Comparison of Chemoreceptive Abilities of the Hydrothermal Shrimp Mirocaris fortunata and the Coastal Shrimp Palaemon elegans.

Chemoreception might play an important role for endemic shrimp that inhabit deep and dark hydrothermal vents to find food sources and to locate active edifices that release specific chemicals. We compared the chemosensory abilities of the hydrothermal shrimp Mirocaris fortunata and the coastal related species, Palaemon elegans. The detection of diverse ecologically relevant chemical stimuli by the antennal appendages was measured with electroantennography. The 2 species can detect food-related odor and sulfide, a short-distance stimulus, via both their antennae and antennules. Neither iron nor manganese, considered as long-distance stimuli, was detected by the antennal appendages. Investigation of the ultrastructure of aesthetasc sensilla revealed no specific features of the hydrothermal species regarding innervation by olfactory sensory neurons. Pore-like structures occurring in the aesthetasc cuticle and dense bacterial covering seem to be unique to hydrothermal species, but their potential link to chemoreception remains elusive.

Comparative Study of Chemosensory Organs of Shrimp From Hydrothermal Vent and Coastal Environments.

The detection of chemical signals is involved in a variety of crustacean behaviors, such as social interactions, search and evaluation of food and navigation in the environment. At hydrothermal vents, endemic shrimp may use the chemical signature of vent fluids to locate active edifices, however little is known on their sensory perception in these remote deep-sea habitats. Here, we present the first comparative description of the sensilla on the antennules and antennae of 4 hydrothermal vent shrimp (Rimicaris exoculata, Mirocaris fortunata, Chorocaris chacei, and Alvinocaris markensis) and of a closely related coastal shrimp (Palaemon elegans). These observations revealed no specific adaptation regarding the size or number of aesthetascs (specialized unimodal olfactory sensilla) between hydrothermal and coastal species. We also identified partial sequences of the ionotropic receptor IR25a, a co-receptor putatively involved in olfaction, in 3 coastal and 4 hydrothermal shrimp species, and showed that it is mainly expressed in the lateral flagella of the antennules that bear the unimodal chemosensilla aesthetascs.

New electroantennography method on a marine shrimp in water.

Antennular chemoreception in aquatic decapods is well studied via the recording of single chemoreceptor neuron activity in the antennule, but global responses of the antennule (or antennae in insects) by electroantennography (EAG) has so far been mainly restricted to aerial conditions. We present here a well-established underwater EAG method to record the global antennule activity in the marine shrimp Palaemon elegans in natural (aqueous) conditions. EAG responses to food extracts, recorded as net positive deviations of the baseline, are reproducible, dose-dependent and exhibit sensory adaptation. This new EAG method opens a large field of possibilities for studying in vivo antennular chemoreception in aquatic decapods, in a global approach to supplement current, more specific techniques.

Characterising multi-level effects of acute pressure exposure on a shallow-water invertebrate: insights into the kinetics and hierarchy of the stress response.

Hydrostatic pressure is an important, ubiquitous, environmental variable of particular relevance in the marine environment. However, it is widely overlooked despite recent evidence that some marine ectotherms may be demonstrating climate-driven bathymetric range shifts. Wide-ranging effects of increased hydrostatic pressure have been observed from the molecular through to the behavioural level. Still, no study has simultaneously examined these multiple levels of organisation in a single experiment in order to understand the kinetics, hierarchy and interconnected nature of such responses during an acute exposure, and over a subsequent recovery period. Here, we quantify the transcription of a set of previously characterised genes during and after acute pressure exposure in adults of the shrimp Palaemonetes varians. Further, we perform respiratory rate and behavioural analysis over the same period. Increases in expression of genes associated with stress and metabolism were observed during and after high-pressure exposure. Respiratory rate increased during exposure and into the recovery period. Finally, differential behaviour was observed under elevated hydrostatic pressure in comparison to ambient pressure. Characterising generalised responses to acute elevated pressure is a vital precursor to longer-term, acclimation-based pressure studies. Results provide a novel insight into what we term the overall stress response (OSR) to elevated pressure; a concept that we suggest to be applicable to other environmental stressors. We highlight the importance of considering more than a single component of the stress response in physiological studies, particularly in an era where environmental multi-stressor studies are proliferating.

Is the deep-sea crab Chaceon affinis able to induce a thermal stress response?

Fluctuations in the stress level of an organism are expressed in behavioural and molecular changes that can affect its ecology and survival. Our knowledge of thermal adaptations in deep-sea organisms is very limited, and this study investigates the critical thermal maximum (CTmax) and the heat-shock response (HSR) in the deep-sea crab Chaceon affinis commonly found in waters of the North East Atlantic. A mild but significant HSR in C. affinis was noted and one of the lowest CTmax known amongst Crustacea was revealed (27.5 °C at 0.1 MPa; 28.5 °C at 10 MPa). The thermal sensitivity of this species appears to be reduced at in situ pressure (10 MPa), given the slightly higher CTmax and the significant 3-fold induction of stress genes hsp70 form 1 and hsp70 form 2. Although C. affinis deep-sea habitat is characterized by overall low temperature this species appears to have retained its ability to induce a HSR. This capability may be linked with C. affinis' occasional exploitation of warmer and thermally instable hydrothermal vent fields, where it has been found foraging for food.

