Pierre Grève

The impact of endosymbionts on the evolution of host sex-determination mechanisms

The past years have revealed that inherited bacterial endosymbionts are important sources of evolutionary novelty for their eukaryotic hosts. In this review we discuss a fundamental biological process of eukaryotes influenced by bacterial endosymbionts: the mechanisms of sex determination. Because they are maternally inherited, several endosymbionts of arthropods, known as reproductive parasites, have developed strategies to convert non-transmitting male hosts into transmitting females through feminization of genetic males and parthenogenesis induction. Recent investigations have also highlighted that endosymbionts can impact upon host sex determination more subtly through genetic conflicts, resulting in selection of host nuclear genes resisting endosymbiont effects. Paradoxically, it is because of their selfish nature that reproductive parasites are such powerful agents of evolutionary change in their host sex-determination mechanisms. They might therefore represent excellent models for studying transitions between sex-determining systems and, more generally, the evolution of sex-determination mechanisms in eukaryotes.

Intense Transpositional Activity of Insertion Sequences in an Ancient Obligate Endosymbiont

The streamlined genomes of ancient obligate endosymbionts generally lack transposable elements, such as insertion sequences (IS). Yet, the genome of Wolbachia, one of the most abundant bacterial endosymbionts on Earth, is littered with IS. Such a paradox raises the question as to why there are so many ISs in the genome of this ancient endosymbiont. To address this question, we investigated IS transpositional activity in the unculturable Wolbachia by tracking the evolutionary dynamics and history of ISWpi1 elements. We show that 1) ISWpi1 is widespread in Wolbachia, being present in at least 55% of the 40 sampled strains, 2) ISWpi1 copies exhibit virtually identical nucleotide sequences both within and among Wolbachia genomes and possess an intact transposase gene, 3) individual ISWpi1 copies are differentially inserted among Wolbachia genomes, and 4) ISWpi1 occurs at variable copy numbers among Wolbachia genomes. Collectively, our results provide compelling evidence for intense ISWpi1 transpositional activity and frequent ISWpi1 horizontal transmission among strains during recent Wolbachia evolution. Thus, the genomes of ancient obligate endosymbionts can carry high loads of functional and transpositionally active transposable elements. Our results also indicate that Wolbachia genomes have experienced multiple and temporally distinct ISWpi1 invasions during their evolutionary history. Such recurrent exposition to new IS invasions may explain, at least partly, the unusually high density of transposable elements found in the genomes of Wolbachia endosymbionts.

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.

Feminizing Wolbachia : a transcriptomics approach with insights on the immune response genes in Armadillidium vulgare

BackgroundWolbachia are vertically transmitted bacteria known to be the most widespread endosymbiont in arthropods. They induce various alterations of the reproduction of their host, including feminization of genetic males in isopod crustaceans. In the pill bug Armadillidium vulgare, the presence of Wolbachia is also associated with detrimental effects on host fertility and lifespan. Deleterious effects have been demonstrated on hemocyte density, phenoloxidase activity, and natural hemolymph septicemia, suggesting that infected individuals could have defective immune capacities. Since nothing is known about the molecular mechanisms involved in Wolbachia-A. vulgare interactions and its secondary immunocompetence modulation, we developed a transcriptomics strategy and compared A. vulgare gene expression between Wolbachia-infected animals (i.e., “symbiotic” animals) and uninfected ones (i.e., “asymbiotic” animals) as well as between animals challenged or not challenged by a pathogenic bacteria.ResultsSince very little genetic data is available on A. vulgare, we produced several EST libraries and generated a total of 28 606 ESTs. Analyses of these ESTs revealed that immune processes were over-represented in most experimental conditions (responses to a symbiont and to a pathogen). Considering canonical crustacean immune pathways, these genes encode antimicrobial peptides or are involved in pathogen recognition, detoxification, and autophagy. By RT-qPCR, we demonstrated a general trend towards gene under-expression in symbiotic whole animals and ovaries whereas the same gene set tends to be over-expressed in symbiotic immune tissues.ConclusionThis study allowed us to generate the first reference transcriptome ever obtained in the Isopoda group and to identify genes involved in the major known crustacean immune pathways encompassing cellular and humoral responses. Expression of immune-related genes revealed a modulation of host immunity when females are infected by Wolbachia, including in ovaries, the crucial tissue for the Wolbachia route of transmission.

Conservation of the Type IV Secretion System throughout Wolbachia evolution.

The Type IV Secretion System (T4SS) is an efficient pathway with which bacteria can mediate the transfer of DNA and/or proteins to eukaryotic cells. In Wolbachia pipientis, a maternally inherited obligate endosymbiont of arthropods and nematodes, two operons of vir genes, virB3-B6 and virB8-D4, encoding a T4SS were previously identified and characterized at two separate genomic loci. Using the largest data set of Wolbachia strains studied so far, we show that vir gene sequence and organization are strictly conserved among 37 Wolbachia strains inducing various phenotypes such as cytoplasmic incompatibility, feminization, or oogenesis in their arthropod hosts. In sharp contrast, extensive variation of genomic sequences flanking the virB8-D4 operon suggested its distinct location among Wolbachia genomes. Long term conservation of the T4SS may imply maintenance of a functional effector translocation system in Wolbachia, thereby suggesting the importance for the T4SS in Wolbachia biology and survival inside host cells.

