fem, which makes female hosts produce all-female offspring without sibling lethality (female drive), is highly prevalent. We found that, with some fluctuations, <italic>w</italic>Fem appeared to be stably maintained for at least 12 years at a high frequency, resulting in the existence of an abnormally high number of virgin females. Interestingly, comparison between sex ratios of captive individuals and sex ratios deduced from <italic>w</italic>Fem frequencies suggested a plastic behavioral change of males and females in response to the shift of sex ratios. <italic>w</italic>Fem presence does not affect brood size but has a slightly negative effect on body size. Stable coexistence of <italic>w</italic>Fem-positive and -negative females in the population may be explained via mate choice by males, which keeps <italic>w</italic>Fem in check. Taken together, this butterfly population is an attractive model for future studies on the population dynamics of sex ratios and mating behavior.

Evolutionary theory predicts that the spread of cytoplasmic sex ratio distorters leads to the evolution of host nuclear suppressors, although there are extremely few empirical observations of this phenomenon. Here, we demonstrate that a nuclear suppressor of a cytoplasmic male killer has spread rapidly in a population of the green lacewing <italic>Mallada desjardinsi</italic> . An <italic>M. desjardinsi</italic> population, which was strongly female-biased in 2011 because of a high prevalence of the male-killing <italic>Spiroplasma</italic> endosymbiont, had a sex ratio near parity in 2016, despite a consistent <italic>Spiroplasma</italic> prevalence. Most of the offspring derived from individuals collected in 2016 had 1 : 1 sex ratios in subsequent generations. Contrastingly, all-female or female-biased broods appeared frequently from crossings of these female offspring with males derived from a laboratory line founded by individuals collected in 2011. These results suggest near-fixation of a nuclear suppressor against male killing in 2016 and reject the notion that a non-male-killing <italic>Spiroplasma</italic> variant has spread in the population. Consistently, no significant difference was detected in mitochondrial haplotype variation between 2011 and 2016. These findings, and earlier findings in the butterfly <italic>Hypolimnas bolina</italic> in Samoa, suggest that these quick events of male recovery occur more commonly than is generally appreciated.

Partner discrimination is crucial in mutualistic interactions between organisms to counteract cheating by the partner. Trophobiosis between ants and aphids is a model system of such mutualistic interaction. To establish and maintain the mutualistic association, ants need to correctly discriminate mutualistic aphids. However, the mechanism by which ants recognize aphids as their partners is poorly understood, despite its ecological and evolutionary importance. Here, we show for the first time the evidence that interaction with nest-mates that have tended aphids ( <italic>Aphis craccivora</italic> ) allows ants ( <italic>Tetramorium tsushimae</italic> ) to learn to recognize the aphid species as their partner. When ants had previously tended aphids, they moderated their aggressiveness towards aphids. More importantly, ants that had interacted with aphid-experienced nest-mates also reduced their aggressiveness towards aphids, even though they had never directly experienced them, indicating that aphid information was transmitted from aphid-experienced ants to inexperienced ants. Furthermore, inhibition of mouth-to-mouth contact (trophallaxis) from aphid-experienced ants to inexperienced ants by providing the inexperienced ants with artificial honeydew solution caused the inexperienced ants to become aggressive towards aphids. These results, with further supporting data, strongly suggest that ants transfer information on their mutualists during trophallactic interactions.

