Daisuke Kageyama

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.

Opposite sex–specific effects of Wolbachia and interference with the sex determination of its host Ostrinia scapulalis

In the adzuki bean borer, Ostrinia scapulalis, the sex ratio in most progenies is 1 : 1. Females from Wolbachia–infected matrilines, however, give rise to all–female broods when infected and to all–male broods when cured of the infection. These observations had been interpreted as Wolbachia–induced feminization of genetic males into functional females. Here, we show that the interpretation is incorrect. Females from both lines have a female karyotype with a WZ sex–chromosome constitution while males are ZZ. At the time of hatching from eggs, WZ and ZZ individuals are present at a 1 : 1 ratio in broods from uninfected, infected and cured females. In broods from Wolbachia–infected females, ZZ individuals die during larval development, whereas in those from cured females, WZ individuals die. Hence, development of ZZ individuals is impaired by Wolbachia but development of WZ females may require the presence of Wolbachia in infected matrilines. Sexual mosaics generated (i) by transfection of uninfected eggs and (ii) by tetracycline treatment of Wolbachia–infected mothers prior to oviposition were ZZ in all tissues, including typically female organs. We conclude that: (i) Wolbachia acts by manipulating the sex determination of its host; and (ii) although sexual mosaics can survive, development of a normal female is incompatible with a ZZ genotype.

Prevalence of Cardinium Bacteria in Planthoppers and Spider Mites and Taxonomic Revision of “Candidatus Cardinium hertigii” Based on Detection of a New Cardinium Group from Biting Midges

ABSTRACT Cardinium bacteria, members of the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), are intracellular bacteria in arthropods that are capable of inducing reproductive abnormalities in their hosts, which include parasitic wasps, mites, and spiders. A high frequency of Cardinium infection was detected in planthoppers (27 out of 57 species were infected). A high frequency of Cardinium infection was also found in spider mites (9 out of 22 species were infected). Frequencies of double infection by Cardinium and Wolbachia bacteria (Alphaproteobacteria capable of manipulating reproduction of their hosts) were disproportionately high in planthoppers but not in spider mites. A new group of bacteria, phylogenetically closely related to but distinct from previously described Cardinium bacteria (based on 16S rRNA and gyrB genes) was found in 4 out of 25 species of Culicoides biting midges. These bacteria possessed a microfilament-like structure that is a morphological feature previously found in Cardinium and Paenicardinium. The bacteria close to the genus Cardinium consist of at least three groups, A, B, and C. Group A is present in various species of arthropods and was previously referred to as “Candidatus Cardinium hertigii,” group B is present in plant parasitic nematodes and was previously referred to as “Candidatus Paenicardinium endonii,” and group C is present in Culicoides biting midges. On the basis of morphological and molecular data, we propose that the nomenclature of these three groups be integrated into a single species, “Candidatus Cardinium hertigii.”

Feminizing Wolbachia in an insect, Ostrinia furnacalis (Lepidoptera: Crambidae)

Wolbachia, which forms a group of maternally inherited bacteria in arthropods, often cause reproduction alterations in their hosts, such as cytoplasmic incompatibility, parthenogenesis, male-killing, hybrid breakdown and feminization. To date, Wolbachia-induced feminization has been reported only in isopods. Here we report that a Wolbachia strain feminizes an insect host, Ostrinia furnacalis. Among 79 wild females of O. furnacalis examined, Wolbachia infection was detected in 13 females. Twelve of the 13 infected females produced all-female progenies, and this trait was maternally inherited. Tetracycline treatment of thelygenic matrilines resulted in the production of all-male progenies. The present findings indicate that the Wolbachiainfection induces feminization of genetic males in O. furnacalis. Differences in the Wolbachia-induced feminization in O. furnacalis and that in isopods are discussed along with the differences in sex determination mechanisms between insects and isopods. Phylogenetic analysis of the wsp gene sequence of Wolbachiasuggests independent evolutionary origins for the Wolbachia-induced feminizations in O. furnacalis and in isopods. Our findings over 5 years suggest that the infection has been maintained at a low prevalence in the O. furnacalis population.

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.

Female-biased sex ratio in the Asian corn borer, Ostrinia furnacalis : evidence for the occurrence of feminizing bacteria in an insect

The maternally inherited, female-biased sex ratio in many arthropods has been attributed to infection with micro-organisms. Male killing, thelytoky and feminization are recognized as the mechanisms of the bacteria-induced sex ratio distortion in arthropods. A sex ratio distortion towards the female has been found in the Japanese population of the Asian corn borer Ostrinia furnacalis (Lepidoptera). In the present study, inheritance of the sex ratio distortion in the Asian corn borer and its underlying mechanism were investigated. Two of the 13 field-collected females produced all-female offspring. The female-biased sex ratio was maternally inherited for three generations. There was no difference in egg hatchability between thelygenic and normal crosses. Treatment with tetracycline for two generations resulted in production of only male offspring. Considering the sex determination system in lepidopteran insects, it was concluded that chromosomal males are feminized by a cytoplasmic agent(s), most probably parasitic bacteria. This is the first report of the occurrence of feminizing bacteria in insects. The sex ratio distorter was considered to occur at low frequency in the Asian corn borer population.

Prevalence of a Non-Male-Killing Spiroplasma in Natural Populations of Drosophila hydei

ABSTRACT Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.

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.

Male killing and incomplete inheritance of a novel spiroplasma in the moth Ostrinia zaguliaevi.

Bacteria of the genus Spiroplasma are widely found in plants and arthropods. Some of the maternally transmitted Spiroplasma endosymbionts in arthropods are known to kill young male hosts (male killing). Here, we describe a new case of Spiroplasma-induced male killing in a moth, Ostrinia zaguliaevi. The all-female trait caused by Spiroplasma was maternally inherited for more than 11 generations but was spontaneously lost in several lineages. Antibiotic treatment eliminated the Spiroplasma infection and restored the 1:1 sex ratio. The survival rates and presence/absence of the W chromosome in the embryonic and larval stages of O. zaguliaevi showed that males were selectively killed, exclusively during late embryogenesis in all-female broods. Based on phylogenetic analyses of 16S rRNA, dnaA and rpoB gene sequences, the causative bacteria were identified as Spiroplasma belonging to the tick symbiont Spiroplasma ixodetis clade. Electron microscopy confirmed bacterial structures in the follicle cells and follicular sheath of adult females. Although many congeneric Ostrinia moths harbor another sex ratio-distorting bacterium (Wolbachia), only O. zaguliaevi harbors Spiroplasma.

Sex-specific death in the Asian corn borer moth (Ostrinia furnacalis) infected with Wolbachia occurs across larval development

Maternally inherited endosymbiotic bacteria of the genus Wolbachia induce various kinds of reproductive alterations in their arthropod hosts. In a Wolbachia-infected strain of the adzuki bean borer moth, Ostrinia scapulalis (Lepidoptera: Crambidae), males selectively die during larval development, while females selectively die when Wolbachia are eliminated by antibiotic treatment. We found that naturally occurring Wolbachia in the congener O. furnacalis caused sex-specific lethality similar to that in O. scapulalis. Cytogenetic analyses throughout the entire larval development clarified that the death of males (when infected) and females (when cured) took place mainly during early larval stages. However, some individuals also died after complete formation of larval bodies but before egg hatching, or at late larval stages, even in the penultimate instar. Although the specific timing was highly variable, death of males and females occurred before pupation without exception. The potential association of sex-specific lethality with the sex determination mechanism was also examined and is discussed.