Terrestrial Isopod Armadillidium Vulgare Research Articles

Aggregation and sheltering behavior of Armadillidium vulgare and Armadillidium nasatum (Isopoda: Armadillidiidae) exposed to fluorescent and ultraviolet light

Terrestrial isopods are gregarious invertebrates, aggregating in colonies. Aggregation reduces risk of predation and desiccation. Isopod aggregation and sheltering behavior have been observed under fluorescent and color-filtered light, but not under ultraviolet (UV) light. With UV levels on the rise, it is important to understand how soil ecosystems might be impacted by human-induced ozone depletion. We examined aggregation and sheltering behaviors of the terrestrial isopods Armadillidium vulgare and Armadillidium nasatum under fluorescent light and under fluorescent light with added UV light. Upon testing, each species was randomly assigned into groups of 10 individuals. Each group was released into a circular arena with a black disk in the middle of the lid which functioned to block light and served as a shelter. Sheltering and aggregation behaviors were recorded every 15 minutes for 45 minutes under the presence versus absence of increased UV light. There was no significant difference in the total number of sheltering events, but positive thigmotaxis (movement toward or away from a physical stimulus) may have influenced sheltering. However, there was a significant increase in the average number of aggregated individuals and the total number of aggregates in groups exposed to increased UV conditions, perhaps reflective of their desiccation avoidance behavior and negative phototaxis. Additionally, A. nasatum individuals were significantly more likely to form aggregates than A. vulgare when exposed to increased UV light conditions, perhaps because A. nasatum is less desiccation-resistant than A. vulgare.

Microstructure, mechanical properties and elemental composition of the terrestrial isopod Armadillidium vulgare cuticle

The exoskeletons of crustaceans are essential for providing protection from predators and other environmental threats. Understanding the structure and mechanical behavior of their natural armor could inspire the design of lightweight and high toughness synthetic materials. Most published work has focused on marine crustacea rather than their terrestrial counterparts, which are exposed to a multitude of unique threats. The interest in the terrestrial isopod Armadillidium vulgare (A. vulgare) has grown but the interrelationship between the microstructure, chemical composition, and mechanical properties has not been thoroughly investigated. Thus, this study aims to elucidate missing details concerning this biological mineralized composite. Exoskeleton specimens were fixated to preserve the intrinsic protein structure. We utilize scanning electron microscopy for microstructure analysis, Raman spectroscopy for elemental analysis, and nanoindentation property mapping to achieve mechanical characterization. The naturally fractured A. vulgare exoskeleton cross-section reveals four subregions with the repeating helicoidal ‘Bouligand’ arrangement most prominent in the endocuticle. The hardness and reduced modulus distributions exhibit a through-thickness exponential gradient with decreasing magnitudes from the outermost to the innermost layers of the exoskeleton. The Raman spectra show a graded spatial distribution of key constituents such as calcium carbonate across the thickness, some of which are consistent with the mechanical property gradient. Potential microstructure, elemental composition, and mechanical property relationships are discussed to explain how the hierarchical structure of this nanolaminate armor protects this species.

Shedding Light on the Antimicrobial Peptide Arsenal of Terrestrial Isopods: Focus on Armadillidins, a New Crustacean AMP Family.

In crustaceans, antimicrobial peptides (AMPs) are clustered into four major groups according to their amino acid composition and structure: (1) single-domain peptides containing cysteine residues such as anti-lipopolysaccharide-factor (ALF), (2) multi-domain or chimeric AMPs such as crustins, (3) non-conventional AMPs, and (4) linear single-domain AMPs. The majority of AMPs has been described in commercially exploited crustaceans, particularly decapods living in aquatic environments (crab, shrimp, lobster, and crayfish). Here, we aimed at establishing the AMPs repertoire of terrestrial isopods (Oniscidea), an original suborder of crustaceans adapted to life outside of the aquatic environment. Using transcriptomic data from 21 species, we identified 110 ALF and 73 crustin sequences. We also characterized the full-length sequence of armadillidins from 17 species, similar to the AMP previously described in the terrestrial isopod Armadillidium vulgare. Furthermore, we tested the antimicrobial activity of three armadillidin peptides characterized from three distantly related species. This analysis revealed similar activity spectra against pathogens, despite extensive structural variation among the tested peptides. In addition to conventional crustacean AMPs, our work highlights armadillidins as a new and independent family of AMPs specific to the Oniscidea, thus opening new perspectives concerning the study of the immune system of terrestrial isopods.

