Wolbachia endosymbiont responsible for cytoplasmic incompatibility in a terrestrial crustacean: effects in natural and foreign hosts.

Wolbachia bacteria are vertically transmitted endosymbionts that disturb the reproduction of many arthropods thereby enhancing their spread in host populations. Wolbachia are often responsible for changes of sex ratios in terrestrial isopods, a result of the feminization of genotypic males. Here we found that the Wolbachia hosted by Cylisticus convexus (wCc) caused unidirectional cytoplasmic incompatibility (CI), an effect commonly found in insects. To understand the diversity of Wolbachia-induced effects in isopods, wCc were experimentally transferred in a novel isopod host, Armadillidium vulgare. wCc conserved the ability to induce CI. However, Wolbachia were not transmitted to the eggs, so the capacity to restore the compatibility in crosses involving two transinfected individuals was lost. The feminizing Wolbachia hosted by A. vulgare was unable to rescue CI induced by wCc. These results showed that Wolbachia in isopods did not evolved broadly to induce feminization, and that CI and the feminizing effect are probably due to different mechanisms. In addition, wCc reduces the mating capacity of infected C. convexus males, suggesting that the bacteria might alter reproductive behaviour. The maintenance of wCc in host populations is discussed.

Wolbachia bacteria effects after experimental interspecific transfers in terrestrial isopods.

Wolbachia bacteria are intracellular parasites, vertically transmitted from mothers to offspring through the cytoplasm of the eggs. They manipulate the reproduction of their hosts to increase in frequency in host populations. In terrestrial isopods for example, Wolbachia are responsible for the full feminization of putative males, therefore increasing the proportion of females, the sex by which they are transmitted. Vertical transmission, however, is not the only means for Wolbachia propagation. Infectious (i.e., horizontal) transmission between different host species or taxa is required to explain the fact that the phylogeny of Wolbachia does not parallel that of their hosts. The aim of this study was to investigate, by experimental transinfections, whether Wolbachia strains could be successfully transferred to a different, previously uninfected isopod host. While Wolbachia survived in all the studied recipient species, vertical transmission was efficient only in cases where donor and recipient species were closely related. Even in this case, Wolbachia strains did not always keep their ability to entirely feminize their host, a deficiency that can be link to a low bacterial density in the host tissues. In addition, Wolbachia infection was associated with a decrease in host fertility, except when the bacterial strain came from the same host population as the recipient animals. This suggest that Wolbachia could be adapted to local host populations. It therefore seems that isopod Wolbachia are highly adapted to their host and can hardly infect another species of hosts. The successful infection of a given Wolbachia strain into a new isopod host species therefore probably requires a strong selection on bacterial variants.

Wolbachia infection in the terrestrial isopod oniscus asellus: sex ratio distortion and effect on fecundity

Maternally inherited Wolbachia bacteria are widespread in arthropods where they are responsible for various reproductive alterations. In terrestrial isopods (woodlice), Wolbachia may induce feminization or cytoplasmic incompatibility (CI), but their effect remains unknown in most host species. To increase our understanding of host/symbiont interactions in terrestrial isopods, the effect of Wolbachia was investigated in the oniscidean Oniscus asellus, mainly to discriminate between feminization and CI. The Wolbachia infection was not linked with a CI phenomenon, but females infected with Wolbachia produced female-biased broods compared with uninfected females. The fecundity of infected females was slightly lower than that of uninfected, but the number of young at the adult stage was similar between the two female categories. The experimental transfer of the symbiont into uninfected strains showed that Wolbachia was responsible for the feminization of a number of genetic males. In female-biased broods, Wolbachia were vertically transmitted to around 88% of the offspring, but the transmission rate was lower in the few male-biased progenies. The feminizing activity of these symbionts was not systematic, as many phenotypic males were infected. These results contrasted with what is known in another woodlouse species, and indicated that feminization has evolved in different ways in terrestrial isopods.

Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization.

