Juliette Ravaux

Heat-shock response and temperature resistance in the deep-sea vent shrimp Rimicaris exoculata.

SUMMARY The shrimp Rimicaris exoculata swarms around hydrothermal black smoker chimneys at most vent sites along the Mid-Atlantic Ridge. This species maintains close proximity to the hydrothermal fluid, where temperatures can reach 350°C and steep thermal and chemical gradients are expected. We performed in vivo experiments in pressurized aquaria to determine the upper thermal limit [critical thermal maximum (CTmax)] of R. exoculata and to investigate some characteristics of the shrimp stress response to heat exposure. These experiments showed that the shrimp does not tolerate sustained exposure to temperatures in the 33-37°C range (CTmax). A heat-inducible stress protein belonging to the hsp70 family was identified in R. exoculata, and its synthesis threshold induction temperature is below 25°C. The R. exoculata optimal thermal habitat may thus be restricted to values lower than previously expected (<25°C).

Defective Laminin 5 Processing in Cylindroma Cells

Cylindromas are benign skin tumors occurring as multiple nodules characteristically well circumscribed by an excess of basement membrane-like material. To determine the molecular defects leading to extracellular matrix accumulation, the ultrastructural, immunological, and biochemical properties of cylindroma tissue and isolated cells were analyzed. In cylindromas, hemidesmosomes are reduced in number, heterogeneous and immature compared to the normal dermal-epidermal junction. Expression of the alpha6beta4 integrin in tumor cells is weaker than in basal keratinocytes of the epidermis. Moreover, although in the epidermis alpha2beta1-integrin expression is restricted to the basal cell layer, it is found in all neoplastic cells within the nodules. Laminin 5 is present throughout the whole thickness of the basement membrane-like zone whereas laminin 10 is restricted to the interface adjacent to the tumor cells. Furthermore, laminin 5 is not properly processed and most of the alpha3A and gamma2 laminin chains remain as 165-kd and 155-kd polypeptides, respectively. Mature laminin 5 is thought to be necessary for correct hemidesmosome and basement membrane formation and its abnormal processing, as well as the low expression of alpha6beta4 integrins, could explain the lack of mature hemidesmosomes. Together, the results show that multiple molecular defects, including alteration of laminin 5 and its integrin receptors, contribute to structural aberrations of the basement membrane and associated structures in cylindromas.

Pressure tolerance of the shallow-water caridean shrimp Palaemonetes varians across its thermal tolerance window

SUMMARY To date, no published study has assessed the full physiological scope of a marine invertebrate species with respect to both temperature and hydrostatic pressure. In this study, adult specimens of the shallow-water shrimp species Palaemonetes varians were subjected to a temperature/pressure regime from 5 to 30°C and from 0.1 to 30 MPa. The rate of oxygen consumption and behaviour in response to varying temperature/pressure combinations were assessed. Rates of oxygen consumption were primarily affected by temperature. Low rates of oxygen consumption were observed at 5 and 10°C across all pressures and were not statistically distinct (P=0.639). From 10 to 30°C, the rate of oxygen consumption increased with temperature; this increase was statistically significant (P<0.001). Palaemonetes varians showed an increasing sensitivity to pressure with decreasing temperature; however, shrimp were capable of tolerating hydrostatic pressures found outside their normal bathymetric distribution at all temperatures. ‘Loss of equilibrium’ (LOE) in ≥50% of individuals was observed at 11 MPa at 5°C, 15 MPa at 10°C, 20 MPa at 20°C and 21 MPa at 30°C. From 5 to 20°C, mean levels of LOE decreased with temperature; this was significant (P<0.001). Low mean levels of LOE were observed at 20 and 30°C and were not distinct (P=0.985). The physiological capability of P. varians to tolerate a wide range of temperatures and significant hydrostatic pressure is discussed.