Maternally inherited <italic>Wolbachia</italic> endosymbionts manipulate arthropod reproduction in various ways. In the butterfly <italic>Eurema mandarina</italic> , a cytoplasmic incompatibility-inducing <italic>Wolbachia</italic> strain <italic>w</italic> CI and the associated mtDNA haplotypes are known to originate from the sister species <italic>Eurema hecabe</italic> , which offered a good case study for microbe-mediated hybrid introgression. Besides <italic>w</italic> CI, some females with the Z0 karyotype harbour a distinct <italic>Wolbachia</italic> strain <italic>w</italic> Fem, which causes all-female production by meiotic drive and feminization. We report that a considerable proportion of <italic>E. mandarina</italic> females (65.7%) were infected with both <italic>w</italic> CI and <italic>w</italic> Fem (CF) on Tanegashima Island. While females singly infected with <italic>w</italic> CI (C) produced offspring at a 1 : 1 sex ratio, CF females produced only females. Although Z-linked sequence polymorphism showed no signs of divergence between C and CF females, mtDNA split into two discrete clades; one consisted of C females and the other CF females, both of which formed a clade with <italic>E. hecabe</italic> but not with uninfected <italic>E. mandarina</italic> . This suggests that CF matrilines also, but independently, experienced a selective sweep after hybrid introgression from <italic>E. hecabe</italic> . Distinct evolutionary forces were suggested to have caused C and CF matrilines to diverge, which would be irreversible because of the particular phenotype of <italic>w</italic> Fem.

Chemical mimicry is an effective strategy when signal receivers recognize and discriminate models by relying on chemical cues. Some aphid enemies mimic the cuticular chemicals of aphids through various means thus avoiding detection and attack by aphid-tending ants. However, because ants have been reported to learn the chemical signatures of aphids in order to distinguish the aphids, the efficacy of chemical mimicry is predicted to depend on the experience of the ants that had tended aphids. The present study tested this hypothesis using two predator species: larvae of the green lacewing Mallada desjardinsi, and larvae of the ladybeetle Scymnus posticalis. Lacewing larvae carry the carcasses of aphids on which they have preyed upon their backs, and these function via chemical camouflage to reduce the aggressiveness of aphid-tending ants toward the larvae. Ladybeetle larvae reportedly produce a covering of wax structures, and their chemicals appear to attenuate ant aggression. We examined whether the behavior of the ant Tetramorium tsushimae toward these predators changed depending on their aphid-tending experience. Ants moderated their aggressiveness toward both predators when they had previously tended aphids, indicating that chemical mimicry by both aphid predators is dependent on previous experience of the ants in tending aphids. Chemical mimicry by the predators of ant-tended aphids is therefore considered to exploit learning-dependent aphid recognition systems of ants.

For maternally transmitted microbes, a female-biased host sex ratio is of reproductive advantage. Here we found a strong female bias in a field population of the green lacewing, Mallada desjardinsi (Insecta; Neuroptera). This bias was attributed to the predominance of individuals harboring a maternally inherited male-killing bacterium that was phylogenetically closely related to the plant-pathogenic Spiroplasma phoeniceum and Spiroplasma kunkelii. Among 35 laboratory-reared broods produced by wild-caught females, 21 broods (60%)-all infected with Spiroplasma-consisted of only females (940 individuals). Among 14 broods consisting of both males and females (516 and 635 individuals, respectively), 4 broods were doubly infected with Spiroplasma and Rickettsia, 6 broods were singly infected with Rickettsia, and 3 broods were uninfected (remaining one brood was unknown). Mortality during embryonic and larval development was prominent in all-female broods but not in normal sex ratio broods. Following antibiotic treatment on all-female broods, mortality was significantly reduced and the sex ratio was restored to 1:1. Strong expression and high prevalence of this male-killer is remarkable considering its low density (~10-5-10-4 cells per host mitochondrial gene copy based on quantitative PCR). In addition, a bacterium closely related to Rickettsia bellii was present in 25 of 34 broods (73.5%), irrespective of the sex ratio, with the infection density comparable to other cases of endosymbiosis (~10-2-10-1 cells per mitochondrial gene copy). Higher density of Rickettsia than Spiroplasma was also demonstrated by electron microscopy which visualized both Spiroplasma-like cells and Rickettsia-like cells inside and outside the ovarian cells.

