Conflict between Feminizing Sex Ratio Distorters and an Autosomal Masculinizing Gene in the Terrestrial Isopod Armadillidium Vulgare Latr

Female sex determination in the pill bug Armadillidium vulgare is frequently under the control of feminizing parasitic sex factors (PSF). One of these PSF is an intracytoplasmic Wolbachia-like bacterium (F), while the other (f) is suspected of being an F-bacterial DNA sequence unstably integrated into the host genome. In most wild populations harboring PSF, all individuals are genetic males (ZZ), and female phenotypes occur only due to the presence of PSF which overrides the male determinant carried by the Z chromosome (females are thus ZZ +F or ZZ +f neo-females). Here we report the effects of the conflict between these PSF and a dominant autosomal masculinizing gene (M) on phenotypes. The M gene is able to override the feminizing effect of the f sex factor and, consequently, male sex may be restored. However, M is unable to restore male sex when competing with the F bacteria. It seems that the main effect of M is to delay the expression of F bacteria slightly, inducing intersex phenotypes. Most of these intersexes are functional females, able to transmit the masculinizing gene. The frequency of M and its effects on the sex ratio in wild populations are discussed.     

Experimental study of temperature effects on the sex ratio of broods in Terrestrial Crustacea Armadillidium vulgare Latr. Possible implications in natural populations

The woodlouse Armadillidium vulgare is characterized by female heterogamety (ZW) and male homogamety (ZZ). However, in several populations, sex determination is influenced by cytoplasmic sex factors (endosymbiotic bacteria = F). At 20 °C these maternally transmitted bacteria reverse genetic males into functional neo-females (ZZ + F) producing highly female broods. When these neo-females were reared at 30° C, the sex ratio of their broods became male-biased. The major process involved in this heat-induced sex ratio inversion was the disappearance of bacteria in embryos in the course of their development, which allowed the young to express a phenotype that conforms with their genotype (i.e. male ZZ). No heat-sensitive stage of development was observed, but at least 35 days at 30° C seem to be necessary to induce F-degradation. The presence of F at 30° C (before its degradation) also induced mortality during vitellogenesis. Daily thermoperiods including a thermophase at 30° C had effects on F similar to that of a constant temperature of 30° C. A. vulgare can live in climates having such thermoperiods (at least during one period of the year), temperature appears to be capable of limiting the presence of F-bacteria in natural populations, and then modifying the evolution of sex-determining mechanisms in such populations.     

Mitochondrial DNA polymorphism and feminizing sex factors dynamics in a natural population of Armadillidium vulgare (Crustacea,Isopoda)

Sex determination in Armadillidium vulgare may be under the control of two parasitic sex factors that reverse genetic males into functional neo-females. The first feminizing factor (F) is a Wolbachia and the other (f) is probably a sequence of the F bacterial DNA unstably integrated into the host genome. Both of these feminizing factors are mainly maternally transmitted. Here we investigate the mitochondrial DNA polymorphism of wild iso-female lineages harbouring either F or f. Among the four haplotypes present in the population, two were the f-harbouring lineages, while two were common to the F- and f-harbouring lineages. This result suggests that there has been an introgression of the f factor into lineages infected by F Wolbachia. Based on previous data, we propose two different ways to account for such introgression. Given the particular dynamics of feminizing factors (f-harbouring lineages increase in populations at the expense of F-harbouring lineages), such an introgression should prevent the replacement of F-linked mitochondrial types by f-linked mitochondrial types in wild populations.     

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.     

