Host tissues as microhabitats for Wolbachia and quantitative insights into the bacterial community in terrestrial isopods.

Animal-bacterial symbioses are highly dynamic in terms of multipartite interactions, both between the host and its symbionts as well as between the different bacteria constituting the symbiotic community. These interactions will be reflected by the titres of the individual bacterial taxa, for example via host regulation of bacterial loads or competition for resources between symbionts. Moreover, different host tissues represent heterogeneous microhabitats for bacteria, meaning that host-associated bacteria might establish tissue-specific bacterial communities. Wolbachia are widespread endosymbiotic bacteria, infecting a large number of arthropods and filarial nematodes. However, relatively little is known regarding direct interactions between Wolbachia and other bacteria. This study represents the first quantitative investigation of tissue-specific Wolbachia-microbiota interactions in the terrestrial isopod Armadillidium vulgare. To this end, we obtained a more complete picture of the Wolbachia distribution patterns across all major host tissues, integrating all three feminizing Wolbachia strains (wVulM, wVulC, wVulP) identified to date in this host. Interestingly, the different Wolbachia strains exhibited strain-specific tissue distribution patterns, with wVulM reaching lower titres in most tissues. These patterns were consistent across different host genetic backgrounds and might reflect different co-evolutionary histories between the Wolbachia strains and A. vulgare. Moreover, Wolbachia-infected females carried higher total bacterial loads in several, but not all, tissues, irrespective of the Wolbachia strain. Taken together, this quantitative approach indicates that Wolbachia is part of a potentially more diverse bacterial community, as exemplified by the presence of highly abundant bacterial taxa in the midgut caeca of several A. vulgare populations.

Effects of parasitic sex-ratio distorters on host genetic structure in the Armadillidium vulgare-Wolbachia association.

In the pill bug Armadillidium vulgare (Crustacea, Oniscidea), Wolbachia facilitates its spread through vertical transmission via the eggs by inducing feminization of genetic males. The spread of feminizing Wolbachia within and across populations is therefore expected to influence mitochondrial DNA (mtDNA) genetic structure by hitchhiking. To test this hypothesis, we analysed nuclear and mtDNA genetic structure, and Wolbachia prevalence in 13 populations of the pill bug host. Wolbachia prevalence (ranging from 0% to 100% of sampled females) was highly variable among populations. All three Wolbachia strains previously observed in A. vulgare were present (wVulC, wVulM and wVulP) with wVulC being the most prevalent (nine of 13 populations). The host showed a genetic structure on five microsatellite loci that is compatible with isolation by distance. The strong genetic structure observed on host mtDNA was correlated with Wolbachia prevalence: three mitotypes were in strong linkage disequilibrium with the three strains of Wolbachia. Neutrality tests showed that the mtDNA polymorphism is not neutral, and we thus suggest that this unusual pattern of mtDNA polymorphism found in A. vulgare was due to Wolbachia.

Evidence for a new feminizing Wolbachia strain in the isopod Armadillidium vulgare: evolutionary implications.

Wolbachia are intracellular maternally inherited alpha-Proteobacteria infecting a wide range of arthropods. In the common pill bug Armadillidium vulgare, the known Wolbachia strain is responsible for feminization of genetic males. We have investigated Wolbachia diversity in 20 populations of A. vulgare from west and east Europe, north Africa and north America. A new Wolbachia strain (wVulM) was identified through the variability of the wsp gene, distantly related to that previously known (wVulC) in this host species. No individual with multiple infections was detected. Inoculation experiments indicated that the new wVulM bacterial strain also induces feminization in A. vulgare. However, the wVulC strain showed a higher transmission rate than the wVulM strain and was the most geographically widespread Wolbachia in A. vulgare populations. Mitochondrial 16SrDNA gene sequencing was conducted in Wolbachia-infected individuals, revealing the occurrence of four host lineages. The comparison of bacterial strains and their respective host mitochondrial phylogenies failed to show concordance, indicating horizontal transmission of the Wolbachia strains within populations of A. vulgare.

