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.

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.”

When the butterfly Eurema hecabe is infected with two different strains ( w HecCI2 and w HecFem2) 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.

In insects, antimicrobial humoral immunity is governed by two distinct gene cascades, IMD pathway mainly targeting Gram-negative bacteria and Toll pathway preferentially targeting Gram-positive bacteria, which are widely conserved among diverse metazoans. However, recent genomic studies uncovered that IMD pathway is exceptionally absent in some hemipteran lineages like aphids and assassin bugs. How the apparently incomplete immune pathways have evolved with functionality is of interest. Here we report the discovery that, in the hemipteran stinkbug Plautia stali , both IMD and Toll pathways are present but their functional differentiation is blurred. Injection of Gram-negative bacteria and Gram-positive bacteria upregulated effector genes of both pathways. Notably, RNAi experiments unveiled significant functional permeation and crosstalk between IMD and Toll pathways: RNAi of IMD pathway genes suppressed upregulation of effector molecules of both pathways, where the suppression was more remarkable for IMD effectors; and RNAi of Toll pathway genes reduced upregulation of effector molecules of both pathways, where the suppression was more conspicuous for Toll effectors. These results suggest the possibility that, in hemipterans and other arthropods, IMD and Toll pathways are intertwined to target wider and overlapping arrays of microbes, which might have predisposed and facilitated the evolution of incomplete immune pathways.

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.

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.