Wolbachia are maternally inherited, intracellular, alpha proteobacteria that infect a wide range of arthropods. They cause three kinds of reproductive alterations in their hosts: cytoplasmic incompatibility, parthenogenesis and feminization. There have been many studies of the distribution of Wolbachia in arthropods, but very few crustacean species are known to be infected. We investigated the prevalence of Wolbachia in 85 species from five crustacean orders. Twenty-two isopod species were found to carry these bacteria. The bacteria were found mainly in terrestrial species, suggesting that Wolbachia came from a continental environment. The evolutionary relationships between these Wolbachia strains were determined by sequencing bacterial genes and by interspecific transfers. All the bacteria associated with isopods belonged to the Wolbachia B group, based on 16S rDNA sequence data. All the terrestrial isopod symbionts in this group except one formed an independent clade. The results of interspecific transfers show evidence of specialization of Wolbachia symbionts to their isopod hosts. They also suggest that host species plays a more important role than bacterial phylogeny in determining the phenotype induced by Wolbachia infection.

Genetic and biochemical studies on ommochrome genesis in an albino strain of a terrestrial isopod, Armadillidium vulgare.

Genetic studies and quantitative determination of levels of 3-hydroxykynurenine and kynurenine were performed in an albino strain of a terrestrial isopod Armadillidium vulgare. From the results of matings between the albino and the albino, the red, the dark red, or the wild type individuals, the albino A. vulgare seems to be regulated by an autosomal gene(s) recessive to its wild allele. Litter mating of F1 progenies obtained by crossing the albino and the red mutant or the albino and the dark red mutant yielded progenies at a ratio of 3:6:3:4 for the red, the dark red, the wild, and the albino phenotypes, respectively. The albino gene(s) seems not to be allelic but to be epistatic to the red gene(s) with respect to ommochrome biosynthesis. Quantitative determination of 3-hydroxykynurenine carried out by high-performance liquid chromatography with electrochemical detection revealed that the 3-hydroxykynurenine content in the albino was significantly lower than that in the wild or the red type. The whole content of 3-hydroxykynurenine after enzymatic conversion of kynurenine to 3-hydroxykynurenine was still considerably lower than that found in the wild type, even though it increased after the conversion. The albino gene(s) seems to be associated with a blockage at distinct level(s) of ommochrome biosynthesis.

Morphological characterization of three phenotypes of the isopod Armadillidium vulgare.

The morphological characteristics and ommochrome quantity in the integument of red, white, and wild type (black-grey) Armadillidium vulgare were studied. The red phenotype was found to possess two kinds of immature ommochrome pigment granules within its pigment cells, in addition to mature pigment granules. The immature granules seemed to contain uniformly distributed fibrilles, or to have an electron-dense central region surrounded by an electron-lucent outer edge. Since these immature pigment granules were typically observed to be distributed along with the mature ones, and were also more easily extractable than the wild type's, it is hypothesized that ommochrome granule maturation in the red phenotype may occur slowly due to a defect in the pigment granule internal process which combines pigments with matrix proteins. Regarding the white phenotype, although its pigment cells were undeveloped, several large-sized vesicles containing a small amount of electron-dense material appeared in the pigment cell cytoplasm. The wild and red type males of A. vulgare were found to have an ommochrome content twice as large as that of the corresponding females, with no ommochrome pigment being detected in the white phenotype. The genetic relationship between the white and red phenotypes was discussed using as a basis the observed pigment granule structure.

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.

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.

Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods.