In ant-aphid associations, many aphid species provide ants with honeydew and are tended by ants, whereas others are never tended and are frequently preyed upon by ants. In these relationships, ants must have the ability to discriminate among aphid species, with mutualistic aphids being accepted as partners rather than prey. Although ants reportedly use cuticular hydrocarbons (CHCs) of aphids to differentiate between mutualistic and non-mutualistic species, it is unclear whether the ability to recognize mutualistic aphid species as partners is innate or involves learning. Therefore, we tested whether aphid recognition by ants depends on learning, and whether the learning behavior is species-specific. When workers of the ant Tetramorium tsushimae had previously tended the cowpea aphid, Aphis craccivora, they were less aggressive toward this species. In addition, ants also reduced their aggressiveness toward another mutualistic aphid species, Aphis fabae, after tending A. craccivora, whereas ants remained aggressive toward the non-mutualistic aphid, Acyrthosiphon pisum, regardless of whether or not they had previous experience in tending A. craccivora. When ants were offered glass dummies treated with CHCs of these aphid species, ants that had tended A. craccivora displayed reduced aggression toward CHCs of A. craccivora and A. fabae. Chemical analyses showed the similarity of the CHC profiles between A. craccivora and A. fabae but not with A. pisum. These results suggest that aphid recognition of ants involves learning, and that the learning behavior may not be species-specific because of the similarity of CHCs between different aphid species with which they form mutualisms.

Ants attack and exclude natural enemies of aphids in ant-aphid mutualisms. However, larvae of the green lacewing, Mallada desjardinsi, prey on the cowpea aphid, Aphis craccivora, without exclusion by aphid-tending ants. Lacewing larvae are protected from ants by carrying aphid carcasses on their backs. Here, we tested whether cuticular hydrocarbons (CHCs) of aphid carcasses affected the aggressiveness of aphid-tending ants. Aphid carcasses were washed with n-hexane to remove lipids. Lacewing larvae with washed aphid carcasses were attacked by aphid-tending ants more frequently than those with untreated aphid carcasses. We measured the aggressiveness of aphid-tending ants to lacewing larvae that were either carrying a piece of cotton wool (a dummy aphid carcass) treated with CHCs from aphids or lacewing larvae, or carrying aphid carcasses. The rates of attack by ants on lacewing larvae carrying CHCs of aphids or aphid carcasses were lower than that of attack on lacewing larvae with conspecific CHCs. Chemical analysis by gas chromatography/mass spectrometry showed similarity of CHCs between aphids and aphid carcasses. These results suggest that aphid carcasses on the backs of lacewing larvae function via chemical camouflage to limit attacks by aphid-tending ants.

黄色LEDパルス光を用いたキクの害虫防除光源装置を開発するために，実際栽培において適用可能な放射照度の範囲を求めた．明期20 ms/暗期80 msの時間構造を有する黄色パルス光をオオタバコガとハスモンヨトウの複眼に照射し，放射照度1～1000 mW m^-2の範囲においてERG信号波形を解析した．その結果，少なくとも1～100 mW m^-2であれば，両種ともに点滅光として常時安定的，かつ持続的に視認していることが明らかとなった．続いて，黄色パルス光の放射照度(0，20，35，50 mW m^-2)が秋ギク'神馬'の開花および切り花形質に及ぼす影響を調査した．35 mW m^-2までの放射照度は，発蕾までの日数，開花までの日数および切り花形質に有意な影響を及ぼさなかった．しかし，50 mW m^-2の放射照度で育てたキクは，他の処理区と比較して，開花までの日数が3日間増加し，切り花長が9 cm大きくなった．以上の結果から，適用できる放射照度の上限値は，放射照度が35～50 mW m^-2の範囲に存在し，少なくとも35 mW m^-2であれば，開花遅延させることなく適用可能であることが明らかとなった

Because of their importance and abundance as predators of pest insects in agroecosystems around the world, eight species of lacewings from four genera (Apertochrysa, Chrysopa, Chrysoperla, and Dichochrysa) were investigated to evaluate their performance when reared on eggs of Ephestia kuehniella Zeller, a factitious prey commonly used in mass rearing systems. In particular, a comparative assessment of trash-carrying versus naked species (ones not carrying trash on their backs) was performed. We measured the developmental times and survival rates of the eight green lacewings and compared these characteristics between trash-carrying and naked lacewing species. Survival during immature development was higher (7787%) for the two naked species (Chrysopa pallens (Rambur) and Chrysoperla suzukii (Okamoto)) than for the trash-carrying species D. alcestes (33%). Immature and larval developmental times were significantly longer among the trash-carrying species than the naked species. The rapid development of naked green lacewings may be a strategy to decrease predation risk by aphid guarding ants and intra-guild predators. © 2012 Entomological Society of America. © 2012 Entomological Society of America.