Changes in cuticle structure during growth in two terrestrial isopods (Crustacea: Isopoda: Oniscidea)

Terrestrial isopods are a successful group of terrestrial crustaceans. Their exoskeletal cuticle has been studied in several species. However, it is not known whether the cuticle of these animals becomes thicker or how its structure changes as the animals grow. We aimed to determine the principles of upscaling of the exoskeletal cuticle during growth in the terrestrial isopods Armadillidium vulgare (Latreille, 1804) and Porcellio scaber Latreille, 1804 with scanning electron microscopy. The tergal cuticle becomes thicker with increasing body length. In A. vulgare, which rolls into a ball in defense against predators, the rate of increase of tergite thickness was greater than in P. scaber, which clings to the substrate when threatened. As the cuticles of both species become thicker, the proportion of the endocuticle in the cuticle increases. There is a strong correlation between cuticle thickness and the thickness of endocuticular lamellae. This indicates that in thicker cuticles, chitin-protein fibers in sequential layers change their orientation by a smaller angle. We found no significant differences in morphometric parameters between the cuticles of A. vulgare and P. scaber when controlling for cuticle thickness. This suggests that known differences in cuticle structure and composition between these two species may result from differences in cuticle thickness.

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.

Survival capacity of the common woodlouse Armadillidium vulgare is improved with a second infection of Salmonella enterica

Immune priming has been widely observed in invertebrates. However, this phenomenon remains incompletely characterized concerning the time course of the protective effect and immune responses. Here, we investigated the existence of a protective effect in the terrestrial isopod Armadillidium vulgare against a second infection with the bacterium Salmonella enterica. We primed animals with a low dose of living bacteria or with the bacterial culture medium. One day, 7 or 15 days later, we measured haemocytes concentration and viability and we injected a LD50 of living S. enterica to monitor the survival rates of infected individuals. We show that A. vulgare is better protected (i.e. survival improvement) upon secondary infection when it has encountered S. enterica 7 days before. This protection, which tends to persist 15 days after the first infection, is however not observed when the priming is performed only 1 day before the LD50 injection. Besides providing a new invertebrate example, we highlight this protection is dynamic and may be partly due to a high haemocyte production.

Joint effects of group sex-ratio and Wolbachia infection on female reproductive success in the terrestrial isopod Armadillidium vulgare

BackgroundIn species that reproduce with sexual reproduction, males and females often have opposite strategies to maximize their own fitness. For instance, males are typically expected to maximize their number of mating events, whereas an excessive number of mating events can be costly for females. Although the risk of sexual harassment by males and resulting costs for females are expected to increase with the proportion of males, it remains unknown whether and how parasitic distorters of a host population’s sex-ratio can shape this effect on the fitness of females. Here, we addressed this question using Armadillidium vulgare and its parasite Wolbachia that alters the sex-ratio of a population. We set up Wolbachia-free and Wolbachia-infected females in experimental groups exhibiting 100, 80, 50% or 20% females for 1 year, during which we measured changes in survival, fertility and fecundity.ResultsWolbachia infection shaped the effects of both population sex-ratio and reproductive season on female fecundity. Compared to Wolbachia-free females, Wolbachia-infected females were less likely to be gravid in populations exhibiting an excess of females and did not exhibit the otherwise negative effect of seasons on this likelihood. Group sex-ratio and Wolbachia infection have independent effects on other measured traits. Male-biased populations had females both exhibiting the lowest survival rate after 6 months and producing the smallest number of offspring, independent of Wolbachia infection. Conversely, Wolbachia-infected females had the lowest likelihood of producing at least one offspring, independent of group sex-ratio. Wolbachia infection had no effect on female survival rate.ConclusionsWe demonstrated that male-biased sex-ratio and the presence of Wolbachia are costly for females due to sexual harassment by males and bacterial infection, respectively. Interestingly, Wolbachia infection triggers another negative effect. This effect does not come from an excess of males and its associated sexual harassment of females but instead from a lack of males and the associated risk for females of remaining unmated. Overall, these findings highlight the importance of social pressures and infection on female fitness and provide insights into our general understanding of the joint and opposite effects of these two parameters in the evolution of reproductive strategies.