Thermal Limit for Metazoan Life in Question: In Vivo Heat Tolerance of the Pompeii Worm

The thermal limit for metazoan life, expected to be around 50°C, has been debated since the discovery of the Pompeii worm Alvinella pompejana, which colonizes black smoker chimney walls at deep-sea vents. While indirect evidence predicts body temperatures lower than 50°C, repeated in situ temperature measurements depict an animal thriving at temperatures of 60°C and more. This controversy was to remain as long as this species escaped in vivo investigations, due to irremediable mortalities upon non-isobaric sampling. Here we report from the first heat-exposure experiments with live A. pompejana, following isobaric sampling and subsequent transfer in a laboratory pressurized aquarium. A prolonged (2 hours) exposure in the 50–55°C range was lethal, inducing severe tissue damages, cell mortalities and triggering a heat stress response, therefore showing that Alvinella’s upper thermal limit clearly is below 55°C. A comparison with hsp70 stress gene expressions of individuals analysed directly after sampling in situ confirms that Alvinella pompejana does not experience long-term exposures to temperature above 50°C in its natural environment. The thermal optimum is nevertheless beyond 42°C, which confirms that the Pompeii worm ranks among the most thermotolerant metazoans.

Bacterial communities associated with the wood-feeding gastropod Pectinodonta sp. (Patellogastropoda, Mollusca)

Even though their occurrence was reported a long time ago, sunken wood ecosystems at the deep-sea floor have only recently received specific attention. Accumulations of wood fragments in the deep sea create niches for a diverse fauna, but the significance of the wood itself as a food source remains to be evaluated. Pectinodonta sp. is a patellogastropod that exclusively occurs on woody substrates, where individuals excavate deep depressions, and is thus a potential candidate for a wood-eating lifestyle. Several approaches were used on Pectinodonta sampled close to Tongoa island (Vanuatu) to investigate its dietary habits. Host carbon is most likely derived from the wood material based on stable isotopes analyses, and high cellulase activity was measured in the digestive mass. Electron microscopy and FISH revealed the occurrence of two distinct and dense bacterial communities, in the digestive gland and on the gill. Gland-associated 16S rRNA gene bacterial phylotypes, confirmed by in situ hybridization, included members of three divisions (Alpha- and Gammaproteobacteria, Bacteroidetes), and were moderately related (90-96% sequence identity) to polymer-degrading and denitrifying bacteria. Gill-associated phylotypes included representatives of the Delta- and Epsilonproteobacteria. The possible involvement of these two bacterial communities in wood utilization by Pectinodonta sp. is discussed.

Sustained hydrostatic pressure tolerance of the shallow water shrimp Palaemonetes varians at different temperatures: insights into the colonisation of the deep sea.

We investigated the tolerance of adult specimens of the shallow-water shrimp Palaemonetes varians to sustained high hydrostatic pressure (10 MPa) across its thermal tolerance window (from 5 to 27 °C) using both behavioural (survival and activity) and molecular (hsp70 gene expression) approaches. To our knowledge, this paper reports the longest elevated hydrostatic pressure exposures ever performed on a shallow-water marine organism. Behavioural analysis showed a 100% survival rate of P. varians after 7 days at 10 MPa and 5 or 10 °C, whilst cannibalism was observed at elevated temperature (27 °C), suggesting no impairment of specific dynamic action. A significant interaction of pressure and temperature was observed for both behavioural and molecular responses. Elevated pressure was found to exacerbate the effect of temperature on the behaviour of the animals by reducing activity at low temperature and by increasing activity at high temperature. In contrast, only high pressure combined with low temperature increased the expression of hsp70 genes. We suggest that the impressive tolerance of P. varians to sustained elevated pressure may reflect the physiological capability of an ancestral species to colonise the deep sea. Our results also support the hypothesis that deep-sea colonisation may have occurred during geological periods of time when the oceanic water column was warm and vertically homogenous.

A tale of two chitons: is habitat specialisation linked to distinct associated bacterial communities?

Although most chitons (Mollusca: Polyplacophora) are shallow-water molluscs, diverse species also occur in deep-sea habitats. We investigated the feeding strategies of two species, Leptochiton boucheti and Nierstraszella lineata, recovered on sunken wood sampled in the western Pacific, close to the Vanuatu Islands. The two species display distinctly different associations with bacterial partners. Leptochiton boucheti harbours Mollicutes in regions of its gut epithelium and has no abundant bacterium associated with its gill. Nierstraszella lineata displays no dense gut-associated bacteria, but harbours bacterial filaments attached to its gill epithelium, related to the Deltaproteobacteria symbionts found in gills of the wood-eating limpet Pectinodonta sp. Stable carbon and nitrogen isotope signatures and an absence of cellulolytic activity give evidence against a direct wood-feeding diet; both species are secondary consumers within the wood food web. We suggest that the distinct associations with bacterial partners are linked to niche specialisations of the two species. Nierstraszella lineata is in a taxonomic family restricted to sunken wood and is possibly adapted to more anoxic conditions thanks to its gill-associated bacteria. Leptochiton boucheti is phylogenetically more proximate to an ancestral form not specialised on wood and may itself be more of a generalist; this observation is congruent with its association with Mollicutes, a bacterial clade comprising gut-associated bacteria occurring in several metazoan phyla.

