Isabelle Marcadé

Widespread Wolbachia infection in terrestrial isopods and other crustaceans

Abstract Wolbachia bacteria are obligate intracellular alpha-Proteobacteria of arthropods and nematodes. Although widespread among isopod crustaceans, they have seldom been found in non-isopod crustacean species. Here, we report Wolbachia infection in fourteen new crustacean species. Our results extend the range of Wolbachia infections in terrestrial isopods and amphipods (class Malacostraca). We report the occurrence of two different Wolbachia strains in two host species (a terrestrial isopod and an amphipod). Moreover, the discovery of Wolbachia in the goose barnacle Lepas anatifera (subclass Thecostraca) establishes Wolbachia infection in class Maxillopoda. The new bacterial strains are closely related to B-supergroup Wolbachia strains previously reported from crustacean hosts. Our results suggest that Wolbachia infection may be much more widespread in crustaceans than previously thought. The presence of related Wolbachia strains in highly divergent crustacean hosts suggests that Wolbachia endosymbionts can naturally adapt to a wide range of crustacean hosts. Given the ability of isopod Wolbachia strains to induce feminization of genetic males or cytoplasmic incompatibility, we speculate that manipulation of crustacean-borne Wolbachia bacteria might represent potential tools for controlling crustacean species of commercial interest and crustacean or insect disease vectors.

Structure and Evolution of the Atypical Mitochondrial Genome of Armadillidium vulgare (Isopoda, Crustacea)

The crustacean isopod Armadillidium vulgare is characterized by an unusual ∼42-kb-long mitochondrial genome consisting of two molecules co-occurring in mitochondria: a circular ∼28-kb dimer formed by two ∼14-kb monomers fused in opposite polarities and a linear ∼14-kb monomer. Here we determined the nucleotide sequence of the fundamental monomeric unit of A. vulgare mitochondrial genome, to gain new insight into its structure and evolution. Our results suggest that the junction zone between monomers of the dimer structure is located in or near the control region. Direct sequencing indicated that the nucleotide sequences of the different monomer units are virtually identical. This suggests that gene conversion and/or replication processes play an important role in shaping nucleotide sequence variation in this mitochondrial genome. The only heteroplasmic site we identified predicts an alloacceptor tRNA change from tRNAAla to tRNAVal. Therefore, in A. vulgare, tRNAAla and tRNAVal are found at the same locus in different monomers, ensuring that both tRNAs are present in mitochondria. The presence of this heteroplasmic site in all sequenced individuals suggests that the polymorphism is selectively maintained, probably because of the necessity of both tRNAs for maintaining proper mitochondrial functions. Thus, our results provide empirical evidence for the tRNA gene recruitment model of tRNA evolution. Moreover, interspecific comparisons showed that the A. vulgare mitochondrial gene order is highly derived compared to the putative ancestral arthropod type. By contrast, an overall high conservation of mitochondrial gene order is observed within crustacean isopods.

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.

A thirty million year-old inherited heteroplasmy.

Due to essentially maternal inheritance and a bottleneck effect during early oogenesis, newly arising mitochondrial DNA (mtDNA) mutations segregate rapidly in metazoan female germlines. Consequently, heteroplasmy (i.e. the mixture of mtDNA genotypes within an organism) is generally resolved to homoplasmy within a few generations. Here, we report an exceptional transpecific heteroplasmy (predicting an alanine/valine alloacceptor tRNA change) that has been stably inherited in oniscid crustaceans for at least thirty million years. Our results suggest that this heteroplasmy is stably transmitted across generations because it occurs within mitochondria and therefore escapes the mtDNA bottleneck that usually erases heteroplasmy. Consistently, at least two oniscid species possess an atypical trimeric mitochondrial genome, which provides an adequate substrate for the emergence of a constitutive intra-mitochondrial heteroplasmy. Persistence of a mitochondrial polymorphism on such a deep evolutionary timescale suggests that balancing selection may be shaping mitochondrial sequence evolution in oniscid crustaceans.