Leaf Endoplasmic Reticulum Bodies Identified in Arabidopsis Rosette Leaves Are Involved in Defense against Herbivory.

ER bodies are endoplasmic reticulum (ER)-derived organelles specific to the order Brassicales and are thought to function in plant defense against insects and pathogens. ER bodies are generally classified into two types: constitutive ER bodies in the epidermal cells of seedlings, and wound-inducible ER bodies in rosette leaves. Herein, we reveal a third type of ER body found in Arabidopsis (Arabidopsis thaliana) rosette leaves and designate them "leaf ERbodies" (L-ER bodies). L-ER bodies constitutively occurred in specific cells of the rosette leaves: marginal cells, epidermal cells covering the midrib, and giant pavement cells. The distribution of L-ER bodies was closely associated with the expression profile of the basic helix-loop-helix transcription factor NAI1, which is responsible for constitutive ER-body formation. L-ER bodies were seldom observed in nai1 mutant leaves, indicating that NAI1 is involved in L-ER body formation. Confocal imaging analysis revealed that L-ER bodies accumulated two types of β-glucosidases: PYK10, the constitutive ER-body β-glucosidase; and BETA-GLUCOSIDASE18 (BGLU18), the wound-inducible ER-body β-glucosidase. Combined with the absence of L-ER bodies in the bglu18 pyk10 mutant, these results indicate that BGLU18 and PYK10 are the major components of L-ER bodies. A subsequent feeding assay with the terrestrial isopod Armadillidium vulgare revealed that bglu18 pyk10 leaves were severely damaged as a result of herbivory. In addition, the bglu18 pyk10 mutant was defective in the hydrolysis of 4-methoxyindol-3-ylmethyl glucosinolate These results suggest that L-ER bodies are involved in the production of defensive compound(s) from 4-methoxyindol-3-ylmethyl glucosinolate that protect Arabidopsis leaves against herbivory attack.

The Genome of Armadillidium vulgare (Crustacea, Isopoda) Provides Insights into Sex Chromosome Evolution in the Context of Cytoplasmic Sex Determination.

The terrestrial isopod Armadillidium vulgare is an original model to study the evolution of sex determination and symbiosis in animals. Its sex can be determined by ZW sex chromosomes, or by feminizing Wolbachia bacterial endosymbionts. Here, we report the sequence and analysis of the ZW female genome of A. vulgare. A distinguishing feature of the 1.72 gigabase assembly is the abundance of repeats (68% of the genome). We show that the Z and W sex chromosomes are essentially undifferentiated at the molecular level and the W-specific region is extremely small (at most several hundreds of kilobases). Our results suggest that recombination suppression has not spread very far from the sex-determining locus, if at all. This is consistent with A. vulgare possessing evolutionarily young sex chromosomes. We characterized multiple Wolbachia nuclear inserts in the A. vulgare genome, none of which is associated with the W-specific region. We also identified several candidate genes that may be involved in the sex determination or sexual differentiation pathways. The A. vulgare genome serves as a resource for studying the biology and evolution of crustaceans, one of the most speciose and emblematic metazoan groups.