Diversification, evolution and sub-functionalization of 70kDa heat-shock proteins in two sister species of Antarctic krill: differences in thermal habitats, responses and implications under climate change

Background A comparative thermal tolerance study was undertaken on two sister species of Euphausiids (Antarctic krills) Euphausia superba and Euphausia crystallorophias. Both are essential components of the Southern Ocean ecosystem, but occupy distinct environmental geographical locations with slightly different temperature regimes. They therefore provide a useful model system for the investigation of adaptations to thermal tolerance. Methodology/Principal Finding Initial CTmax studies showed that E. superba was slightly more thermotolerant than E. crystallorophias. Five Hsp70 mRNAs were characterized from the RNAseq data of both species and subsequent expression kinetics studies revealed notable differences in induction of each of the 5 orthologues between the two species, with E. crystallorophias reacting more rapidly than E. superba. Furthermore, analyses conducted to estimate the evolutionary rates and selection strengths acting on each gene tended to support the hypothesis that diversifying selection has contributed to the diversification of this gene family, and led to the selective relaxation on the inducible C form with its possible loss of function in the two krill species. Conclusions The sensitivity of the epipelagic species E. crystallorophias to temperature variations and/or its adaptation to cold is enhanced when compared with its sister species, E. superba. These results indicate that ice krill could be the first of the two species to be impacted by the warming of coastal waters of the Austral ocean in the coming years due to climate change.

Thermal biology of the deep-sea vent annelid Paralvinella grasslei:in vivo studies

SUMMARY The annelid Paralvinella grasslei is a deep-sea vent endemic species that colonizes the wall of active chimneys. We report here the first data on its thermal biology based on in vivo experiments in pressurized aquaria. Our results demonstrate that P. grasslei survives a 30 min exposure at 30°C, and suggest that the upper thermal limit of this species is slightly above this temperature. The first signs of stress were noticed at 30°C, such as a significant increase in the animals activity and the expression of HSP70 stress proteins. A preliminary investigation of the kinetics of stress protein expression surprisingly showed high levels of HSP70 proteins as late as 3.5 h after the heat shock. Finally, we provide here the first sequences for vent annelid hsp70 (P. grasslei, Hesiolyra bergi and Alvinella pompejana). These constitute valuable tools for future studies on the thermal biology of these annelids.

Development of an ecotoxicological protocol for the deep-sea fauna using the hydrothermal vent shrimp Rimicaris exoculata

In light of deep-sea mining industry development, particularly interested in massive-sulphide deposits enriched in metals with high commercial value, efforts are increasing to better understand potential environmental impacts to local fauna. The aim of this study was to assess the natural background levels of biomarkers in the hydrothermal vent shrimp Rimicaris exoculata and their responses to copper exposure at in situ pressure (30MPa) as well as the effects of depressurization and pressurization of the high-pressure aquarium IPOCAMP. R. exoculata were collected from the chimney walls of the hydrothermal vent site TAG (Mid Atlantic Ridge) at 3630m depth during the BICOSE cruise in 2014. Tissue metal accumulation was quantified in different tissues (gills, hepatopancreas and muscle) and a battery of biomarkers was measured: metal exposure (metallothioneins), oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase) and oxidative damage (lipid peroxidation). Data show a higher concentration of Cu in the hepatopancreas and a slight increase in the gills after incubations (for both exposed groups). Significant induction of metallothioneins was observed in the gills of shrimps exposed to 4μM of Cu compared to the control group. Moreover, activities of enzymes were detected for the in situ group, showing a background protection against metal toxicity. Results suggest that the proposed method, including a physiologically critical step of pressurizing and depressurizing the test chamber to enable the seawater exchange during exposure to contaminants, is not affecting metal accumulation and biomarkers response and may prove a useful method to assess toxicity of contaminants in deep-sea species.