Physiological and behavioral responses of Orchestia gammarellus (Amphipoda, Talitridae) towards trace elements contamination soil.

Effects of trace elements on the hepatopancreas ultrastructure and on the locomotor activity rhythm were investigated in the amphipod talitrid Orchestia gammarellus collected from Bizerte lagoon banks situated in the north of Tunisia. Animals were exposed to a series of contaminated soil with different concentrations of cadmium and zinc. The locomotor activity rhythm was studied under constant darkness. Histopathological analysis showed that the trace elements, especially the Cd, induced significant changes in the morphology and in the ultrastructural organization of hepatopancreatic cells. The significant alterations obtained were dose dependent. Concerning the behavioral response, results revealed the presence of two components whatever the experimental conditions. In addition, a great inter-individual variability of the locomotor rhythm was observed. Patterns were in majority bimodal for the control individuals and became unimodal and multimodal when exposed under Cd and Zn, respectively. Furthermore, ultradian and circadian periods were determined. The circadian period lengthened after Zn exposure. In addition, the locomotor activity rhythm was more stable for control individuals. However, those exposed to Cd were less active.

Physiological and histopathological responses of Porcellio laevis (Isopoda, Crustacea) as indicators of metal trace element contamination.

This study was designed to assess the impact of the mixture of cadmium (Cd) and zinc (Zn) on the bioaccumulation and the ultrastructural changes in the hepatopancreas of Porcellio laevis (Latreille, 1804) after 4 weeks of exposure to contaminated Quercus leaves under laboratory conditions. For each metal, four concentrations were used with four replicates for each concentration. Metal concentrations in the hepatopancreas and the rest of the body were determined using atomic absorption spectrometry. From the first week until the end of the experiment, a weight gain in P. laevis was observed particularly between the first and the end of exposure from 93.3 ± 18.22 mg fw to 105.22 ± 16.16 mg fw and from 106.4 ± 22.67 mg fw to 125.9 ± 23.9 mg fw for Mix1 and Mix4, respectively. Additionally, the determined metal trace elements (MTE) concentrations in the hepatopancreas were considerably higher compared to those in the rest of the body and seem to be dose-dependent. Using transmission electron microscopy (TEM), some alterations were highlighted in the hepatopancreas. The main observed alterations were (a) the destruction of the microvilli border in a considerable portion of cells, (b) the increase of the lipid droplets with different shapes and sizes, (c) the increase in the number of the mitochondria, and (d) the appearance of TE in the form of B-type granules. The obtained results confirmed the ability of P. laevis to deal with high amounts of MTE, suggesting its possible use in future soil's biomonitoring programs.

The induced damage in the hepatopancreas of Orchestia species after exposure to a mixture of Cu/Zn-An ultrastructural study.

The hepatopancreas of crustaceans species has been recognized as an essential target organ to assess trace elements' effects. Due to its dynamic and capability of detoxifying trace metal, this organ often indicates distinct pathological disturbances. In the present work, we intend to evaluate the bioaccumulation of trace metal in three Orchestia species (Orchestia montagui, Orchestia gammarellus, and Orchestia mediterranea) living in symmetry in the banks of Bizerte lagoon (37°13'8″N 09°55'1″E) after their exposure during 14 days to a mixture of copper and zinc, and to highlight the effect of these metals on their hepatopancreas ultrastructure using transmission electron microscopy. At the end of the experiment, results showed that the mortality and the body mass varied according to the used nominal concentrations. Significant alterations were noted in all the treatment groups. The degree of these alterations depends on the used concentration, and they are represented especially by the cells remoteness and the border lyses, the reduction of the nuclear volume, the increase in the cytoplasm density with the presence of trace metal in the nucleus as well as in the vacuole, the disorganization and the destruction of microvilli, the condensation of the majority of cellular organelles and mitochondria swelling. Through this study, Orchestia genus could be an attractive candidate for the biochemical study of trace metal toxicity in Tunisian wetlands.

Evaluation of trace element contamination using Armadillo officinalis Duméril, 1816 (Crustacea, Isopoda) as a tool: An ultrastructural study.

To estimate trace element bioaccumulation in Armadillo officinalis, specimens were collected from Ghar El Melh lagoon then exposed for 3 weeks in contaminated sediments with copper, zinc, and cadmium. From the first week until the end of the experiment, a decrease in A. officinalis growth related to the increase of Cd concentration in the sediment was recorded. However, a mass gain was highlighted under Cu and Zn exposures. At the end of experiment, body metal concentrations were measured using flame atomic emission spectrometry. Results of the bioaccumulation factor showed that the species could be considered as a macroconcentrator of copper (BAF > 2) and a deconcentrator of zinc (BAF < 2). Microscopy observations of hepatopancreas cells showed morphological and histological changes even at the lowest concentration. They consisted in the microvillus border destruction, lipid droplets modifications, trace element accumulation, and the condensation of the majority of cellular organelles. The degree of these alterations was found to be dose-dependent. Through these results, the isopod A. officinalis could be used as relevant monitor organisms for soil metal contamination.