How do familiarity and relatedness influence mate choice in Armadillidium vulgare?

Mate choice is an important process in sexual selection and usually prevents inbreeding depression in populations. In the terrestrial isopod Armadillidium vulgare, the close physical proximity between individuals may increase the risk of reproducing with siblings. Moreover, individuals of this species can be infected with the feminizing bacteria of Wolbachia, which influence male mate choice. However, little is known about the kinship or familiarity assessment of the selected partner that occurs when a male can choose between females with or without Wolbachia. To investigate the potential mechanisms leading to mate choice and the potential impact of the parasite, we performed behavioral choice tests on males where they could choose between sibling vs. nonsibling females, familiar vs. unfamiliar females, and sibling familiar vs. unfamiliar nonsibling females. To investigate the costs of inbreeding, we compared the reproductive success of both sibling and nonsibling mates. Our results revealed that male copulation attempts were higher for familiar females and for nonsibling females when both females were Wolbachia-infected, but the duration was longer when both females were Wolbachia-free. When males mated with a sibling female, their fecundity was severely decreased, consistent with inbreeding depression. Overall, we observed copulations with all types of females and demonstrated discrimination capacities and potential preferences. We highlight the complexity of the tradeoff between kinship, familiarity and parasite transmission assessment for mate choice.

Nitrogen excretion during marsupial development in the terrestrial isopod Armadillidium vulgare

Marsupial embryos of Armadillidium vulgare (Isopoda: Oniscidea) were collected at different stages of development and assayed for products of nitrogen excretion. Stages were classified as early stage one, late stage one (clear embryo and somite differentiation), early stage two (chorion shed, prior to blastokinesis), late stage two (following blastokinesis), and mancae (vitelline membrane shed; second embryonic molt). Stage one and stage two embryos were primarily ammonotelic. Mancae showed a significant increase in stored uric acid and decrease in ammonia production, in most cases to undetectable levels. The increased metabolic rate of mancae, and the fact that they imbibe marsupial fluid prior to exiting the marsupium, may have favored a switch from ammonotely to uricotely to avoid ammonia toxicity. Protein metabolism, estimated from ammonia production, accounted for 7% of the measured catabolic rate in Stage 2 embryos. Newly emerged juveniles showed a >2-fold increase in metabolism relative to mancae, accompanying the transition from aquatic to aerial respiration. Following 48 h post-emergence, juveniles resumed ammonia excretion, volatilizing the base (NH3) as in later instars. Elevated ammonia excretion in early juveniles may derive from the catabolism of remaining yolk protein. A sharp increase in whole-animal glutamine in juveniles is consistent with its role as an intermediary nitrogen store during periodic ammonia excretion. Total ammonia concentration in the marsupial fluid fluctuated but did not increase significantly over time and ammonia was not volatilized across the oostegites, indicating that embryo ammonia is transported into the maternal hemolymph for excretion.

Genetic variability in populations of the terrestrial isopod Armadillidium vulgare living in soils with different land uses

The Argentine Pampas region is recognized by its high productivity and fertility, which make its soils suitable for agricultural use. However, the intensification of agricultural activities over the last forty years has led to an intense disturbance regime, characterized by an increase in the soil degradation rate. Soil degradation and its relationship with soil fauna communities are crucial issues in resource management. In this study, we investigated the effects of land-use change on the genetic variability of the terrestrial isopod Armadillidium vulgare, chosen as a biological model. The diversity and population genetic structure of this species were analyzed using inter-simple sequence repeat (ISSR)-PCR markers, in three land uses in two localities of Luján, Buenos Aires, Argentina. Genetic variability was high in natural grassland populations and lower in agricultural land uses. Both conventional FST analysis and Bayesian approach for dominant markers showed significant genetic differences between land uses within each locality. The loss of genetic variability and the population genetic structure can be used as indicators of system disturbance. Thus, in the soils studied, the degree of genetic variability of representative populations of the soil fauna can be a good indicator of the disturbance degree.

