Hyper-accumulation of vanadium in animals: Two sponges compete with urochordates.

Vanadium (V) concentrations in organisms are usually very low. To date, among animals, only some urochordate and annelid species contain very high levels of V in their tissues. A new case of hyper-accumulation of V in a distinct animal phylum (Porifera), namely, the two homoscleromorph sponge species Oscarella lobularis and O. tuberculata is reported. The measured concentrations (up to 30 g/kg dry weight) exceed those reported previously and are not found in all sponge classes. In both Oscarella species, V is mainly accumulated in the surface tissues, and in mesohylar cells, as V(IV), before being partly reduced to V(III) in the deeper tissues. Candidate genes from Bacteria and sponges have been identified as possibly being involved in the metabolism of V. This finding provides clues for the development of bioremediation strategies in marine ecosystems and/or bioinspired processes to recycle this critical metal.

Methylmercury exposure of the sponge O. lobularis induces strong tissue and cell defects.

Mediterranean marine biota suffers from various anthropogenic threats. Among them, pollutants such as mercury (Hg) represent important environmental issues that are exacerbated by bioaccumulation and bioamplification along food webs via its organic form, monomethylmercury (MMHg). To date, very little is known regarding the impact of mercury on Porifera and the few available studies have been exclusively focused on Demospongiae. This work studies the effect of MMHgCl at different biological levels of Oscarella lobularis (Porifera, Homoscleromorpha). Bioaccumulation assays show that MMHgCl significantly accumulated in sponge tissues after a 96-h exposure to 0.1 µg L-1. Toxicity assays (LC5096h) show a sensibility that depends on life-stage (adult vs bud). Additionally, we show that the exposure to 1 µg L-1 MMHgCl negatively impacts the epithelial integrity and the regeneration process in buds, as shown by the loss of cell-cell contacts and the alteration of osculum morphogenesis. For the first time in a sponge, a whole set of genes classically involved in metal detoxification and in antioxidant response were identified. Significant changes in catalase, superoxide dismutase and nuclear factor (erythroid-derived 2)-like 2 expressions in exposed juveniles were measured. Such an integrative approach from the physiological to the molecular scales on a non-model organism expands our knowledge concerning sensitivity and toxicity mechanisms induced by MMHg in Porifera, raising new questions regarding the possible defences used by marine sponges.

The sponge Oscarella lobularis (Porifera, Homoscleromorpha) as a suitable biomonitor of metallic contamination in Mediterranean coastal ecosystems.

The biomonitoring of metallic contamination in marine ecosystems is often focused on animal species of commercial interest and in lesser extent on non-model marine invertebrates. The aim of this study was to compare the metal concentrations (Li, Al, Ti, Cr, Fe, Ni, Cu, Zn, As, Ag, Cd, Hg, Pb) in seven marine sponges with a particular interest in the homoscleromorph sponge Oscarella lobularis at different sites of the Bay of Marseille, France. Inter-species variabilities suggest that the seven sponge species studied accumulate metals differently. In O. lobularis, a multi-site analysis shows different bioaccumulation between the eight sampled populations. These inter-site differences may reflect differences in the hydrodynamic features and in past and present industrial activities. Because Oscarella lobularis shows a homogeneous metal accumulation pattern in comparison with the other tested species, it appears to be suitable for metal contamination biomonitoring in Mediterranean coastal waters, in particular of the coralligenous communities.

Extreme lifespan of the human fish (Proteus anguinus): a challenge for ageing mechanisms.

Theories of extreme lifespan evolution in vertebrates commonly implicate large size and predator-free environments together with physiological characteristics like low metabolism and high protection against oxidative damages. Here, we show that the 'human fish' (olm, Proteus anguinus), a small cave salamander (weighing 15-20 g), has evolved an extreme life-history strategy with a predicted maximum lifespan of over 100 years, an adult average lifespan of 68.5 years, an age at sexual maturity of 15.6 years and lays, on average, 35 eggs every 12.5 years. Surprisingly, neither its basal metabolism nor antioxidant activities explain why this animal sits as an outlier in the amphibian size/longevity relationship. This species thus raises questions regarding ageing processes and constitutes a promising model for discovering mechanisms preventing senescence in vertebrates.