Double trouble: combined action of meiotic drive and Wolbachia feminization in Eurema butterflies

Arthropod sex ratios can be manipulated by a diverse range of selfish genetic elements, including maternally inherited Wolbachia bacteria. Feminization by Wolbachia is rare but has been described for Eurema mandarina butterflies. In this species, some phenotypic and functional females, thought to be ZZ genetic males, are infected with a feminizing Wolbachia strain, wFem. Meanwhile, heterogametic WZ females are not infected with wFem. Here, we establish a quantitative PCR assay allowing reliable sexing in three Eurema species. Against expectation, all E. mandarina females, including wFem females, had only one Z chromosome that was paternally inherited. Observation of somatic interphase nuclei confirmed that W chromatin was absent in wFem females, but present in females without wFem. We conclude that the sex bias in wFem lines is due to meiotic drive (MD) that excludes the maternal Z and thus prevents formation of ZZ males. Furthermore, wFem lines may have lost the W chromosome or harbour a dysfunctional version, yet rely on wFem for female development; removal of wFem results in all-male offspring. This is the first study that demonstrates an interaction between MD and Wolbachia feminization, and it highlights endosymbionts as potentially confounding factors in MD of sex chromosomes.     

Sexual selection in an isopod with Wolbachia‐induced sex reversal: males prefer real females

A variety of genetic elements encode traits beneficial to their own transmission. Despite their ‘selfish’ behaviour, most of these elements are often found at relatively low frequencies in host populations. This is the case of intracytoplasmic Wolbachia bacteria hosted by the isopod Armadillidium vulgare that distort the host sex ratio towards females by feminizing the genetic males they infect. Here we tested the hypothesis that sexual selection against Wolbachia‐infected females could maintain a polymorphism of the infection in populations. The infected neo‐females (feminized males) have lower mating rates and received less sperm relative to uninfected females. Males exhibited an active choice: they interacted more with uninfected females and made more mating attempts. A female behavioural difference was also observed in response to male mating attempts: infected neo‐females more often exhibited behaviours that stop the mating sequence. The difference in mating rate was significant only when males could choose between the two female types. This process could maintain a polymorphism of the infection in populations. Genetic females experimentally infected with Wolbachia are not exposed to the same sexual selection pressure, so the infection alone cannot explain these differences.     

Sex determination in crayfish: are intersex Cherax quadricarinatus (Decapoda, Parastacidae) genetically females?

In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae), a gonochoristic species, seven different combinations of intersex individuals (with both male and female genital openings) have been described. However, to date, the genetic basis for this phenomenon has not been investigated. This study was designed to test a simple chromosome-based sex-determination model for C. quadricarinatus that assumes the male to be the homogametic (ZZ) sex. According to our model, intersex individuals that are functionally males are genetically females (WZ). Individual crosses were performed between intersex and female crayfish, with control crosses being performed between normal males and females. The control crosses yielded, in most cases, the expected 1:1 sex ratio in the F1 progeny. Crosses between intersex individuals and females yielded a 1:3 (male:female) sex ratio in most crosses. According to our hypothesis, one-third of the females produced in a cross of a female with an intersex animal should be WW females. The hypothesis was tested by crossing normal males with F1 females, which were progeny of intersex fathers. These crosses yielded almost 100% females, a finding that conforms to the above-suggested sex determination model for C. quadricarinatus and the female WZ genotype of intersex individuals.     

Rapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvae

Two different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female‐specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W‐specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2‐EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2‐EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co‐amplified the CpW2‐EcoRI sequence to identify the W chromosome and the Z‐linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth.     

Production of WW super females by diploid gynogenesis in Xenopus laevis

Summary In Xenopus laevis, which does not show sex chromosomal dimorphism, the female is heterogametic (WZ) and the male is homogametic (ZZ). By activating eggs with UV-irradiated spermatozoa, and by inhibiting the formation of the second polar body gynogenetic diploids were obtained, including some WW females. These super-females are fertile and not sublethal; by gynogenetic reproduction they in turn generate only WW females, while after mating with a male they give rise to WZ females exclusively.From the sex ratio of the gynogenetic progeny of normal WZ females, the map distance between the centromere and the sex determining gene(s) has been calculated. By examining the sex ratio and the distribution of individuals exhibiting the phenotype of periodic albinism in the gynogenetic offspring of a^p/+females, it has been demonstrated that the a^p gene and the sex determining gene(s) are not linked.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