Investigating the Molecular Genetic Basis of Cytoplasmic Sex Determination Caused by Wolbachia Endosymbionts in Terrestrial Isopods.

In animals, sexual differences between males and females are usually determined by sex chromosomes. Alternatively, sex may also be determined by vertically transmitted intracellular microbial endosymbionts. The best known cytoplasmic sex manipulative endosymbiont is Wolbachia which can, for instance, feminize genetic males into phenotypic females in the terrestrial isopod Armadillidium vulgare. However, the molecular genetic basis of cytoplasmic sex determination is unknown. To identify candidate genes of feminization induced by Wolbachia strain wVulC from A. vulgare, we sequenced the genome of Wolbachia strain wCon from Cylisticus convexus, the most closely related known Wolbachia strain to wVulC that does not induce feminization, and compared it to the wVulC genome. Then, we performed gene expression profiling of the 216 resulting wVulC candidate genes throughout host developmental stages in A. vulgare and the heterologous host C. convexus. We identified a set of 35 feminization candidate genes showing differential expression during host sexual development. Interestingly, 27 of the 35 genes are present in the f element, which is a piece of a feminizing Wolbachia genome horizontally transferred into the nuclear genome of A. vulgare and involved in female sex determination. Assuming that the molecular genetic basis of feminization by Wolbachia and the f element is the same, the 27 genes are candidates for acting as master sex determination genes in A. vulgare females carrying the f element.

Estimation of the walking ability of an exotic terrestrial isopod Armadillidium vulgare Latreille by field and laboratory measurements

Estimation of the walking ability of an exotic terrestrial isopod Armadillidium vulgare Latreille by field and laboratory measurements

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.

Males prefer virgin females, even if parasitized, in the terrestrial isopod Armadillidium vulgare.

In many species, males increase their reproductive success by choosing high‐quality females. In natural populations, they interact with both virgin and mated females, which can store sperm in their spermatheca. Therefore, males elaborate strategies to avoid sperm competition. In the terrestrial isopod Armadillidium vulgare, females can store sperm and produce several clutches. Moreover, this species can be parasitized by Wolbachia, which feminizes genetic males, transforming them into functional females. Our study compared attractiveness and mate choice when a male is exposed to both virgin and experienced females (i.e., females who have produced offspring and rested for 6 months), with or without Wolbachia. Our results revealed that males are more attracted to virgin females than experienced females, even if these virgin females are parasitized. Moreover, the chemical analysis highlighted different odors in females according to their reproductive and infection (Wolbachia‐free or vertically Wolbachia‐infected) status. Males attempted copulation more frequently and for longer with virgin females, even if Wolbachia‐infected, while experienced females refused further copulation. The evolutionary consequences of both male choice and female resistance on their fitness are discussed in this study.

Symbiotic Bacteria Influence the Odor and Mating Preference of Their Hosts

In many species, chemical communication is a determining factor in mate choice. Some species use the composition of cuticular compounds to discriminate between potential mates. Moreover, the presence of parasites can also influence mate choice and alter the odor of an individual. In the current study, we tested the effect of the endosymbiont Wolbachia, which is naturally present in the terrestrial isopod Armadillidium vulgare and can induce male feminization in the species. We compared male attraction to different types of females (Wolbachia-free, females naturally infected by Wolbachia, Wolbachia-injected females, and females injected with a bacteria-free suspension). Our behavioral assays revealed that males consistently preferred Wolbachia-free females and that this preference correlates to changes in cuticular compounds. Males also preferred naturally infected females over Wolbachia-injected females. The ability of males to discriminate between females could result in Wolbachia impacting its host phenotype, thus affecting the odor of the host. Female odor is the result of an honest signal that makes it possible for males to discriminate between Wolbachia-infected and non-infected females. The male preference for Wolbachia-free females is adaptive, and long-term studies have demonstrated that such choices confer improved fitness.