Extreme longevity in trees: live slow, die old?

We have examined the extreme longevity displayed by trees in relation to a theory mainly developed in animals, namely, the controversial rate of living (ROL) theory of aging which proposes that longevity is negatively correlated to metabolic rate. Plant metabolism implies respiration and photosynthesis; both are susceptible to negatively impact longevity. The relationship between longevity and metabolism was studied in leaves and stems of several species with the aim of challenging the ROL theory in trees. Leaf and stem life spans were found to be highly correlated to metabolism (R(2) = 0.97), and stems displayed a much lower metabolism than leaves. Analysis of covariance (ANCOVA), with metabolism as the covariate, revealed no difference between mean leaf and stem life spans, which would appear to conform to the expectations of the ROL theory. Consequently, the extremely high longevity of trees may be explained by the lower metabolism displayed by the stems. These results clearly reflect different energy allocation and energy expenditure rate strategies between leaves and stems, which may result in different senescence rates (and life spans) in these organs. They also suggest that, in contrast to animals, the ROL theory of aging may apply to woody plants at the organ level, thereby opening a promising new line of research to guide future studies on plant senescence.

Selection of physiological and metabolic adaptations to food deprivation in the Pyrenean newt Calotriton asper during cave colonisation.

Food restriction is one of the major and most common constraints that subterranean animals face in their biotope. Cave-dwelling organisms thus have to cope with fasting periods that can extend from a month to a year. However, adaptive fasting resistance previously found in subterranean fauna has only been highlighted by direct comparisons with phylogenetically distant epigean organisms, which could severely impact conclusions. Here we report physiological and metabolic responses to 42 days of fasting followed by 10 days of refeeding in two populations (one subterranean and one epigean) of Calotriton asper. In the fed state (control), the hypogean population exhibited a hypometabolism together with higher glycogen (+25% in liver and muscles) and triglyceride stores (+50% in muscles). During the fasting period, cave individuals exhibited a 20% decrease in VO(2) whereas epigean individuals experienced no significant change. In addition, the energetic reserves always remained higher in the hypogean population. According to phylogenic and biogeographic data, cave colonization by this species dates back to less than 10,000 years, suggesting a rapid selection of adaptive traits related to fasting. This study strongly suggests that cave colonization induces a decrease in metabolism together with a higher capacity to accumulate energy reserves and therefore to withstand unpredictable fasting periods.

How can a rare protected plant cope with the metal and metalloid soil pollution resulting from past industrial activities? Phytometabolites, antioxidant activities and root symbiosis involved in the metal tolerance of Astragalus tragacantha.

Astragalus tragacantha is a protected plant species in France that grows even in the trace metal and metalloid (TMM) polluted soils of the Calanques National Park (PNCal). Soils are mainly contaminated by lead, copper, zinc and arsenic. An ex situ experiment was conducted, firstly to determine the molecular responses and root traits involved in the TMM tolerance of this plant species by growing individuals in a soil from the surroundings of one of the brownfields of the PNCal, known as l'Escalette, where this plant species grows spontaneously. Secondly, in order to determine the plasticity of these responses, seeds were collected from three different populations, at l'Escalette (polluted site), one from the Frioul archipelago (non-polluted, insular site) and one from La Seyne (non-polluted, littoral site). The results of this study confirmed the capacity of A. tragacantha to germinate and grow in TMM contaminated soils. Only moderate significant variations in chlorophyll and flavonol indices, proline content and antioxidant activities were detected between polluted and control soil conditions for all populations. The main driver for A. tragacantha TMM tolerance seemed to be its ability to be associated with root symbionts i.e. arbuscular mycorrhizal fungi and dark septate endophytes, corresponding to a nutrient-uptake strategy trait. This work provides support for the challenge of A. tragacantha conservation along the littoral of the PNCal, because increasing the number of A. tragacantha individuals would both increase vegetation cover of the polluted soils to reduce the pollution transfer and reinforce the populations of this species.