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.

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

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.

Do Hydrothermal Shrimp Smell Vents?

Deep-sea species endemic to hydrothermal vents face the critical challenge of detecting active sites in a vast environment devoid of sunlight. This certainly requires specific sensory abilities, among which olfaction could be a relevant sensory modality, since chemical compounds in hydrothermal fluids or food odors could potentially serve as orientation cues. The temperature of the vent fluid might also be used for locating vent sites. The objective of this study is to observe the following key behaviors of olfaction in hydrothermal shrimp, which could provide an insight into their olfactory capacities: (1) grooming behavior; (2) attraction to environmental cues (food odors and fluid markers). We designed experiments at both deep-sea and atmospheric pressure to assess the behavior of the vent shrimp Rimicaris exoculata and Mirocaris fortunata, as well as of the coastal species Palaemon elegans and Palaemon serratus for comparison. Here, we show that hydrothermal shrimp groom their sensory appendages similarly to other crustaceans, but this does not clean the dense bacterial biofilm that covers the olfactory structures. These shrimp have previously been shown to possess functional sensory structures, and to detect the environmental olfactory signals tested, but we do not observe significant attraction behavior here. Only temperature, as a signature of vent fluids, clearly attracts vent shrimp and thus is confirmed to be a relevant signal for orientation in their environment.

Assessing a species thermal tolerance through a multiparameter approach: the case study of the deep-sea hydrothermal vent shrimp Rimicaris exoculata.

Assessing species thermal tolerance requires identification of their thermal strategies and evaluation of their ability to cope with temperature fluctuations. The mobilization of the molecular heat stress response (HSR), which is a proxy for the thermal tolerance, would be part of the strategy of species colonizing highly variable thermal environments. We here investigate multiple parameters of the HSR in the deep-sea vent shrimp Rimicaris exoculata that colonizes such environments. The set points of the HSR induction, compared to those of the coastal species Palaemonetes varians, clearly reflect a high thermotolerance in this species, while the HSR is proved to be rarely mobilized in the R. exoculata natural populations. Finally, the compilation of multiple parameters such as the upper thermal limit and several thresholds of the HSR, as well as thermal behavior observations, allows us to provide a more accurate picture of the combination and complementarity of strategies that can account for the overall thermal tolerance of the species.

Neuroanatomy of a hydrothermal vent shrimp provides insights into the evolution of crustacean integrative brain centers.

Alvinocaridid shrimps are emblematic representatives of the deep hydrothermal vent fauna at the Mid-Atlantic Ridge. They are adapted to a mostly aphotic habitat with extreme physicochemical conditions in the vicinity of the hydrothermal fluid emissions. Here, we investigated the brain architecture of the vent shrimp Rimicaris exoculata to understand possible adaptations of its nervous system to the hydrothermal sensory landscape. Its brain is modified from the crustacean brain ground pattern by featuring relatively small visual and olfactory neuropils that contrast with well-developed higher integrative centers, the hemiellipsoid bodies. We propose that these structures in vent shrimps may fulfill functions in addition to higher order sensory processing and suggest a role in place memory. Our study promotes vent shrimps as fascinating models to gain insights into sensory adaptations to peculiar environmental conditions, and the evolutionary transformation of specific brain areas in Crustacea.

Immune failure reveals vulnerability of populations exposed to pollution in the bioindicator species Hediste diversicolor.

Human activities on the shoreline generate a growing pollution, creating deleterious habitats in coastal zones. Some species nevertheless succeed in such harsh milieus, raising the question of their tolerance to environmental stress. The annelid Hediste diversicolor lives buried in the sediments, directly exposed to contaminants trapped in the mud. After verifying the similarity of their genetic contexts, we compared reproductive output and individual immune resistance measures of populations living in polluted vs. 'clean' sediments, and related these assessments with measures of phthalates and metal pollution, and associated toxicity indices. Chemical analyses predicted no toxicity to the local infauna, and phenological studies evidenced no direct cost of living in noxious habitats. However, populations exposed to pollutants showed a significantly reduced survival upon infection with a local pathogen. Surprisingly, physiological studies evidenced a basal overinflammatory state in the most exposed populations. This over-activated baseline immune phenotype likely generates self-damage leading to enhanced immune cell death rate and immune failure. Monitoring the immune status of individual worms living in anthropic areas could thus be used as a reliable source of information regarding the actual health of wild populations.

First hsp70 from two hydrothermal vent shrimps, Mirocaris fortunata and Rimicaris exoculata: characterization and sequence analysis.