Evolutionary Significance of Wolbachia-to-Animal Horizontal Gene Transfer: Female Sex Determination and the f Element in the Isopod Armadillidium vulgare.

An increasing number of horizontal gene transfer (HGT) events from bacteria to animals have been reported in the past years, many of which involve Wolbachia bacterial endosymbionts and their invertebrate hosts. Most transferred Wolbachia genes are neutrally-evolving fossils embedded in host genomes. A remarkable case of Wolbachia HGT for which a clear evolutionary significance has been demonstrated is the “f element”, a nuclear Wolbachia insert involved in female sex determination in the terrestrial isopod Armadillidium vulgare. The f element represents an instance of bacteria-to-animal HGT that has occurred so recently that it was possible to infer the donor (feminizing Wolbachia closely related to the wVulC Wolbachia strain of A. vulgare) and the mechanism of integration (a nearly complete genome inserted by micro-homology-mediated recombination). In this review, we summarize our current knowledge of the f element and discuss arising perspectives regarding female sex determination, unstable inheritance, population dynamics and the molecular evolution of the f element. Overall, the f element unifies three major areas in evolutionary biology: symbiosis, HGT and sex determination. Its characterization highlights the tremendous impact sex ratio distorters can have on the evolution of sex determination mechanisms and sex chromosomes in animals and plants.

Paternity success depends on male genetic characteristics in the terrestrial isopod Armadillidium vulgare

Mate choice for compatible partners has been gaining interest during the past years, which implies that what represents a suitable male varies between females and that genetic diversity in offspring should be increased. In this context, mechanisms of mate choice for heterozygous and/or genetically dissimilar partners can be expected. To test for the presence of such mechanisms in the gregarious crustacean Armadillidium vulgare, we performed experimental crosses allowing a female to reproduce with two males. More heterozygous males had a higher paternity success whatever the number of males (only one or both males) participating in reproduction, which could result from better competing abilities or from female preference for such males. When only one male fathered a brood, this male was the most genetically dissimilar to the female, suggesting the existence of a female choice for dissimilarity. Additionally, the more genetically similar both males were to the female, the fewer offspring were produced per brood. Genetic diversity is thus likely to be involved in both pre- and post-copulatory processes, as well as to have an impact on fitness in this terrestrial isopod. This work improves our understanding of the genetic characteristics involved in mating behaviours, and provides insights on the mechanisms at the basis of genetic diversity maintenance. Amongst the many parameters that can be taken into account during mate choice, genetic characteristics must not be neglected, as more and more studies highlight that what is best for one female may not be best for another. Here, we show that both heterozygosity and genetic similarity can influence paternity success in a gregarious terrestrial isopod. This could have strong implications in terms of fitness as a high genetic similarity has a negative impact on offspring number in this species. This work reflects the growing interest for genetic variables, and is positioned at the interface between molecular biology and behavioural ecology.

How Do Females’ Genetic Characteristics Influence Male Mate Preference in the Terrestrial Isopod Armadillidium vulgare?

AbstractGenetic diversity is a key factor that can influence mate choice in many species. We experimentally determined the influence of this factor on mate preference in the crustacean terrestrial isopod Armadillidium vulgare. This biological model is gregarious which could increase the risk of inbreeding by mating with closely related partners. Mechanisms of inbreeding avoidance during mate choice can thus be expected. Moreover, previous studies predict that males would be the choosy sex. We performed Y‐choice tests giving males the choice between two females presenting different levels of heterozygosity and genetic similarity to the male. Our results show potential inbreeding avoidance according to the genetic characteristics of females presented to males. The higher the variation in genetic similarity to the male between females is, the higher the preference of the male towards the most dissimilar female is. Hence, male preferences may only be detectable when the difference between females’ genetic characteristics is large enough. If heterozygosity is associated with fitness in A. vulgare (as in many organisms), the patterns of mate preference we observe may be adaptive.