Three plusiine species, Autographa nigrisigna, Macdunnoughia confusa, and Thysanoplusia intermixta (Lepidoptera: Noctuidae), are commonly found together in lettuce, Lactuca sativa L., fields in Japan. Given the marked morphological similarities between these species and the difficulty associated with discriminating between them using only visual cues, we used multiplex polymerase chain reaction (PCR) assay to distinguish between the three target species. Multiplex PCR uses four primers to simultaneously amplify a specific region of the mitochondrial DNA and produce species-specific banding patterns. The stringency of the method was tested using specimens of different sex, location, and developmental stage, and consistent results were obtained for all samples. Indeed, our method has the potential to clarify the species structure of plusiine species in lettuce fields. © 2011 Entomological Society of America.

Larvae of the green lacewing Mallada desjardinsi Navas are known to place dead aphids on their backs. To clarify the protective role of the carried dead aphids against ants and the advantages of carrying them for lacewing larvae on ant-tended aphid colonies, we carried out some laboratory experiments. In experiments that exposed lacewing larvae to ants, approximately 40% of the larvae without dead aphids were killed by ants, whereas no larvae carrying dead aphids were killed. The presence of the dead aphids did not affect the attack frequency of the ants. When we introduced the lacewing larvae onto plants colonized by ant-tended aphids, larvae with dead aphids stayed for longer on the plants and preyed on more aphids than larvae without dead aphids. Furthermore, the lacewing larvae with dead aphids were attacked less by ants than larvae without dead aphids. It is suggested that the presence of the dead aphids provides physical protection and attenuates ant aggression toward lacewing larvae on ant-tended aphid colonies. © 2011 The Japanese Society of Applied Entomology and Zoology.

We describe the complete mitochondrial genomes of the green lacewing species Chrysoperla nipponensis (Okamoto, 1914) and Apochrysa matsumurae Okamoto 1912 (Neuroptera: Chrysopidae). The genomes were 16,057 and 16,214 bp in size, respectively, and comprised 37 genes (13 protein coding genes, 22 tRNA genes and two rRNA genes). A major noncoding (control) region was 1,244 bp in C. nipponensis and 1,407 in A. matsumurae, and the structure was simpler than that reported in other Neuroptera, lacking conserved blocks or long tandem repeats. The overall arrangement of genes was almost the same as that found in most arthropod mitochondrial genomes, with the one exception of a tRNA rearrangement to tRNA-Cys-tRNA-Trp-tRNA-Tyr, rather than the plesiomorphic tRNA-Trp-tRNA-Cys-tRNA-Tyr. A high A + T content (78.89 and 79.02%, respectively), A + T-rich codon bias, and a mismatch between the mostused codon and its corresponding tRNA anticodon were observed as a typical feature of the insect mitochondrial genome. © Springer Science+Business Media B.V. 2010.