The evolution of sex determination in isopod crustaceans

Sex is determined by non-Mendelian genetic elements overriding the sex factors carried by the heterochromosomes in some species of terrestrial isopods. A bacterium Wolbachia and a non-bacterial feminizing factor (f) can both force chromosomal males of Armadillidium vulgare to become phenotypic functional females. The f factor is believed to be a genetic element derived from the Wolbachia genome that becomes inserted into the host nuclear genome. The feminizing factors can be considered to be selfish genetic elements because they bias their host's sex ratio to increase their own transmission. New sex-determining genes are selected (genes resisting the feminizing effects, or the transmission of feminizing elements) as a consequence of the conflict between these elements and the rest of the host's genome. These events drive the sex-determining mechanisms to evolve, and may explain the polymorphism of sex factors and the poor differentiation of the heterochromosomes in isopods.     

Misdirection of dosage compensation underlies bidirectional sex-specific death in Wolbachia-infected Ostrinia scapulalis

Endosymbiotic bacteria of the genus Wolbachia often manipulate the reproductive system of their hosts to propagate themselves in host populations. Ostrinia scapulalis moths infected with Wolbachia (wSca) produce female-only progeny (sex chromosomes: ZW), whereas females cured of the infection by antibiotic treatment produce male-only progeny (ZZ). The occurrence of female- and male-only progeny has been attributed to the specific death of the opposite sex during embryonic and larval development. In this bidirectional sex-specific lethality, embryos destined to die express a phenotypic sex opposite to their genotypic sex. On the basis of these findings, we suggested that wSca carries a genetic factor that feminizes the male host, the W chromosome of the host has lost its feminizing function, and discordance between the genotypic and phenotypic sexes underlies this sex-specific death. In the present study, we examined whether the failure of dosage compensation was responsible for this sex-specific mortality. Quantitative PCRs showed that Z-linked gene expression levels in embryos destined to die were not properly dosage compensated; they were approximately two-fold higher in the male progeny of wSca-infected females and approximately two-fold lower in the female progeny of infected-and-cured females. These results support our hypothesis that misdirection of dosage compensation underlies the sex-specific death.     

Induction par la température d'une physiologie mâle chez les néo-femelles et les intersexués du Crustacé Oniscoïde Armadillidium vulgare Latr., hébergeant un bactéroïde à action féminisante

In the Oniscoid Armadillidium vulgare, inside the thelygenic line of the Niort population, intersexed females, whatever their sizes, and most of the young neo-females from 5 to 6 mm long (males feminized by a polytropic intracytoplasmic bacteroid kept for 13— 47 days at 35°C, and then replaced at 20°C, are masculinized. The masculinization of their external sexual characters is more or less complete, and the ovary is changed into a functional testicle with one or several utricles, each of them having an androgenic neogland. This masculinization, which restores a phenotype corresponding with the genotype, goes with the disappearance of the typical forms of the bacteroid, such as they are observed in neo-females and the intersexed individuals kept at 20° C.

Yet, this male physiology is only temporary: a female physiology is restored after the animals have been kept at 20° C for 2–4 mth, but the acquired male differentiation is maintained. This implies that special forms of bacteroids continue to exist and that, when the host is again kept at 20° C, they produce the typical feminizing factors.

The absence of masculinization in neo-female adults is not due to the maintenance of the bacteroid, but to the impossibility of inducing the differentiation of an androgenic neo-gland after the 6th molt of the postembryonic development; the cells of the primal androgenic gland—which exist in all females—have then completely disappeared, or have definitively turned into conjunctival cells. Masculinization does not occur either in real young females (genetic females), which proves that temperature is only an indirect cause, and acts by inhibition of the feminization action of the bacteroid.     

