Satoko Narita

Genetic structure of sibling butterfly species affected by Wolbachia infection sweep: evolutionary and biogeographical implications

It was recently recognized that in Japan, the common yellow butterfly, Eurema hecabe, consists of two sibling species, which have been unnamed yet and tentatively called yellow (Y) type and brown (B) type. We investigated the diversity of nuclear and mitochondrial genes in Japanese populations of Y type and B type of E. hecabe. The phylogeny based on nuclear genes agreed with the distinction between Y type and B type, which had been also supported by a wide array of biological data. However, the phylogeny based on mitochondrial genes did not reflect the distinction. PCR survey of Wolbachia revealed that B‐type populations were all infected while Y‐type populations contained both infected and uninfected individuals. A single genotype of Wolbachia, which was inferred to be a CI‐inducing strain from their wsp gene sequence, was prevalent in these populations. Notably, the mitochondrial phylogeny was in perfect agreement with the pattern of Wolbachia infection, suggesting that the Wolbachia infection had affected the mitochondrial genetic structure of the host insects. Probably, the Wolbachia strain and the associated mitochondrial genomes have been occasionally introduced from B‐type populations to Y‐type populations through migration and subsequent interspecific hybridization, and CI‐driven population sweep has been spreading the Wolbachia strain and the particular mitochondrial haplotypes, which originated from B‐type populations, into Y‐type populations. On the basis of these results together with the geological and biogeographical knowledge of the Japanese Archipelago, we proposed an evolutionary hypothesis on the invasion and spread of Wolbachia infection in B‐type and Y‐type of E. hecabe.

Unexpected Mechanism of Symbiont-Induced Reversal of Insect Sex: Feminizing Wolbachia Continuously Acts on the Butterfly Eurema hecabe during Larval Development

ABSTRACT When the butterfly Eurema hecabe is infected with two different strains (wHecCI2 and wHecFem2) of the bacterial endosymbiont Wolbachia, genetic males are transformed into functional females, resulting in production of all-female broods. In an attempt to understand how and when the Wolbachia endosymbiont feminizes genetically male insects, larval insects were fed an antibiotic-containing diet beginning at different developmental stages until pupation. When the adult insects emerged, strikingly, many of them exhibited sexually intermediate traits in their wings, reproductive organs, and genitalia. The expression of intersexual phenotypes was strong in the insects treated from first instar, moderate in the insects treated from third instar, and weak in the insects treated from fourth instar. The insects treated from early larval instar grew and pupated normally but frequently failed to emerge and died in the pupal case. The dead insects in the pupal case contained lower densities of the feminizing Wolbachia endosymbiont than the successfully emerged insects, although none of them were completely cured of the symbiont infection. These results suggest the following: (i) the antibiotic treatment suppressed the population of feminizing Wolbachia endosymbionts; (ii) the suppression probably resulted in attenuated feminizing activity of the symbiont, leading to expression of intersexual host traits; (iii) many of the insects suffered pupal mortality, possibly due to either intersexual defects or Wolbachia-mediated addiction; and hence (iv) the feminizing Wolbachia endosymbiont continuously acts on the host insects during larval development for expression of female phenotypes under a male genotype. Our finding may prompt reconsideration of the notion that Wolbachia-induced reproductive manipulations are already complete before the early embryonic stage and provide insights into the mechanism underlying the symbiont-induced reversal of insect sex.

Insect Sex Determination Manipulated by Their Endosymbionts: Incidences, Mechanisms and Implications

The sex-determining systems of arthropods are surprisingly diverse. Some species have male or female heterogametic sex chromosomes while other species do not have sex chromosomes. Most species are diploids but some species, including wasps, ants, thrips and mites, are haplodiploids (n in males; 2n in females). Many of the sexual aberrations, such as sexual mosaics, sex-specific lethality and conversion of sexuality, can be explained by developmental defects including double fertilization of a binucleate egg, loss of a sex chromosome or perturbation of sex-determining gene expression, which occur accidentally or are induced by certain environmental conditions. However, recent studies have revealed that such sexual aberrations can be caused by various groups of vertically-transmitted endosymbiotic microbes such as bacteria of the genera Wolbachia, Rickettsia, Arsenophonus, Spiroplasma and Cardinium, as well as microsporidian protists. In this review, we first summarize the accumulated data on endosymbiont-induced sexual aberrations, and then discuss how such endosymbionts affect the developmental system of their hosts and what kinds of ecological and evolutionary effects these endosymbionts have on their host populations.

Gynandromorphs and intersexes: potential to understand the mechanism of sex determination in arthropods

Arthropods are sexually dimorphic. An arthropod individual usually differentiates into a male or a female. With very low frequencies, however, individuals with both male and female morphological characters have repeatedly been found in natural and laboratory populations of arthropods. Gynandromorphs (i.e., sexual mosaics) are genetically chimeric individuals consisting of male and female tissues. On the other hand, intersexes are genetically uniform (i.e., complete male, complete female or intermediate in every tissue) but all or some parts of their tissues have either a sexual phenotype opposite to their genetic sex or an intermediate sexual phenotype. Possible developmental processes (e.g., double fertilization of a binucleate egg, loss of a sex chromosome or upregulation/downregulation of sex-determining genes) and causal factors (e.g., mutations, genetic incompatibilities, temperatures or endosymbionts) for the generation of gynandromorphs and intersexes are reviewed and discussed.