Hydroxyindole-O-methyltransferase in the Chicken Retina: Immunocytochemical- Localization and Daily Rhythm of mRNA

In the vertebrate retina and pineal gland, melatonin production displays diurnal variations with high levels at night. Hydroxyindole‐O‐methyltransferase (HIOMT, EC 2.1.1.4) catalyses the last step of melatonin biosynthesis. In the present study, a cDNA encoding chicken HIOMT was used to examine the effects of environmental lighting on HIOMT mRNA expression in the chicken retina. A day/night rhythm of HIOMT mRNA level was observed, with an average 5‐fold increase during the night. Light strongly suppressed the night‐time rise in HIOMT mRNA concentration while darkness prevented its daytime fall. An antibody directed against chicken HIOMT was used for immunocytochemical identification of retinal melatoninergic cells. HIOMT immunoreactivity could be observed in rods as well as in cones. However, the lowest levels of HIOMT immunoreactivity were always observed in the accessory cones of double cones. A few HIOMT‐positive cell bodies could also be observed in the inner nuclear layer. Altogether, these data indicate that HIOMT gene expression in the retina is organized on a daily basis as a direct response to light, and that the different types of photoreceptors may not be equally involved in melatonin production.

The Immune Cellular Effectors of Terrestrial Isopod Armadillidium vulgare: Meeting with Their Invaders, Wolbachia

Background Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes. Methodology/Principal Findings In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×103 Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearsons test = 12 822.98, df = 2, p<0.001). Conclusions/Significance The characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals.

Birth of a W sex chromosome by horizontal transfer of Wolbachia bacterial symbiont genome

Significance Sex determination is an evolutionarily ancient, key developmental pathway governing sexual differentiation in animals. Sex determination systems are remarkably variable between species or groups of species, however, and the evolutionary forces underlying transitions between these systems are poorly understood. Here we provide evidence indicating that the genome of Wolbachia bacterial endosymbionts was horizontally transferred into a chromosome of the common pillbug Armadillidium vulgare, which resulted in this chromosome evolving as a new female (W) sex chromosome. This represents a remarkable mechanism underpinning the birth of sex chromosomes. We conclude that bacteria can be powerful agents of evolutionary transitions in sex determination systems in animals. Sex determination is a fundamental developmental pathway governing male and female differentiation, with profound implications for morphology, reproductive strategies, and behavior. In animals, sex differences between males and females are generally determined by genetic factors carried by sex chromosomes. Sex chromosomes are remarkably variable in origin and can differ even between closely related species, indicating that transitions occur frequently and independently in different groups of organisms. The evolutionary causes underlying sex chromosome turnover are poorly understood, however. Here we provide evidence indicating that Wolbachia bacterial endosymbionts triggered the evolution of new sex chromosomes in the common pillbug Armadillidium vulgare. We identified a 3-Mb insert of a feminizing Wolbachia genome that was recently transferred into the pillbug nuclear genome. The Wolbachia insert shows perfect linkage to the female sex, occurs in a male genetic background (i.e., lacking the ancestral W female sex chromosome), and is hemizygous. Our results support the conclusion that the Wolbachia insert is now acting as a female sex-determining region in pillbugs, and that the chromosome carrying the insert is a new W sex chromosome. Thus, bacteria-to-animal horizontal genome transfer represents a remarkable mechanism underpinning the birth of sex chromosomes. We conclude that sex ratio distorters, such as Wolbachia endosymbionts, can be powerful agents of evolutionary transitions in sex determination systems in animals.

Variations of immune parameters in terrestrial isopods: a matter of gender, aging and Wolbachia

Ecological factors modulate animal immunocompetence and potentially shape the evolution of their immune systems. Not only environmental parameters impact on immunocompetence: Aging is one major cause of variability of immunocompetence between individuals, and sex-specific levels of immunocompetence have also been frequently described. Moreover, a growing core of data put in light that vertically transmitted symbionts can dramatically modulate the immunocompetence of their hosts. In this study, we addressed the influence of gender, age and the feminising endosymbiont Wolbachia (wVulC) on variations in haemocyte density, total PO activity and bacterial load in the haemolymph of the terrestrial isopod Armadillidium vulgare. This host–symbiont system is of particular interest to address this question since: (1) wVulC was previously shown as immunosuppressive in middle-aged females and (2) wVulC influences sex determination. We show that age, gender and Wolbachia modulate together immune parameters in A. vulgare. However, wVulC, which interacts with aging, appears to be the prominent factor interfering with both PO activity and haemocyte density. This interference with immune parameters is not the only aspect of wVulC virulence on its host, as reproduction and survival are also altered.

Characterization and transcriptional analysis of two gene clusters for type IV secretion machinery in Wolbachia of Armadillidium vulgare

Wolbachia are maternally inherited alpha-proteobacteria that induce feminization of genetic males in most terrestrial crustacean isopods. Two clusters of vir genes for a type IV secretion machinery have been identified at two separate loci and characterized for the first time in a feminizing Wolbachia. Furthermore, we demonstrated that these operons are transcriptionally active in ovaries and in all other tissues tested, suggesting that T4SS has a significant role in Wolbachia biology. These observations and the identification of homologous vir genes in Wolbachia strains infecting insects or nematodes show that vir genes are conserved among Wolbachia strains whatever the phenotype induced by the bacteria.