Osmoregulatory responses to cadmium in reference and historically metal contaminated Gammarus fossarum (Crustacea, Amphipoda) populations.

In order to better understand the variable sensitivities of crustaceans to metals, we investigated the impact of cadmium exposure in 3 populations of Gammarus fossarum from different rivers of France. The first population lives in a Cd-contaminated river from a geochemical background, while the others inhabit Cd-free sites. Osmoregulation, a relevant biomarker to evaluate crustacean health following metal contamination, was used as a proxy to evaluate the intra- and inter-populationnal sensitivities to Cd. Specimens from each population were experimentally exposed to 9 µg Cd2+/L Cd for 7 days and hemolymph osmolality (HO) was then individually measured. In exposed populations, high inter-individual variations in HO values were noted, resulting in their separation into non-impacted and slightly or highly Cd-impacted (with lower HO) animals. In gills of impacted organisms, deep histopathological alterations and protein overexpression of Na+/K+-ATPase and V-H+-ATPase were observed through histology and immunolocalization, while non-impacted animals showed profiles comparable to controls. Moreover, the osmoregulatory processes in the population living in the Cd-contaminated site were impacted by acute Cd exposure in the laboratory as much as for one of the two populations originating from Cd-free sites. The observed changes did not reveal any obvious adaptive osmoregulatory phenomena at the population scale, but they may be due to differences in fitness between individuals and between populations in relation to the features of their respective environments, unrelated with the presence of the metal.

An adaptable mesocosm platform for performing integrated assessments of nanomaterial risk in complex environmental systems.

Physical-chemists, (micro)biologists, and ecologists need to conduct meaningful experiments to study the environmental risk of engineered nanomaterials with access to relevant mechanistic data across several spatial and temporal scales. Indoor aquatic mesocosms (60L) that can be tailored to virtually mimic any ecosystem appear as a particularly well-suited device. Here, this concept is illustrated by a pilot study aimed at assessing the distribution of a CeO2-based nanomaterial within our system at low concentration (1.5 mg/L). Physico-chemical as well as microbiological parameters took two weeks to equilibrate. These parameters were found to be reproducible across the 9-mesocosm setup over a 45-day period of time. Recovery mass balances of 115 ± 18% and 60 ± 30% of the Ce were obtained for the pulse dosing and the chronic dosing, respectively. This demonstrated the relevance of our experimental approach that allows for adequately monitoring the fate and impact of a given nanomaterial.

Exposure to cerium dioxide nanoparticles differently affect swimming performance and survival in two daphnid species.

The CeO2 NPs are increasingly used in industry but the environmental release of these NPs and their subsequent behavior and biological effects are currently unclear. This study evaluates for the first time the effects of CeO2 NPs on the survival and the swimming performance of two cladoceran species, Daphnia similis and Daphnia pulex after 1, 10 and 100 mg.L⁻¹ CeO2 exposures for 48 h. Acute toxicity bioassays were performed to determine EC50 of exposed daphnids. Video-recorded swimming behavior of both daphnids was used to measure swimming speeds after various exposures to aggregated CeO2 NPs. The acute ecotoxicity showed that D. similis is 350 times more sensitive to CeO2 NPs than D. pulex, showing 48-h EC50 of 0.26 mg.L⁻¹ and 91.79 mg.L⁻¹, respectively. Both species interacted with CeO2 NPs (adsorption), but much more strongly in the case of D. similis. Swimming velocities (SV) were differently and significantly affected by CeO2 NPs for both species. A 48-h exposure to 1 mg.L⁻¹ induced a decrease of 30% and 40% of the SV in D. pulex and D. similis, respectively. However at higher concentrations, the SV of D. similis was more impacted (60% off for 10 mg.L⁻¹ and 100 mg.L⁻¹) than the one of D. pulex. These interspecific toxic effects of CeO2 NPs are explained by morphological variations such as the presence of reliefs on the cuticle and a longer distal spine in D. similis acting as traps for the CeO2 aggregates. In addition, D. similis has a mean SV double that of D. pulex and thus initially collides with twice more NPs aggregates. The ecotoxicological consequences on the behavior and physiology of a CeO2 NPs exposure in daphnids are discussed.