Rickettsia-like maternally inherited bacteria have been shown to be involved in a variety of alterations of arthropod sexuality, such as female-biased sex ratios, parthenogenesis, and sterility of crosses either between infected males and uninfected females or between infected individuals (cytoplasmic incompatibility). We have characterized several of these microorganisms through partial sequences of the small (16S) and large (23S) subunit ribosomal DNA. All the symbionts identified, which include several cytoplasmic incompatibility microorganisms, several endosymbionts of terrestrial isopods, and symbionts of two thelytokous Trichogramma wasp species, belong to a monophyletic group of related symbionts, some of which have previously been detected in several insects exhibiting cytoplasmic incompatibility. Three molecular lineages can be identified on the basis of 16S as well as 23S sequences. Although they are only known as endocellular symbionts, Wolbachia spread by horizontal transfer across host lineages as evidenced by their diversification which occurred long after that of their hosts, and by the non-congruence of the phylogenetic relationships of symbionts and their hosts. Indeed, symbionts of two different lineages have been found in the same host species, whereas closely related endosymbionts are found in distinct insect orders. Isopod endosymbionts form a separate lineage, and they can determine feminization as well as cytoplasmic incompatibility. The ability to determine cytoplasmic incompatibility, found in all lineages, is probably ancestral to this group.

Masculinization of female isopods (Crustacea) correlated with non-Mendelian inheritance of cytoplasmic viruses.

When in genetic females external male characters differentiate, the phenomenon is called "male pseudohermaphroditism." This male differentiation occurs in terrestrial isopods (Crustacea, suborder Oniscoidea) and sometimes involves only some epithelial areas (gynandromorphous mosaics). It is not induced by male hormones or by abnormal ovary function. This intersexuality is transmitted maternally (by the intersex females) or paternally (by the brothers of intersex females) to between 30% and 60% of their offspring. Although it occurs at 20 degrees C, the male differentiation disappears when breeding takes place at 27 degrees C. Male characters differentiate in normal females--even in other Oniscoidea species (Porcellio dilatatus, Porcellio laevis, Armadillidium vulgare)--after injection of a 0.22-micron filtered tissue extract. Since an inhibitor of bacterial protein synthesis (gentamycin) does not inhibit this masculinizing effect, we infer that neither organelles nor bacteria are involved. Intersexuality is always correlated with the presence of cytoplasmic viral particles in both intersex-female and transmitter-male tissues. Striking similarities to the Drosophila S virus are noted. A reovirus-like Oniscoidea masculinizing virus, which probably acts only on the epithelial areas sensitive to the male hormones, is most likely the causative agent of this intersexuality. Here we report the conversion of secondary sexual characters putatively caused by a virus.

Purification and characterization of androgenic hormone from the terrestrial isopod Armadillidium vulgare Latr. (Crustacea, Oniscidea).

Androgenic hormone (AH) was purified from hypertrophied androgenic glands of intersexed Armadillidium vulgare (genetic males feminized by symbiotic endocellular bacteria). Two isohormones labeled AH1 and AH2 with similar molecular weights in the range 17,000-18,000 were isolated. Amino acid analysis showed the absence of cysteine in these two forms. A polyclonal antiserum was raised which recognized AH1 and AH2. The physiological significance of this polymorphism is still not known.

[Study of the synthesis of vitellogenin in intersexual males of Armadillidium vulgare Latreille (oniscoid isopod crustacean): comparison with males and with intact or ovariectomized females]. / Etude de la synthèse de la vitellogénine chez les mâles intersexués d'Armadillidium vulgare Latreille (Crustacé Isopode Oniscoïde): comparison avec les mâles et les femelles intactes ou ovariectomisées.

In some natural populations of Armadilidium vulgare, intersex animals are genetic males which are feminized by maternally transmitted symbiotic bacteria. In these intersex males (iM) the fat body synthesizes vitellogenin, although their gonads are testes with hypertrophied--but nonfunctional--androgenic glands. Vitellogenin is present in the hemolymph of males changed experimentally into iM 90 days after inoculation of the feminizing bacteria. During the molting cycle, vitellogenin synthesis in iM varies as in ovariectomized females or in vitellogenic females, with a peak at the stage D1." In A. vulgare, vitellogenin synthesis is a neutral character since it can be observed in a genetic male or in an ovariectomized female; however, it is inhibited by the androgenic hormone. In intersex males, vitellogenin synthesis is the result of their refractoriness to androgenic hormone.