本研究は, LEDのもつ優れた応答性に着目して, キクに開花遅延させることなく, ヤガ類に対して高い防除効果を発揮する物理的防除装置の開発を目指している. 黄色LEDを特定の明期と暗期の時間構造によって終夜パルス点灯させることで, 当初の目標をほぼ達成することができた. 以下に得られた結論を示す.<BR>(1) ヤガ類 (ハスモンヨトウおよびオオタバコガ) 成虫において, 光強度1.2 mW m^-2 (1 lx相当), 明期10 msおよび暗期10 msのパルス光照射に対して, ERG信号の応答特性を確認した.<BR>(2) 当該2種のヤガ類に対する光防除にあたっては, 同一の時間構造のパルス光で対応できる可能性が高いことが示唆された.<BR>(3) ERG信号の応答特性を踏まえ, 終夜照明下の秋ギクは, 光強度が小さい場合, 明期と暗期が共に10 msのパルス光を採用すれば, 開花遅延を回避できることが明らかとなった.<BR>(4) 光強度が19 m W m^-2と大きい場合は, 明期が10 msであれば 50 ms以上の暗期を確保することで, 秋ギクの開花遅延を回避できることが明らかとなった.<BR>(5) 明期20 ms, 暗期80 msとする終夜のパルス点灯下で, キクに寄生したオオタバコガの幼虫数を予備的に調査した結果, 無処理 (無照明) と比較して寄生幼虫数は少ないことを確認した.

A maternally-inherited spiroplasma endosymbiont of Drosophila hydei does not exert apparent phenotypes on both sexes of its host and is prevalent in natural populations of D. hydei. Our previous experiments using a laboratory stock of D. hydei revealed that low temperatures (such as 15°C and 18°C) dramatically lower the vertical transmission rates of this spiroplasma. Therefore, we hypothesized that, in temperate regions, the infection frequencies may decrease in cool seasons but increase in the summer season. To clarify the temporal population dynamics of the spiroplasma infection, D. hydei were collected from two Japanese populations in 2006-2008 from May to early August, representing the only period when a number of D. hydei are collectable in Japan, and examined for spiroplasma infection. Within each year, the frequency of spiroplasma infection fluctuated considerably in both populations. Consistent with our hypothesis, the infection frequency showed an increasing trend in both populations in 2007. However, the data in 2006 and 2008 did not show consistent patterns of increase. The population dynamics of spiroplasma infection may be affected but not critically determined by temperature. Moreover, despite the fluctuation within each year, the infection frequencies seemed to be stable across the years. The frequencies of spiroplasma infection in D. hydei populations may be stabilized by multiple factors. One of these factors may involve a context-dependent positive effect of spiroplasma on the fitness of D. hydei, as was recently observed in laboratory experiments. © 2010 Springer Science+Business Media B.V.

The substrate-borne vibrational courtship songs of type A and type B Chrysoperla nipponensis (Okamoto) (Neuroptera: Chrysopidae) are described in detail, from populations sampled throughout Japan and near Beijing, China. The song of type A is long (≈ 5-6 s) and is made up of numerous (four to 12) volleys of four distinct forms that differ in their carrier frequencies. The song of type B is shorter (≈2 s) and functions as a repeated single-volley song, but each volley is shown here to consist of four to six distinct subsections. During heterosexual duets, partners of type A exchange their long multi-volley songs, whereas partners of type B repeatedly exchange short single-volley songs. Geographical variation within each song type is low and largely insignificant. In addition, previously recognized mitochondrial (cytochrome oxidase subunit I) haplotypes Al and A2 of type A C. nipponensis do not have different songs. These results support the hypothesis that types A and B C. nipponensis are distinct species, with type A corresponding to the nominate species (i.e., C. nipponensis s. str.) and type B as yet unnamed. Their songs are among the most complex yet found in the Chrysoperla carnea swarm of cryptic species. © 2009 Entomological Society of America.

Wolbachia, belonging to Alphaproteobacteria, is ubiquitously found in arthropods and filarial nematodes, and is known to manipulate the reproduction of its hosts in various ways, such as feminization, male killing, induction of parthenogenesis or induction of cytoplasmic incompatibility. We found that the Wolbachia infection frequencies of the butterfly Colias erate poliographus were high (85.7100%) in seven Japanese populations. Crossing experiments and rearing revealed that the Wolbachia strain exhibited strong cytoplasmic incompatibility and perfect vertical transmission in C. erate poliographus. Moreover, a comparison of the survival rates between infected and cured broods suggested that Wolbachia infection had beneficial effects on host fitness. Our findings suggested that the high infection frequencies in Japanese populations have been accomplished by these advantageous traits of the Wolbachia strain. Furthermore, the multilocus sequence typing (MLST) scheme revealed that the Wolbachia in C. erate poliographus is a novel strain (ST141), belonging to supergroup B. © 2008 Cambridge University Press.