Effects of trace metal elements on ultrastructural features of hepatopancreas of Armadillidium granulatum Brandt, 1833 (Crustacea, Isopoda).

This study was conducted to compare metals bioaccumulation in the terrestrial isopod Armadillidium granulatum collected from Ghar El Melh lagoon. We focused on recognizing the effects of trace elements on hepatopancreas functional role. To this end, isopod specimens were exposed for 3 weeks to sediments contaminated with cadmium, copper, zinc, mercury, and nickel. Three concentrations were used in duplicate for each experimental condition. At the end of the experiment, metal body burdens were determined using flame atomic absorption spectrometry. Results of the bioaccumulation factor (BAF) showed that the species A. granulatum was classified as a Cu macroconcentrator (BAF > 2) and a Zn deconcentrator (BAF < 2). Dose dependent morphological and histological changes were observed in the hepatopancreas cells using transmission electron microscopy. The predominant features were: microvillus border disruption, condensation of the cytoplasm with increasing endoplasmic reticulum, mitochondria, lysosomes and granules that accumulated metals in B and S cells. The number of lipid droplets decreased especially after Cd, Zn, Hg, and Ni treatments. This study demonstrated that the terrestrial isopod A. gramulatum could be a good indicator of soil metal contamination.

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

BACKGROUND: In 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. RESULTS: Wolbachia 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. CONCLUSIONS: We 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.

Ecotoxicological effects of trace element contamination in talitrid amphipod Orchestia montagui Audouin, 1826.

This study deals with the evaluation of trace element bioaccumulation and histological alterations in the hepatopancreas of the supralittoral amphipod Orchestia montagui Audouin, 1826 due to the exposure to cadmium, copper, and zinc. Orchestia montagui individuals were maintained during 14 days in soils contaminated with different trace elements namely cadmium, copper, and zinc; a control was also prepared. Our results show that the mortality and the body mass vary according to the metal and the nominal concentration used. In general, the mortality increases from the seventh day. However, the body mass shows a decrease with cadmium exposure and an increase with copper and zinc exposures. Furthermore, the concentration factor highlights that this species is considered a macroconcentrator for copper and zinc. The hepatopancreas of unexposed and exposed animals were compared to detect histological changes. Our results show significant alterations in the hepatopancreas of the exposed animals after the experiment. The degree of these alterations was found to be dose-dependent. Among the histological changes in the hepatopancreas in O. montagui, a loss of cell structure was noted, especially cell remoteness and border lyses, the reduction of nuclear volume, an increase in the cytoplasm density with the presence of trace element deposits in both the nucleus and vacuoles, a disorganization and destruction of microvilli, and a condensation of the majority of cell organelles and mitochondria swelling. Through this study, we have confirmed that O. montagui can be a relevant model to assess trace metal element pollution in Tunisian coastal lagoons with the aim of using it in future biomonitoring programs.

Effect of agricultural practices on terrestrial isopods: a review.

Terrestrial isopods (approximately 3700 known species in the world) are encountered in temperate and tropical regions, from the seashore to high altitudes and from floodplain forests to deserts. They are known to contribute to soil biodiversity. Environmental factors and anthropogenic actions, particularly land use changes such as primarily agricultural practices, and urbanization affect soil biodiversity and their functions. Human practices, such as soil tillage, pesticide application, chemical pollution, along with soil acidification adversely affect isopod abundance and diversity. It is thus important to recognise the vital contributions of soil biodiversity in support of environmental quality protection through maintaining soil functions and their significance to sustainable land use. This review will also deal with recent studies attempting to evaluate the impact of returning to an environmentally friendly agriculture by restoring refuge habitats such as grass strips, hedges, and woodlands for terrestrial isopods.

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.

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.

Hierarchical organization of the cuticle of the subsocial desert isopod, Hemilepistus reaumurii.

The crustacean cuticle is a hierarchically organised material which provides protection and sites for muscle attachment. The physical properties of this exoskeleton envelope are adapted to the function and the eco-physiological requirements of the species. This paper aimed to study, using the TEM, the structure of the tubercle and the tergite cuticle of the arid species Hemilepistus reaumurii in a comparison with a subhumid isopod in order to relate some peculiar features to an adaptive process to environmental constraints. Results showed that wild H. reaumurii cuticles were twice as thick in comparison with Porcellio variabilis which is a subhumid zone isopod. It is suggested therefore that the thick cuticle of wild H. reaumurii can be an adaptation to terrestrial life and a protection against osmotic stress and water loss in an arid environment. In addition the inside of the tubercle showed a high number of lipid droplets stacked into an adipose tissue which suggest that tubercles were used for storage for nutritive material in wild H. reaumurii.

Multiple drivers of decline in the global status of freshwater crayfish (Decapoda: Astacidea).