[Mechanism of the refractory state of androgen hormone in Armadillidium vulgare Latr. (crustacean, isopod, oniscoid) harboring a feminizing bacteria]. / Contribution à l'étude du mécanisme de l'état réfractaire à l'hormone androgène chez les Armadillidium vulgare Latr. (crustacé, isopode, oniscoïde) hébergeant une bactérie féminisante.

In thelygenous lines of Armadillidium vulgare, neo-females and intersex males (iM) with feminizing symbiotic bacteria are not masculinized by an extract from iM androgenic gland, which, however, masculinizes bacterialess genetic females. Injection of iM hemolymph extract masculinizes these genetic females. This indicates that androgenic hormone is present in iM hemolymph. Lack of androgenic hormone activity in thelygenous lines is supposed to result from the action of bacteria on the androgenic hormone receptors. Since a temporary recovery of the male differentiation of iM can be induced by implantation of different parts of central nervous system, bacteria effect is probably indirect, through an action on a neurosecretory system, perhaps one of those controlling the functioning of the androgenic gland.

[Masculinization of prepubertal and pubertal females of Sphaeroma serratum Fabr., (Crustacea Isopoda Flabellifera) by implantation of an androgenic gland of pubertal male]. / Masculinisation des femelles prépubères et pubères de Sphaeroma serratum Fabr., (Crustacé Isopode Flabellifère) par implantation d'une glande androgène de mâle pubère.

In Sphaeroma serratum, the differentiation of the male external sexual characteristics, as a result of an androgenic gland implant, proceeds more easily in females in vitellogenesis than in immature females. On the contrary, the transformation of the gonads is quicker and more obvious in immature females than in mature ones. This transformation which leads, in all cases, to an inversion of the ovary to a functional testicle able to produce spermatozoa, always occurs without any differentiation of an androgenic gland, contrary to what can be observed with Oniscoïds. The details of the external sexual differentiation of the grafted females can be related to the functioning of a protocerebral neurosecretory center having, as in males, an androinhibitory effect on the androgenic gland implant; the activity of this center, which seems to correspond to the center secreting VIH, would be particularly high with immature females and would become very low--or nonexistent--in females in vitellogenesis.

[Neuroendocrine control of reproduction in Crustacea]. / Contrôle neuroendocrine de la reproduction chez les Crustacés.

The neuroendocrine factors synthetized by various regions of the nervous system and which have inhibiting or stimulating effects can control the male and female physiology of malacostraca via sexual hormones and moulting hormone. There are between decapods and peracarids functional homologies but some differences can be noticed as to the localization of these antagonistic neurosecretory systems. The functioning of these systems is regulated by external factors such as the photoperiod and the temperature. The interactions between these different neurohormones, the sexual hormones and the moulting hormone are discussed.

Ultrastructural development of the neurohemal organ joined to the ecdysial gland after imaginal moulting in the male isopod Sphaeroma serratum Fab. (Crustacea Flabellifera).

The present ultrastructural study deals with the lateral cephalic nerve plexus of Sphaeroma serratum, a neurohemal organ joined to the Y organ (ecdysial gland). This plexus acts as a storage centre for neurosecretory products from two sources: the two autochtonous cells (plexus cells) within the plexus itself, and the neurosecretory cells in various parts of the central nervous system, particulary the "mandibular ganglion" (A-cells). In prepuberal animals, plexus cells and subesophageal A-cells produce neurosecretory granules of two types measuring 1550 +/- 50 A and 1570 +/- 40 A respectively. Five categories of axon terminals were distinguished in the plexus. The granules found in two of these terminal types are believed to come from the plexus cells and from the "mandibular ganglion" A-cells. Cessation of production of neurosee plexus with concomitant depletion and disappearance of different granule categories. The first axon terminals affected by this process are the two categories containing granules originating in the plexus and "mandibular ganglion" A-cells. Degeneration of the ecdysial gland in male Sphaeroma serratum might be connected with the cessation of granule formation in these two types of cell.