Agrobacterium-mediated genetic transformation was applied to produce transgenic plants of spinach (Spinacia oleracea) resistant to 2 pest species, Trichoplusia ni and Autographa nigrisigna. Leaf segments from in vitro spinach plants of cultivar 'Glory' were co-cultivated with A. tumefaciens strain EHA105, which harbored the plasmid pBE2111FMB containing a synthetic cry1Ac gene encoding an insecticidal crystal protein of Bacillus thuringiensis, and neomycin phosphotransferase II and bialaphos resistance genes as selectable marker genes. Kanamycin (Km)-resistant calluses were obtained from co-cultivated leaf segments 1 month after selection on 2.5 g l-1 gellan gum-solidified MS medium containing 30 g l-1 sucrose, 0.5 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D), 2mg l-1 kinetin, 100 mg l-1 Km and 20 mg l-1 meropenem trihydrate. Regeneration of adventitious shoots from Km-resistant calluses was performed on 2.5 g l -1 gellan gum-solidified MS medium containing 30 g l-1 sucrose, 0.01 mg l-1 2,4-D, 1mg l-1 kinetin, 1mg l -1 gibberellic acid, 100 mg l-1 Km and 10 mg l -1 meropenem trihydrate at 14°C. PCR and Southern blot analyses of genomic DNA isolated from T1 plants confirmed the successful integration of T-DNA into the plant genome. Expression of Cry1Ac protein was confirmed in leaves of transgenic plants by Western blot analysis. Insect bioassays against T. ni and A. nigrisigna performed with T1 plants showed more than 93.3% insect mortality within 1 week. These results suggest that the cry1Ac gene was effectively expressed in spinach.

We surveyed a 1,033-bp fragment of the cytochrome oxidase subunit I (COI) sequence to look for a useful diagnostic marker for discrimination between the Japanese cryptic species Chrysoperla nipponensis (Okamoto) (Neuroptera: Chrysopidae) (types A and B) and the commercially introduced Chrysoperla carnea s. str. from Germany. Phylogenetic analysis showed four distinct haplotype groups: one group corresponded to C. nipponensis type B and one group to C. carnea s. str., and the remaining two distinct groups, A1 and A2, corresponded to the same song phenotype, C. nipponensis type A. A2 was linked with the group of C. carnea s. str. and A1 was linked with the group of C. nipponensis type B. Cross-testing between A1 and A2 revealed their reproductive compatibility. Mitochondrial polyphyly within C. nipponensis type A may, therefore, originate from introgression or incomplete lineage sorting. Within the mitochondrial COI region presented here, there were 17 variable nucleotide sites useful for discriminating the four haplotype groups from each other. © 2008 Entomological Society of America.

Maternally transmitted endosymbionts of the genus Spiroplasma infecting several species of Drosophila are known to cause selective death of male offspring (male killing). The male-killing trait is considered to be advantageous for maternally transmitted endosymbionts. However, a non-male-killing spiroplasma is present in Japanese populations of Drosophila hydei at high frequencies (23-66%). This spiroplasma is phylogenetically closely related to the male-killing spiroplasma infecting other Drosophila species. It is unknown why this spiroplasma is maintained in its host populations despite its inability to cause male killing. We examined the susceptibilities of the spiroplasma in D. hydei to four different temperatures (28, 25, 18, and 15 degrees C). Diagnostic PCR revealed that vertical transmission of the spiroplasma was nearly perfect at 28 and 25 degrees C, partially suppressed at 18 degrees C, and completely blocked at 15 degrees C. Furthermore, quantitative PCR demonstrated that offspring treated at 18 degrees C exhibited dramatically lower densities of spiroplasma (i.e., approximately one-tenth) compared to offspring treated at 28 and 25 degrees C. Considering the low temperatures during winter in Japan, some unknown advantageous effects of the spiroplasma that compensate for the failure of vertical transmission are suggested to act in natural populations of D. hydei.