The vent shrimps, Mirocaris fortunata and Rimicaris exoculata, live in a highly fluctuating thermal environment and undergo frequent temperature bursts. As a first step in the investigation of the response to heat stress, this work aimed to characterize stress proteins in these two species. Complementary deoxyribonucleic acid (cDNA) clones encoding a 70-kDa heat shock protein (HSP) were isolated and characterized from M. fortunata and R. exoculata. The cDNA clones were of 2055 and 1941 base pairs in length, and contained a 2018-bp complete open reading frame (ORF) and a 1785-bp partial coding sequence, respectively. The amino acid sequences corresponding to these ORF are 645 residues in length for M. fortunata and 595 for R. exoculata, and were clearly characterized as members of the HSP70 family. The C-terminal extremity would identify R. exoculata sequence as a cytoplasm HSP70. The relationships between the crustacean HSP70 sequences were examined by two phylogenetic methods, i.e. Maximum Likelihood and Bayesian methods. The resulting trees suggested that M. fortunata sequence may correspond to constitutively expressed HSP70, named HSC70, whereas R. exoculata sequence may correspond to an inducible form of HSP70. The HSP70 sequences from the hydrothermal shrimps proved to be very similar to the other homologous shrimp sequences, except for the presence of an insertion of unknown function in the ATPase domain of R. exoculata sequence.

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.

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.

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.

Adaptation to thermally variable environments: capacity for acclimation of thermal limit and heat shock response in the shrimp Palaemonetes varians.

In the context of climate change, there is a sustained interest in understanding better the functional mechanisms by which marine ectotherms maintain their physiological scope and define their ability to cope with thermal changes in their environment. Here, we present evidence that the variable shrimp Palaemonetes varians shows genuine acclimation capacities of both the thermal limit (CT(max)) and the heat shock response (hsp70 induction temperature). During cold acclimation to 10 °C, the time lag to adjust the stress gene expression to the current environmental temperature proved to exceed 1 week, thereby highlighting the importance of long-term experiments in evaluating the species' acclimation capacities. Cold and warm-acclimated specimens of P. varians can mobilise the heat shock response (HSR) at temperatures above those experienced in nature, which suggests that the species is potentially capable of expanding its upper thermal range. The shrimp also survived acute heat shock well above its thermal limit without subsequent induction of the HSR, which is discussed with regard to thermal adaptations required for life in highly variable environments.

Identification of differentially expressed genes in the hydrothermal vent shrimp Rimicaris exoculata exposed to heat stress.

The deep-sea vent shrimp Rimicaris exoculata dominates the vagile megafauna at most vent sites along the Mid-Atlantic Ridge. This shrimp swarms around the hot end of the hydrothermal biotope where temperature can exceed its critical maximal temperature (33-38.5 ± 2°C). It may therefore be subjected to a thermal regime that is assumed to be stressful for animals. In this study, we used a global transcriptomic approach by constructing suppression subtractive hybridization cDNA libraries in order to identify specific up- and down-regulated genes in R. exoculata exposed to a severe heat stress (1h at 30°C). A total of 218 sequences representing potentially highly expressed genes in thermally stressed shrimp were obtained. Expression of 11 genes involved in various cell functions was quantified in control and heat shocked specimens using real-time PCR. Differential expression was observed for some specific genes such as mannose receptor C1, metalloprotease, histone H1, and hemocyanin with a strong up-regulation of several genes encoding heat shock proteins. These results suggest that R. exoculata is affected at both cellular and molecular levels by sustained exposure at 30°C. The sequenced ESTs presented here will provide an excellent basis for future thermal stress studies on deep-sea vent fauna.

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.

Survey of genome size in 28 hydrothermal vent species covering 10 families.

Knowledge of genome size is a useful and necessary prerequisite for the development of many genomic resources. To better understand the origins and effects of DNA gains and losses among species, it is important to collect data from a broad taxonomic base, but also from particular ecosystems. Oceanic thermal vents are an interesting model to investigate genome size in very unstable environments. Here we provide data estimated by flow cytometry for 28 vent-living species among the most representative from different hydrothermal vents. We also report the genome size of closely related coastal decapods. Haploid C-values were compared with those previously reported for species from corresponding orders or infraorders. This is the first broad survey of 2C values in vent organisms. Contrary to expectations, it shows that certain hydrothermal vent species have particularly large genomes. The vent squat lobster Munidopsis recta has the largest genome yet reported for any anomuran: 2C=31.1 pg=30.4x10(9) bp. In several groups, such as Brachyura, Phyllodocida, and Veneroida, vent species have genomes that clearly rank at the high end of published values for each group. We also describe the highest DNA content yet recorded for the Brachyura (coastal crabs Xantho pilipes and Necora puber). Finally, analysis of genome size variation across populations revealed unexpected intraspecific variation in the vent shrimp Mirocaris fortunata that could not be attributed simply to ploidy changes.

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

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 degrees 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 degrees C, such as a significant increase in the animal's 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.