Wild Sex in Zebrafish: Loss of the Natural Sex Determinant in Domesticated Strains

Sex determination can be robustly genetic, strongly environmental, or genetic subject to environmental perturbation. The genetic basis of sex determination is unknown for zebrafish (Danio rerio), a model for development and human health. We used RAD-tag population genomics to identify sex-linked polymorphisms. After verifying this “RAD-sex” method on medaka (Oryzias latipes), we studied two domesticated zebrafish strains (AB and TU), two natural laboratory strains (WIK and EKW), and two recent isolates from nature (NA and CB). All four natural strains had a single sex-linked region at the right tip of chromosome 4, enabling sex genotyping by PCR. Genotypes for the single nucleotide polymorphism (SNP) with the strongest statistical association to sex suggested that wild zebrafish have WZ/ZZ sex chromosomes. In natural strains, “male genotypes” became males and some “female genotypes” also became males, suggesting that the environment or genetic background can cause female-to-male sex reversal. Surprisingly, TU and AB lacked detectable sex-linked loci. Phylogenomics rooted on D. nigrofasciatus verified that all strains are monophyletic. Because AB and TU branched as a monophyletic clade, we could not rule out shared loss of the wild sex locus in a common ancestor despite their independent domestication. Mitochondrial DNA sequences showed that investigated strains represent only one of the three identified zebrafish haplogroups. Results suggest that zebrafish in nature possess a WZ/ZZ sex-determination mechanism with a major determinant lying near the right telomere of chromosome 4 that was modified during domestication. Strains providing the zebrafish reference genome lack key components of the natural sex-determination system but may have evolved variant sex-determining mechanisms during two decades in laboratory culture.     

A cost of Wolbachia-induced sex reversal and female-biased sex ratios: decrease in female fertility after sperm depletion in a terrestrial isopod

A number of parasites are vertically transmitted to new host generations via female eggs. In such cases, host reproduction is an intimate component of parasite fitness and no cost of the infection on host reproduction is expected to evolve. A number of these parasites distort host sex ratios towards females, thereby increasing either parasite fitness or the proportion of the host that transmit the parasite. In terrestrial isopods (woodlice), Wolbachia bacteria are responsible for sex reversion and female-biased sex ratios, changing genetic males into functional neo-females. Although sex ratio distortion is a powerful means for parasites to increase in frequency in host populations, it also has potential consequences on host biology, which may, in turn, have consequences for parasite prevalence. We used the woodlouse Armadillidium vulgare to test whether the interaction between Wolbachia infection and the resulting excess of females would limit female fertility through the reduction in sperm number that they receive from males. We showed that multiple male mating induces sperm depletion, and that this sperm depletion affects fertility only in infected females. This decrease in fertility, associated with male mate choice, may limit the spread of Wolbachia infections in host populations.     

Mechanisms that influence sex ratio variation in the invasive hymenopteran Sirex noctilio in South Africa

Sirex noctilio is an economically important invasive pest of commercial pine forestry in the Southern Hemisphere. Newly established invasive populations of this woodwasp are characterized by highly male‐biased sex ratios that subsequently revert to those seen in the native range. This trend was not observed in the population of S. noctilio from the summer rainfall regions in South Africa, which remained highly male‐biased for almost a decade. The aim of this study was to determine the cause of this persistent male bias. As an explanation for this pattern, we test hypotheses related to mating success, female investment in male versus female offspring, and genetic diversity affecting diploid male production due to complementary sex determination. We found that 61% of females in a newly established S. noctilio population were mated. Microsatellite data analysis showed that populations of S. noctilio from the summer rainfall regions in South Africa are far less genetically diverse than those from the winter rainfall region, with mean Nei's unbiased gene diversity indexes of 0.056 and 0.273, respectively. These data also identified diploid males at low frequencies in both the winter (5%) and summer (2%) rainfall regions. The results suggest the presence of a complementary sex determination mechanism in S. noctilio, but imply that reduced genetic diversity is not the main driver of the male bias observed in the summer rainfall region. Among all the factors considered, selective investment in sons appears to have the most significant influence on male bias in S. noctilio populations. Why this investment remains different in frontier or early invasive populations is not clear but could be influenced by females laying unfertilized eggs to avoid diploid male production in populations with a high genetic relatedness.     