Rates of biodiversity loss are higher in freshwater ecosystems than in most terrestrial or marine ecosystems, making freshwater conservation a priority. However, prioritization methods are impeded by insufficient knowledge on the distribution and conservation status of freshwater taxa, particularly invertebrates. We evaluated the extinction risk of the world's 590 freshwater crayfish species using the IUCN Categories and Criteria and found 32% of all species are threatened with extinction. The level of extinction risk differed between families, with proportionally more threatened species in the Parastacidae and Astacidae than in the Cambaridae. Four described species were Extinct and 21% were assessed as Data Deficient. There was geographical variation in the dominant threats affecting the main centres of crayfish diversity. The majority of threatened US and Mexican species face threats associated with urban development, pollution, damming and water management. Conversely, the majority of Australian threatened species are affected by climate change, harvesting, agriculture and invasive species. Only a small proportion of crayfish are found within the boundaries of protected areas, suggesting that alternative means of long-term protection will be required. Our study highlights many of the significant challenges yet to come for freshwater biodiversity unless conservation planning shifts from a reactive to proactive approach.

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.

Widespread Wolbachia infection in terrestrial isopods and other crustaceans.

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.

Widespread atypical mitochondrial DNA structure in isopods (Crustacea, Peracarida) related to a constitutive heteroplasmy in terrestrial species.

Metazoan mitochondrial DNA (mtDNA) is generally composed of circular monomeric molecules. However, a few exceptions do exist and among them two terrestrial isopods Armadillidium vulgare and Porcellionides pruinosus have an atypical mtDNA composed of linear monomers associated with circular "head-to-head" dimers: a very unusual structure for animal mtDNA genome. To assess the distribution of this atypical mtDNA among isopods, we performed RFLP and Southern blot analyses on mtDNA of 16 terrestrial (Oniscidea family) and two aquatic isopod species: the marine Sphaeroma serratum (suborder Flabellifera, sister group of Oniscidea) and the freshwater Asellus aquaticus (Asellota, early derived taxon of isopod). The atypical mtDNA structure was observed in 15 terrestrial isopod species and A. aquaticus, suggesting a wide distribution of atypical mtDNA among isopods. However, a typical metazoan mtDNA structure was detected in the marine isopod S. serratum and the Oniscidea Ligia oceanica . Our results suggest two possible scenarios: an early origin of the atypical mtDNA in isopods followed by reversion to the typical ancestral mtDNA structure for several species, or a convergent appearance of the atypical mtDNA structure in two isopod suborders. We compare this distribution of the atypical mtDNA structure with the presence of a heteroplasmy also observed in the mtDNA of several terrestrial isopod species. We discuss if this transmitted heteroplasmy is vectored by the atypical mtDNA and its impact on the maintenance of the atypical mtDNA in isopods.

Growth and metal accumulation in Porcellio scaber exposed to poplar litter from Cd-, Pb-, and Zn-contaminated sites.

This study aimed at determining, in the laboratory, the effects of poplar litter collected in woody habitats contaminated by heavy metals on growth and metal accumulation in the terrestrial isopod Porcellio scaber. Cd, Pb, and Zn pseudototal and CaCl(2)-extractable concentrations in litter types were determined using AAS. Juveniles were fed ad libitum, individually, for 28 days with four litter types presenting an increasing gradient of metal contamination. Individuals were weighed every week and metal body burdens were determined at the end of the experiment. From the first week until the end of the experiment, a decrease in P. scaber growth related to the increase of metal concentration in litter types was recorded. Significant correlations were observed between metal body burdens and metal concentrations in litter types. However, Cd accumulation in woodlice appeared to be related to the Cd/Zn concentration ratio in litter types. All these results showed the potential of weight gain in P. scaber as a suitable indicator for litter quality assessment with ecological relevance.

Chiral ligand-exchange chromatography of amino acids using porous graphitic carbon coated with a dinaphthyl derivative of neamine.

In this paper, we describe the preparation and the evaluation of a porous graphitic carbon (PGC) column coated with a new dinaphthyl derivative of neamine for chiral ligand-exchange (LE) chromatography. It was shown that the graphitic surface/dinaphthyl anchor system efficiently (1.15 micromol/m(2)) and stably (three months of intensive use) adsorbs the neamine template onto the chromatographic support. The resulting coated PGC stationary phase showed appreciable LE-based enantioselective properties towards several native amino acids.

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.

Chiral resolution of histidine using an anti-D-histidine L-RNA aptamer microbore column.

In this paper, we report a new anti-amino acid aptamer chiral stationary phase (CSP). The enantiomers of histidine were separated using an immobilized histidine-specific L-RNA aptamer (40-mer) and an aqueous buffer as mobile phase. The effects of the variation of different operating parameters, including the mobile phase pH and the MgCl2 concentration as well as the column temperature, on the solute retention were assessed. The results suggested that (i) the protonated form of histidine was involved in the stereospecific RNA binding and (ii) Mg2+ was essential for the target enantiomer binding to the specific aptamer sites. From a practical point of view, it appeared that the baseline resolution in a minimum analysis time can be achieved at a column temperature of 35 degrees C for an eluent containing 10 mM of MgCl2, pH 5.5.