Wolbachia diversity in the Porcellionides pruinosus complex of species (Crustacea: Oniscidea): evidence for host-dependent patterns of infection.

Porcellionides pruinosus is a cosmopolitan woodlouse. It is known to exhibit patterns of geographical variation between populations, and has been suspected to consist of several very closely related species. This species was found to carry Wolbachia endosymbionts, alpha-proteobacteria which are known to modify the reproduction of their crustacean hosts by inducing cytoplasmic incompatibility or feminization. In the P. pruinosus complex, Wolbachia induced feminization, but two different patterns of infection were reported: high prevalence and presence of infected males, or low prevalence and absence of infected males. In this study we investigated nine populations described as Porcellionides pruinosus carrying different Wolbachia strains, with the aim of investigating the possibility of coevolution between symbionts and hosts. Molecular analyses were carried out on both Wolbachia DNA (wsp gene) and host mitochondrial DNA (mt LSU rDNA). We show that (1) the nine host populations exhibited a high genetic differentiation so that they have to be split into two sibling species; (2) three different Wolbachia strains were found in the host complex but were not species-specific; and (3) the pattern of infection by Wolbachia was more host-dependent than symbiont-dependent.

Population genetic structure of the endangered freshwater crayfish Austropotamobius pallipes, assessed using RAPD markers.

Random amplified polymorphic DNA (RAPD) analysis was performed to characterize the genetic diversity of Austropotamobius pallipes, a threatened freshwater crayfish native to Europe. Four decamer primers which generated six unambiguous polymorphic bands were used to analyse crayfish from 21 populations sampled in the major part of its range. Genetic diversity within populations of A. pallipes, estimated by Shannon's diversity index, ranged from 0 to 0.446 with a mean of 0.159. A UPGMA dendrogram constructed from pairwise PhiST values between populations, revealed three clusters corresponding to populations sampled in the southern, northwestern and eastern part of its range. AMOVA analysis revealed a high genetic structure of A. pallipes populations PhiST=0.814, with 73.11% of the genetic variation distributed between these clusters. It suggests a historical geographical separation of these groups into three refugial areas, probably in the Rhine, Mediterranean and Atlantic basins during recent glaciations. The close genetic relationships between English and western French populations are in accordance with a natural postglacial origin of English populations from individuals having survived in an Atlantic refugium. However, the present results suggest that the Irish stock originated from a human translocation of individuals from an Atlantic refugium.

Phylogenetic analysis of mitochondrial LSU rRNA in oniscids.

The phylogenetic relationships among oniscids (Crustacea, Isodopa) remain contradictory despite numerous morphological studies. We have investigated them using molecular data. Partial sequences of the mitochondrial LSU rRNA gene were obtained from 42 species of aquatic and terrestrial crustaceans from 31 genera. This gene provided well-supported information, notwithstanding the high taxonomic level of this study, indicating a useful amount of variation despite the noise due to multiple substitutions. The phylogenetic inferences demonstrated that a) Crinocheta and Synocheta sections are monophyletic and sister-groups, b) Ligiidae and Tylidae representatives are in a basal position compared to other oniscids, c) Helleria brevicornis, the only representative of the Helleriinae subfamily, has undergone different evolution, d) the relationships between aquatic isopods and ancient groups of Oniscidea are not resolved, probably due to fast radiation not discriminated by the molecular phylogeny.

Ultrastructural characterisation and molecular taxonomic identification of Nosema granulosis n. sp., a transovarially transmitted feminising (TTF) microsporidium.

