Investigating Wolbachia symbiont-mediated host protection against a bacterial pathogen using a natural Wolbachia nuclear insert.

Wolbachia bacterial endosymbionts provide protection against pathogens in various arthropod species but the underlying mechanisms remain misunderstood. By using a natural Wolbachia nuclear insert (f-element) in the isopod Armadillidium vulgare, we explored whether Wolbachia presence is mandatory to observe protection in this species or the presence of its genes is sufficient. We assessed survival of closely related females carrying or lacking the f-element (and lacking Wolbachia) challenged with the bacterial pathogen Salmonella enterica. Despite marginal significant effects, the f-element alone did not appear to confer survival benefits to its host, suggesting that Wolbachia presence in cells is crucial for protection.

Immune priming depends on age, sex and Wolbachia in the interaction between Armadillidium vulgare and Salmonella.

The protection conferred by a first infection upon a second pathogenic exposure (i.e. immune priming) is an emergent research topic in the field of invertebrate immunity. Immune priming has been demonstrated in various species, but little is known about the intrinsic factors that may influence this immune process. In this study, we tested whether age, gender and the symbiotic bacterium Wolbachia affect the protection resulting from immune priming in A. vulgare against S. enterica. We firstly primed young and old, symbiotic and asymbiotic males and females, either with a non-lethal low dose of S. enterica, LB broth or without injection (control). Seven days post-injection, we performed a LD50 injection of S. enterica in all individuals and we monitored their survival rates. We demonstrated that survival capacities depend on these three factors: young and old asymbiotic individuals (males and females) expressed immune priming (S. enterica-primed individuals survived better than LB-primed and non-primed), with a general decline in the strength of protection in old females, but not in old males, compared to young. When Wolbachia is present, the immune priming protection was observed in old, but not in young symbiotic individuals, even if the Wolbachia load on entire individuals is equivalent regardless to age. Our overall results showed that the immune priming protection in A. vulgare depends on individuals' states, highlighting the need to consider these factors both in mechanistical and evolutionary studies focusing on invertebrate's immunity.

Reproductive senescence and parental effects in an indeterminate grower.

Reproductive senescence is the decrease of reproductive performance with increasing age and can potentially include trans-generational effects as the offspring produced by old parents might have a lower fitness than those produced by young parents. This negative effect may be caused either by the age of the father, mother or the interaction between the ages of both parents. Using the common woodlouse Armadillidium vulgare, an indeterminate grower, as a biological model, we tested for the existence of a deleterious effect of parental age on fitness components. Contrary to previous findings reported from vertebrate studies, old parents produced both a higher number and larger offspring than young parents. However, their offspring had lower fitness components (by surviving less, producing a smaller number of clutches or not reproducing at all) than offspring born to young parents. Our findings strongly support the existence of trans-generational senescence in woodlice and contradict the belief that old individuals in indeterminate growers contribute the most to recruitment and correspond thereby to the key life stage for population dynamics. Our work also provides rare evidence that the trans-generational effect of senescence can be stronger than direct reproductive senescence in indeterminate growers.

Producing offspring in Armadillidium vulgare: Effects of genetic diversity and inbreeding.

Genetic diversity is known to be correlated to fitness traits, and inbred individuals often display lower values for life history traits. In this study, we attempt to quantify how inbreeding affects such traits in the terrestrial isopod Armadillidium vulgare by performing inbred and non-inbred crosses under laboratory conditions. We estimated genetic characteristics of parents and offspring, and related them to fecundity and fertility measures, as well as offspring growth and survival. Our study shows that a decrease in offspring number might result from mortality around birth, but not to changes in fecundity, fertilization rate, or developmental failure between inbred and non-inbred crosses. More heterozygous females tended to be bigger and had a higher fecundity, which could have implications in mate choice. No effect of inbreeding was detected on offspring growth and survival. These results can be related to previously observed effects of genetic characteristics on mating strategies in A. vulgare, and could shed light on mechanisms of inbreeding avoidance in this species.