Cellular and molecular osmoregulatory responses to cadmium exposure in Gammarus fossarum (Crustacea, Amphipoda).

Osmoregulation represents a reliable indicator of the physiological state of crustaceans. It is mainly effected in gills via Na(+)/K(+)-ATPase (NKA) providing the major driving force for ion transport. In the present study conducted in the freshwater amphipod Gammarus fossarum, the impact of an exposure to 15 µg Cd L(-1) for 3 and 7d was investigated on the haemolymph osmolality (HO), gill structure, NKA localization in gills and its relative expression. In Cd-exposed G. fossarum, mean HO significantly decreased compared to controls. In animals exposed for 3 and 7d, high inter-individual variations in HO values were noted, resulting in their separation into unimpacted, slightly impacted and impacted animals. In unimpacted individuals, gills retained their organization, showing a thicker gill epithelium than in controls; NKA fluorescence was continuously observed along the gill epithelium and was distributed on a broader area than in controls. In slightly impacted individuals, a thinner epithelium, a slight collapse of the gill and a lower NKA fluorescence were observed compared to unimpacted specimens. In impacted individuals, dramatic alterations of the gill structure, including hyperplasia and alteration of the pillars, resulting in the collapse of the gill and the disappearance of the haemolymphatic canals were observed, as well as very limited NKA fluorescence. Therefore, the degree of gill alteration and the intensity of NKA fluorescence observed in the different groups were correlated with their respective HO levels. The relative amount of the NKA α-subunit mRNA significantly increased in specimens exposed to Cd for 3d compared to controls, and then returned to control level after 7d. The relationships between the changes in HO values, NKA immunostaining and mRNA relative expression are discussed. These results confirm that HO represents a valuable biomarker to evaluate crustacean health, and they underline the interest to assess individual responses to contaminants.

High anoxia tolerance in the subterranean salamander Proteus anguinus without oxidative stress nor activation of antioxidant defenses during reoxygenation.

The present study describes a high anoxia tolerance in an amphibian at high temperature. Indeed, the subterranean salamander Proteus anguinus survived 12 h under anoxia at 12 degrees C. Surprisingly, such experimental conditions did not affect P. anguinus oxidative status while muscles and liver antioxidant enzymes activities decreased under 8 h anoxia and only return to basal level during reoxygenation. To test if such adaptation is common in Urodels, equivalent experimentations have been conducted on another newt: the stream-dwelling Calotriton asper. This latter species exhibited only 1.5 h survival under anoxia in spite of higher antioxidant enzymes activities than P. anguinus. Furthermore, aerobic recovery after 1 h anoxia induced a 30% increase of oxidative damage partly explained by SOD and CAT activities that did not return to control values during reoxygenation, demonstrating a lower capacity to counteract ROS overproduction than P. anguinus. In addition, uncoupling protein (UCP) transcript was for the first time detected, partly sequenced and quantified in amphibian muscles and liver. UCP may be considered as a ROS production attenuator by mediating a discharge of the proton gradient generated by the respiratory chain. The putative role of UCP in post-anoxic oxidative status of both species is discussed.

Freezing or supercooling: how does an aquatic subterranean crustacean survive exposures at subzero temperatures?