柏市のこんぶくろ池周辺に生育しているズミ集団の遺伝的劣化が懸念されているが，その実態究明と保全方法の検討のために，こんぶくろ池を含む関東地域7集団の遺伝的構造と花の形態変異を調べた。アロザイム分析の結果，全集団のヘテロ接合度期待値は0.22と双子葉植物としては高い値を示したが，こんぶくろ池ズミ集団のそれは0.247でもっとも高かった。遺伝的距離より作成した系統図からは，こんぶくろ池集団は東関東グループに属すると考えられた。しかし，花の形態は，アロザイム分析より得られた地理的グループにとらわれない独特な特徴を持っていた。これらのことから，こんぶくろ池ズミ集団の遺伝的劣化は起きておらず，個体群再生のためには，他地域集団からの移植によらず当地自生集団より苗木を育成することが適当といえる。

Systematic relationships among higher taxa within Chrysopidae, a large and agriculturally significant neuropteran family, are poorly understood. A molecular phylogenetic survey of Chrysopidae was performed with three nuclear genes, namely wingless (546 bp), phosphoenolpyruvate carboxykinase (483 bp), and sodium/potassium ATPase alpha subunit (410 bp). We examined 83 species in 24 genera, mainly from Japan, Eurasia and Africa. Parsimony and Bayesian analyses of combined datasets of a total of 1439 bp demonstrated that (1) monophyly of the subfamily Chrysopinae was supported but the relationship between Nothochrysinae and Apochrysinae was unclear, although the two subfamilies together may constitute the sister taxon of Chrysopinae; (2) of the three tribes examined within Chrysopinae (Ankylopterygini, Belonopterygini and Chrysopini), monophyly of Ankylopterygini and Belonopterygini was supported, but the relationships among the three remain unclear; (3) seven sub-clades in Chrysopini were indicated, namely (i) Brinckochrysa, (ii) Chrysemosa + Suarius, (iii) Chrysotropia + Nineta, (iv) Mallada + Chrysoperla + Peyerimhoffina, (v) Cunctochrysa + Meleoma + Nipponochrysa + Apertochrysa albolineatoides, (vi) Chrysopa + Plesiochrysa, and (vii) Dichochrysa + Apertochrysa eurydera; and (4) most genera were monophyletic, except for Apertochrysa and Cunctochrysa, each of which was shown to have two distinct origins. Our molecular analysis allowed the assignment of several species of uncertain affinities to known genera. There was some disagreement between the molecular and previously published morphological phylogenies, but in general our results confirmed existing morphological hypotheses of evolution within the family.

In butterflies, the adult sex ratio observed in the field is usually male-biased, although the sex ratio of their progeny is 1:1. This is due to the higher motility and larger behavioral range of males than females. As expected, the sex ratio of Eurema hecabe butterflies collected at 6 localities throughout Japan was male-biased. However, in Tsukuba, located in the central part of Japan, the sex ratio was found to be biased toward females. Their progeny reared in the laboratory also exhibited a female-biased sex ratio. A single strain of Wolbachia is considered to be the cause of the sex-ratio distortion, because antibiotic treatment reversed the sex ratio to 1:1, and only a single nucleotide sequence of wsp, a highly variable Wolbachia gene, was detected by molecular analysis. Cytogenetic analysis excluded the possibility of feminization as the underlying mechanism. In addition, when the wild-caught females that had already mated in nature were treated with antibiotics before oviposition, egg-hatch rates were extremely low, suggesting that the same Wolbachia strain also caused cytoplasmic incompatibility. Our findings suggest the possibility that a single strain of Wolbachia induces 2 distinct reproductive manipulations in the same host.