Z LINKAGE OF FEMALE PROMISCUITY GENES IN THE MOTH UTETHEISA ORNATRIX: SUPPORT FOR THE SEXY‐SPERM HYPOTHESIS?

Female preference genes for large males in the highly promiscuous moth Utetheisa ornatrix (Lepidoptera: Arctiidae) have previously been shown to be mostly Z‐linked, in accordance with the hypothesis that ZZ–ZW sex chromosome systems should facilitate Fisherian sexual selection. We determined the heritability of both female and male promiscuity in the highly promiscuous moth U. ornatrix (Lepidoptera: Arctiidae) through parent–offspring and grandparent–offspring regression analyses. Our data show that male promiscuity is not sex‐limited and either autosomal or sex‐linked whereas female promiscuity is primarily determined by sex‐limited, Z‐linked genes. These data are consistent with the “sexy‐sperm hypothesis,” which posits that multiple‐mating and sperm competitiveness coevolve through a Fisherian‐like process in which female promiscuity is a kind of mate choice in which sperm‐competitiveness is the trait favored in males. Such a Fisherian process should also be more potent when female preferences are Z‐linked and sex‐limited than when autosomal or not limited.     

Cytoplasmic feminizing elements in a two-population model: infection dynamics, gene flow modification, and the spread of autosomal suppressors

We investigate the dynamics of a cytoplasmic parasitic element with feminizing effect in a two-population model. We assume that the host species has a ZZ/ZW sex determination system. Our analysis reveals that the feminizer and the W chromosome can stably coexist by dominating different populations if the transmission rate differs significantly between the populations and migration is sufficiently weak. In the equilibrium of coexistence, genetic influx at any host autosomal locus is strongly enhanced in the population where infection is prevalent but not modified in the other population. We further explore conditions for the spread of autosomal suppressor genes that reduce transmission of feminizing elements to the cost of host viability, and compute their equilibrium frequencies. Our results confirm the hypothesis that selfish genetic elements convert infected host populations into genetic sinks, thereby restricting the spread of transmission suppressors.     

The effect of temperature on sex ratio in the isopod Porcellionides pruinosus: Environmental sex determination or a by-product of cytoplasmic sex determination?

The sex ratios of the progenies of woodlice Porcellionides pruinosus (Crustacea, Isopoda) raised at different temperatures were studied. Females from three French populations sampled in the wild produced highly female-biased broods at 20°C and male-biased broods above 30°C. The effect of high temperature was not due to selective mortality of females. Sex determination was thus sensitive to temperature in P. pruinosus. We also found an interpopulation variability of sex ratio thermosensitivity and a weak inheritance of male-biased sex ratios at high temperatures. Samples taken from a wild population throughout the year showed that while the thermal conditions required for changes in the sex ratio occurred, there was no significant variation in the sex ratio. On the other hand, almost all the females and many males in the four populations studied harboured intracytoplasmic bacteria. These maternally inherited symbionts belong to the genus Wolbachia and are known to possess a feminizing effect. While in other arthropods Wolbachia are destroyed at high temperatures, the symbionts of P. pruinosus were detected by a PCR procedure whatever the rearing temperatures. In light of these results, we propose that the thermosensitivity of sex determination in P. pruinosus could reflect the removal of the cytoplasmic effect on sex determination rather than environmental sex determination sensu stricto. The reduction in the amount of bacteria (but not their entire elimination), or the inhibition of bacterial metabolism, may be responsible for sex ratio variations relating to temperature. The incomplete inheritance of male-biased sex ratios at high temperatures might reflect a selection of thermo-tolerant bacterial strains.