Inverted Repeats and Genome Architecture Conversions of Terrestrial Isopods Mitochondrial DNA

Mitochondrial DNA (mtDNA) is usually depicted as a circular molecule, however, there is increasing evidence that linearization of mtDNA evolved independently many times in organisms such as fungi, unicellular eukaryotes, and animals. Recent observations in various models with linear mtDNA revealed the presence of conserved inverted repeats (IR) at both ends that, when they become single-stranded, may be able to fold on themselves to create telomeric-hairpins involved in genome architecture conversions. The atypical mtDNA of terrestrial isopods (Crustacea: Oniscidea) composed of linear monomers and circular dimers is an interesting model to study genome architecture conversions. Here, we present the mtDNA control region sequences of two species of the genus Armadillidium: A. vulgare and A. pelagicum. All features of arthropods mtDNA control regions are present (origin of replication, poly-T stretch, GA and TA-rich blocks and one variable domain), plus a conserved IR. This IR can potentially fold into a hairpin structure and is present in two different orientations among the A. vulgare populations: either in one sense or in its reverse complement. This polymorphism, also observed in a single individual (heteroplasmy), might be a signature of genome architecture conversions from linear to circular monomeric mtDNA via successive opening and closing of the molecules.

Bidirectional cytoplasmic incompatibility caused by Wolbachia in the terrestrial isopod Porcellio dilatatus.

In the terrestrial isopod species Porcellio dilatatus, unidirectional Cytoplasmic Incompatibility (CI) between two morphs (P. d. dilatatus and P. d. petiti) caused by a Wolbachia strain (wPet) infecting the morph P. d. petiti has been previously described by experiments initiated four decades ago. Here, we studied another Wolbachia that has been recently detected in a population of the morph P. d. dilatatus. The MLST markers reveal that this Wolbachia is a new strain called wDil distinct from wPet also belonging to the isopod clade of Wolbachia. Quantifications of both Wolbachia strains in the gonads of the two P. dilatatus morphs revealed that all males exhibit similar Wolbachia titers while the titers in females depend on the Wolbachia strain they host. Crossing experiments showed that both wDil and wPet induced partial unidirectional CI with different intensities. Moreover, these two strains induced bidirectional CI when individuals were both infected with one of the two different Wolbachia strains. This way, we demonstrated that P. dilatatus can be infected by two closely related Wolbachia strains (wDil and wPet), that seem to have different modification-rescue systems.

Modulation of host immunity and reproduction by horizontally acquired Wolbachia

The Wolbachia are symbiotic bacteria vertically transmitted from one host generation to another. However, a growing amount of data shows that horizontal transfers of Wolbachia also frequently occur within and between host species. The consequences of the arrival of new symbionts on host physiology can be studied by their experimental introduction in asymbiotic hosts. After experimental transfers of the eight major isopod Wolbachia strains in the isopod Porcellio dilatatus only two of them (wCon and wDil) were found to (1) have no pathogenic effect on the host and (2) be able to pass vertically to the host offspring. In the present work, we studied the influence of these two strains, able to complete an horizontal transfer, on immunity and reproduction of P. dilatatus at two stages of the transfer: (1) in recipient hosts that encounter the symbionts: to test the influence of symbiont when acquired during host life and (2) in vertically infected offspring: to test the influence of a symbiotic interaction occurring all lifelong. The impact of Wolbachia varied depending on the stage: there were clearer effects in vertically infected individuals than in those that acquired the symbionts during their lives. Moreover, the two Wolbachia strains showed contrasted effects: the strain wCon tended to reduce the reproductive investment but to maintain or increase immune parameters whilst wDil had positive effects on reproductive investment but decreased the investment in some immune parameters. These results suggest that horizontally acquisition of Wolbachia can influence the balance between host immune and reproductive traits.