Molecular phylogeography of two sibling species of Eurema butterflies

The common yellow butterfly Eurema hecabe is widely distributed in East Asia, and is one of the most burdensome species for taxonomists due to the numerous geographic and seasonal wing colour patterns. Moreover, within this species, individuals with a yellow wing fringe that occur in temperate regions of Japan (Y type) proved to be biologically different from others that occur widely in subtropical regions of Japan and all over East Asia (B type). To unveil the genetic variation within and between the two types, a total of 50 butterflies collected at 18 geographic localities in East Asia were examined for nucleotide sequence variation of three mitochondrial regions: cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and NADH dehydrogenase subunit 5 (ND5). In addition, they were also examined for infection status with the endosymbiotic bacteria Wolbachia. The three mitochondrial sequences consistently showed that (i) Y type and B type were highly divergent, (ii) nucleotide variation within B type was very small although sampled from a geographically wide range, and (iii) a weak association existed between mitochondrial DNA haplotypes and Wolbachia infection status.

Strong cytoplasmic incompatibility and high vertical transmission rate can explain the high frequencies of Wolbachia infection in Japanese populations of Colias erate poliographus (Lepidoptera: Pieridae).

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 (85.7-100%) 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.

Wolbachia-induced feminisation newly found in Eurema hecabe, a sibling species of Eurema mandarina (Lepidoptera: Pieridae)

1. Complete feminisation of genetic males into functional females, a unique case among insects, is known in Eurema mandarina (former Eurema hecabe Y type) that are infected with two strains of Wolbachia, wCIEm and wFemEm.

A natural population of the butterfly Eurema hecabe with Wolbachia-induced female-biased sex ratio not by feminization

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.

A GYNANDROMORPH OF EUREMA HECABE (LEPI-DOPTERA: PIERIDAE) FROM JAPAN

ABSTRACT A gynandromorph of a pierid butterfly, Eurema hecabe, caught at Mt. Tsukuba in Central Japan is described here. Wings were apparently bilaterally asymmetric, while the external and internal genitalia clearly showed the morphology and genotype of the female. The fact that this individual showed a female genotype excluded the possibility of incomplete feminization due to Wolbachia infection. Presence of a spermatophore in the bursa copulatrix and the difference in number of matured eggs between the right and left ovaries indicated that this gynandromorphic individual had copulated and oviposited in the field.

Master manipulation continued: feminizing Wolbachia endosymbiont distorts sex chromosome inheritance

Wolbachia is a maternally inherited ubiquitous endosymbiotic bacterium of arthropods that displays a diverse repertoire of host reproductive manipulations. For the first time, we demonstrate that Wolbachia manipulates sex chromosome inheritance in a sexually reproducing insect. Eurema mandarina butterfly females on Tanegashima Island, Japan, are infected with the wFem Wolbachia strain and produce all-female offspring, while antibiotic treatment results in male offspring. Fluorescence in situ hybridization (FISH) revealed that wFem-positive and wFem-negative females have ZO and WZ sex chromosome sets, respectively, demonstrating the predicted loss of the W chromosome from wFem-infected lineages. Genomic quantitative polymerase chain reaction (qPCR) analysis showed that wFem-positive females lay only ZO eggs that carry a paternal Z, whereas females from lineages that are naturally wFem-negative lay both WZ and ZZ eggs. In contrast, antibiotic treatment of adult wFem females resulted in the production of ZO and ZZ eggs, suggesting that this Wolbachia strain can induce meiotic drive. Moreover, most male offspring produced by antibiotic-treated wFem females had a ZZ karyotype, implying reduced survival of ZO individuals in the absence of feminizing effects of Wolbachia. Antibiotic treatment of wFem-infected larvae induced male-specific splicing of the doublesex (dsx) gene transcript, causing an intersex phenotype. Thus, the loss of the female-determining W chromosome in ZO individuals is functionally compensated by Wolbachia feminization. We discuss how Wolbachia may manipulate oogenesis to cause meiotic drive and that Wolbachia may have acquired this coordinated dual mode of reproductive manipulation first by the evolution of feminization and then cytoplasmically induced meiotic drive. Significance Statement Genomes are vulnerable to selfish genetic elements that enhance their own transmission often at the expense of host fitness. These include cytoplasmic elements such as endosymbiotic bacteria that cause feminization, male-killing, parthenogenesis and cytoplasmic incompatibility. We demonstrate, for the first time, that meiotic drive, a phenomena so far seen only for nuclear genetic elements, can also be caused by the ubiquitous endosymbiotic bacterium Wolbachia. In female butterflies with a ZO sex chromosome constitution, Wolbachia prevents the production of ZZ zygotes. Wolbachia also compensates for the female-determining function of the W chromosome lost from infected lineages, thereby causing the production of all-female progeny. Our findings highlight that cytoplasmic elements play an important role in sex determination systems and sex chromosome evolution.Genomes are vulnerable to selfish genetic elements that enhance their own transmission often at the expense of host fitness. Examples are cytoplasmic elements such as maternally inherited bacteria that cause feminization, male-killing, parthenogenesis and cytoplasmic incompatibility. We demonstrate, for the first time, that segregation distortion, a phenomenon so far seen only for nuclear genetic elements, can also be caused by a cytoplasmic element, the ubiquitous endosymbiotic bacterium Wolbachia . For Eurema mandarina butterfly lineages with a Z0 sex chromosome constitution, we provide direct and conclusive evidence that Wolbachia induces production of all-female progeny by a dual role: the compensation for the female-determining function that is absent in Z0 lineages (feminization) and the prevention of maternal sex chromosome inheritance to offspring as a specific type of segregation distortion. Therefore, our findings highlight that both sex determination and chromosome inheritance - crucially important developmental processes of higher eukaryotes - can be manipulated by cytoplasmic parasites.