Crystallization temperature (T(c)), resistance to inoculative freezing (IF), ice contents, bound water, protein and glycogen body contents were measured in the aquatic subterranean crustacean Niphargus rhenorhodanensis and in the morphologically close surface-dwelling aquatic crustacean Gammarus fossarum, both acclimated at 12 degrees C, 3 degrees C and -2 degrees C. Cold acclimation induced an increase in the T(c) values in both species but no survival was observed after thawing. However, after inoculation at high sub-zero temperatures, cold-acclimated N. rhenorhodanensis survived whereas all others, including the 3 degrees C and -2 degrees C acclimated G. fossarum died. In its aquatic environment, N. rhenorhodanensis is likely to encounter inoculative freezing before reaching the T(c) and IF tolerance appears as a highly adaptive trait in this species. Bound water and glycogen were found to increase in the 3 degrees C and -2 degrees C acclimated N. rhenorhodanensis, whereas no variation was observed in G. fossarum. Considering the hydrophilic properties of glycogen, such a rise may be correlated with the increased bound water measured in cold-acclimated N. rhenorhodanensis, and may be linked to the survival of this species when it was inoculated. The ecological significance of the survival of the aquatic subterranean crustacean to inoculative freezing is paradoxical, as temperature is currently highly buffered in its habitat. However, we assume that past geographical distribution and resulting life history traits of N. rhenorhodanensis are key parameters in the current cold-hardiness of the species.

Metabolic responses to cold in subterranean crustaceans.

Changes in polyol, sugar and free amino acid (FAA) body contents were investigated in the aquatic, subterranean (i.e. hypogean) crustaceans Niphargus rhenorhodanensis and Niphargus virei and in a morphologically close aquatic, surface-dwelling (i.e. epigean) crustacean Gammarus fossarum acclimated to 12 degrees C, 3 degrees C and -2 degrees C. With decreasing temperature, G. fossarum significantly increased its alanine and glutamine levels, while trehalose body content was found to increase above control levels only at -2 degrees C. N. virei showed moderate increases of alanine and glycine, and no change in trehalose level was observed in this species. By contrast, N. rhenorhodanensis was the only species showing a significant rise in its total FAA pool, mainly explained by alanine, glycine, arginine and glutamine accumulations. This species also gradually increased its trehalose body content with decreasing temperature. Several cold-hardy ectotherms show metabolic responses to cold that are identical to those observed in N. rhenorhodanensis. A previous comparative study showed that the hypogean N. rhenorhodanensis exhibited a survival time (Lt50) at -2 degrees C that was 26.3 times and 2.6 times higher than the hypogean N. virei and the epigean G. fossarum, respectively. Thus, crustacean levels of FAA and trehalose were correlated with their respective cold tolerances. Such differences in metabolic responses to cold in both hypogean organisms were unexpected since they both live in thermally buffered biotopes. Considering the current distribution areas of the two subterranean crustaceans studied, we assume that the cold hardiness found in the hypogean N. rhenorhodanensis could be correlated with its biogeography history during the quaternary glaciations.

Behavioural, ventilatory and respiratory responses of epigean and hypogean crustaceans to different temperatures.

Impact of temperature (from -2 to 28 degrees C) on survival, oxygen consumption, locomotory and ventilatory activities was measured in two aquatic subterranean crustaceans (Niphargus rhenorhodanensis and Niphargus virei) and in a morphologically close surface-dwelling crustacean (Gammarus fossarum). The hypogean N. virei presented all characteristics of a stenothermal organism: it showed small thermal plasticity and optimised its performance on a narrow range of temperature. In contrast, the epigean G. fossarum and more surprisingly the hypogean N. rhenorodanensis can be both characterized as eurythermal organisms: they exhibited important survival times and conserved their performance optimum throughout a large range of temperature. Such differences of survival and performance patterns in two hypogean organisms were unexpected since they both live in very thermally buffered biotopes. Our data suggest fresh hypotheses about the role of glaciations in the history and adaptation of hypogean crustaceans.