When GIS zooms in: spatio-genetic maps of multipaternity in Armadillidium vulgare.

Geographic information system (GIS) tools are designed to illustrate, analyse and integrate geographic or spatial data, usually on a macroscopic scale. By contrast, genetic tools focus on a microscopic scale. Because in reality, landscapes have no predefined scale, our original study aims to develop a new approach, combining both cartographic and genetic approaches to explore microscopic landscapes. For this, we focused on Armadillidium vulgare, a terrestrial isopod model in which evolutionary pressures imposed by terrestrial life have led to the development of internal fertilisation and, consequently, to associated physiological changes. Among these, the emergence of internal receptacles, found in many taxa ranging from mammals to arthropods, allowed females to store sperm from several partners, enabling multipaternity. Among arthropods, terrestrial isopods like the polygynandrous A. vulgare present a female structure, the marsupium, in which fertilised eggs migrate and develop into mancae (larval stage). To test our innovative combined approach, we proposed different males to four independent females, and at the end of incubation in the marsupium, we mapped (using GIS methods) and genotyped (using 12 microsatellite markers) all the incubated mancae. This methodology permitted to obtain spatio-genetic maps describing heterozygosity and spatial distribution of mancae and of multipaternity within the marsupial landscape. We discussed the interest of this kind of multidisciplinary approach which could improve in this case our understanding of sexual selection mechanisms in this terrestrial crustacean. Beyond the interesting model-focused insights, the main challenge of this study was the transfer of GIS techniques to a microscopic scale and our results appear so as pioneers rendering GIS tools available for studies involving imagery whatever their study scale.

Isolation, characterization and PCR multiplexing of microsatellite loci for two sub-species of terrestrial isopod Porcellio dilatatus (Crustacea, Oniscidea).

Several microsatellite markers have already been developed for different terrestrial isopod species such as Armadillidium vulgare, A. nasatum and Porcellionides pruinosus. In all these species, the endosymbiont Wolbachia has a feminizing effect that generates a female bias in sex ratio and reduces the number of reproductive males. Thus this can potentially decrease the genetic diversity of host populations. However, in some other isopod species, Wolbachia induces cytoplasmic incompatibility (CI); the most commonly described effect of Wolbachia in arthropods. The CI by rendering some crossings incompatible can reduce the gene flow and strengthen genetic differentiation between isopod populations. To date, the influence of Wolbachia inducing CI on population structure of terrestrial isopods has never been investigated. In this study, we developed 10 polymorphic microsatellite markers shared by two sub-species of Porcellio dilatatus. Crossings between the two sub-species are partially incompatible due to two CI-inducing Wolbachia strains. These new microsatellite markers will allow us to investigate the effect of CI on host genetic differentiation in this species complex.

Why and how do protective symbionts impact immune priming with pathogens in invertebrates?

Growing evidence demonstrates that invertebrates display adaptive-like immune abilities, commonly known as "immune priming". Immune priming is a process by which a host improves its immune defences following an initial pathogenic exposure, leading to better protection after a subsequent infection with the same - or different - pathogens. Nevertheless, beneficial symbionts can enhance similar immune priming processes in hosts, such as when they face repeated infections with pathogens. This "symbiotic immune priming" protects the host against pathogenic viruses, bacteria, fungi, or eukaryotic parasites. In this review, we explore the extent to which protective symbionts interfere and impact immune priming against pathogens from both a mechanical (proximal) and an evolutionary (ultimate) point of view. We highlight that the immune priming of invertebrates is the cornerstone of the tripartite interaction of hosts/symbionts/pathogens. The main shared mechanism of immune priming (induced by symbionts or pathogens) is the sustained immune response at the beginning of host-microbial interactions. However, the evolutionary outcome of immune priming leads to a specific discrimination, which provides enhanced tolerance or resistance depending on the type of microbe. Based on several studies testing immune priming against pathogens in the presence or absence of protective symbionts, we observed that both types of immune priming could overlap and affect each other inside the same hosts. As protective symbionts could be an evolutionary force that influences immune priming, they may help us to better understand the heterogeneity of pathogenic immune priming across invertebrate populations and species.