A novel microsporidian parasite is described, which infects the crustacean host Gammarus duebeni. The parasite was transovarially transmitted and feminised host offspring. The life cycle was monomorphic with three stages. Meronts were found in host embryos, juveniles, and in the gonadal tissue of adults. Sporoblasts and spores were restricted to the gonad. Sporogony was disporoblastic giving rise to paired sporoblasts, which then differentiated to form spores. Spores were not found in regular groupings and there was no interfacial envelope. Spores were approximately 3.78 x 1.22 microns and had a thin exospore wall, a short polar filament, and an unusual granular polaroplast. All life cycle stages were diplokaryotic. A region from the parasite small subunit ribosomal RNA gene was amplified and sequenced. Phylogenetic analysis based on these data places the parasite within the genus Nosema. We have named the species Nosema granulosis based on the structure of the polaroplast.

Mitochondrial DNA variability and wolbachia infection in two sibling woodlice species

Several morphological races and subspecies have been described and later included within the terrestrial isopod species Porcellionides pruinosus. During our study of this species, we have worked on specimens from France, Greece, Tunisia and Reunion island. Laboratory crosses have revealed two separate groups of populations: French populations (four localities) in one group, and those from Tunisia, Reunion island and Greece in the other. French individuals were reproductively isolated from those of the other populations. We have undertaken a survey of mitochondrial DNA (mtDNA) polymorphism in these seven populations. We observed two groups of mitotypes corresponding to the two groups of populations. Interfertility experiments between populations and the mitochondrial genetic distances between mitotypes both suggest the presence of two different species, one in France and one in Greece, Tunisia and Reunion island. The two species harbour, respectively, two different Wolbachia lines. Another feature of the molecular genetic analysis was the apparent mitochondrial monomorphism in the French populations and the low variability in the other three populations. The result can be related to the possibility of Wolbachia-induced genetic hitchhiking in these populations.

Mitochondrial DNA polymorphism, sex ratio distorters and population genetics in the isopod Armadillidium vulgare.

Two maternally inherited sex ratio distorters (SRD) impose female-biased sex ratios on the wood louse Armadillidium vulgare by feminizing putative males. These SRD are (i) an intracytoplasmic bacterium of the genus Wolbachia, and (ii) another non-Mendelian element of unknown nature: the f element. Mitochondrial DNA variation was investigated in A. vulgare field populations to trace the evolution of host-SRD relationships and to investigate the effect of SRD on host cytoplasmic polymorphism. The Wolbachia endosymbionts showed no polymorphism in their ITS2 sequence and were associated with two closely related mitochondrial types. This situation probably reflects a single infection event followed by a slight differentiation of mitochondria. There was no association between the f element and a given mitochondrial type, which may confirm the fact that this element can be partially paternally transmitted. The spreading of a maternally inherited SRD in a population should reduce the mitochondrial diversity by a hitchhiking process. In A. vulgare, however, a within-population mtDNA polymorphism was often found, because of the deficient spread of Wolbachia and the partial paternal inheritance of the f element. The analysis of molecular variance indicated that A. vulgare populations are genetically structured, but without isolation by distance.

Organization of the large mitochondrial genome in the isopod Armadillidium vulgare.

The mitochondrial DNA (mtDNA) in animals is generally a circular molecule of approximately 15 kb, but there are many exceptions such as linear molecules and larger ones. RFLP studies indicated that the mtDNA in the terrestrial isopod Armadillidium vulgare varied from 20 to 42 kb. This variation depended on the restriction enzyme used, and on the restriction profile generated by a given enzyme. The DNA fragments had characteristic electrophoretic behaviors. Digestions with two endonucleases always generated fewer fragments than expected; denaturation of restriction profiles reduced the size of two bands by half; densitometry indicated that a number of small fragments were present in stoichiometry, which has approximately twice the expected concentration. Finally, hybridization to a 550-bp 16S rDNA probe often revealed two copies of this gene. These results cannot be due to the genetic rearrangements generally invoked to explain large mtDNA. We propose that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer. Densitometry suggested that these two molecular structures were present in different proportions within a single individual. The absence of mutations within the dimers also suggests that replication occurs during the monomer phase.

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.

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.