Immune priming in Armadillidiumvulgare against Salmonellaenterica: direct or indirect costs on life history traits?

Invertebrate immune priming is defined as an enhanced protection against secondary pathogenic infections when individuals have been previously exposed to the same or a different pathogen. Immune priming can be energetically costly for individuals, thus impacting trade-offs between life-history traits, like reproduction, growth, and lifetime. Here, the reproductive cost(s) and senescence patterns of immune priming against S.enterica in the common woodlouse A.vulgare (Crustacea, Isopoda) were investigated. Four different groups of females were used that either (1) have never been injected (control), (2) were injected twice with S.enterica (7 days between infections), (3) were firstly injected with LB-broth, then with S.enterica, and (4) females injected only once with S.enterica. All females were allowed to breed with one non-infected male and were observed for eight months. Then, the number of clutches produced, the time taken to produce the clutch(es), the number of offspring in each clutch, the senescence biomarkers of females, and parameters of their haemocytes were compared. The result was that immune priming did not significantly impact reproductive abilities, senescence patterns, and haemocyte parameters of female A.vulgare, but had an indirect effect through body weight. The lighter immune primed females took less time to produce the first clutch, which contained less offspring, but they were more likely to produce a second clutch. The opposite effects were observed in the heavier immune primed females. By highlighting that immune priming was not as costly as expected in A.vulgare, these results provide new insights into the adaptive nature of this immune process.

Experimental evidence of Wolbachia introgressive acquisition between terrestrial isopod subspecies.

Wolbachia are the most widespread endosymbiotic bacteria in animals. In many arthropod host species, they manipulate reproduction via several mechanisms that favor their maternal transmission to offspring. Among them, cytoplasmic incompatibility (CI) promotes the spread of the symbiont by specifically decreasing the fertility of crosses involving infected males and uninfected females, via embryo mortality. These differences in reproductive efficiency may select for the avoidance of incompatible mating, a process called reinforcement, and thus contribute to population divergence. In the terrestrial isopod Porcellio dilatatus, the Wolbachia wPet strain infecting the subspecies P. d. petiti induces unidirectional CI with uninfected individuals of the subspecies P. d. dilatatus. To study the consequences of CI on P. d. dilatatus and P. d. petiti hybridization, mitochondrial haplotypes and Wolbachia infection dynamics, we used population cages seeded with different proportions of the 2 subspecies in which we monitored these genetic parameters 5 and 7 years after the initial setup. Analysis of microsatellite markers allowed evaluating the degree of hybridization between individuals of the 2 subspecies. These markers revealed an increase in P. d. dilatatus nuclear genetic signature in all mixed cages, reflecting an asymmetry in hybridization. Hybridization led to the introgressive acquisition of Wolbachia and mitochondrial haplotype from P. d. petiti into nuclear genomes dominated by alleles of P. d. dilatatus. We discuss these results with regards to Wolbachia effects on their host (CI and putative fitness cost), and to a possible reinforcement that may have led to assortative mating, as possible factors contributing to the observed results.

Promiscuity and sex ratio in the terrestrial isopod Armadillidium vulgare and consequences on genetic diversity.

In promiscuous mating systems, both males and females mate with several partners. While the benefits of multiple mating are well recognized for males, there are several non-mutually exclusive hypotheses to explain multiple mating for females. Promiscuity is widespread in terrestrial isopods. Here, we placed experimental populations of the terrestrial isopod Armadillidium vulgare under varying sex ratios to manipulate the number of available partners, and better characterise the mating system in this species by performing paternity tests using microsatellite markers. We observed that females usually mate multiply with up to 5 males in a single event of reproduction. A higher number of fathers in broods did not increase brood size nor heterozygosity, but increased allelic richness. Promiscuity seems to be asymmetric in this species, with females being less affected by changes in sex ratio than were males and would be an adaptive mating system maximising the offspring genetic diversity.