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Simple Summary Chemical senses play a crucial role in mediating fundamental behaviors in most animals, including habitat selection and navigation. In the darkness of the deep sea, do shrimp endemic to hydrothermal vents use these senses to locate active emissions? Here, we examine the olfactory behaviors of two species of vent shrimp and one coastal species for comparison, to determine whether hydrothermal species have functional olfactory capacities and respond to environmental cues. Among these cues, food odors and vent fluid markers (chemicals, as well as temperature) were tested. Such in vivo experiments on deep-sea fauna are challenging to conduct because the animals sampled at depth may suffer from decompression. We, therefore, used dedicated pressurized equipment, and designed experiments at both deep-sea and atmospheric pressure. Vent shrimp groom their olfactory organs similarly to other crustaceans, yet they bear a dense bacterial cover, raising questions about the role of bacteria in shrimp olfaction. Whilst hydrothermal shrimp have been shown to possess a functional sense of smell, none of our test setups allowed us to demonstrate significant attraction to odors. Both hydrothermal species, however, showed attraction to warm temperature emissions, supporting the hypothesis that temperature is a major cue for orientation in the hydrothermal environment. Abstract 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 capaci

Rimicaris exoculata is one of the most well-known and emblematic species of endemic vent fauna. Like many other species from these ecosystems, Rimicaris shrimps host important communities of chemosynthetic bacteria living in symbiosis with their host inside the cephalothorax and gut. For many of these symbiotic partners, the mode of transmission remains to be elucidated and the starting point of the symbiotic relationship is not yet defined, but could begin with the egg. In this study, we explored the proliferation of microbial communities on R. exoculata broods through embryonic development using a combination of NGS sequencing and microscopy approaches. Variations in abundance and diversity of egg microbial communities were analyzed in broods at different developmental stages and collected from mothers at two distinct vent fields on the Mid-Atlantic Ridge (TAG and Snake Pit). We also assessed the specificity of the egg microbiome by comparing communities developing on egg surfaces with those developing on the cuticle of pleopods, which are thought to be exposed to similar environmental conditions because the brood is held under the female’s abdomen. In terms of abundance, bacterial colonization clearly increases with both egg developmental stage and the position of the egg within the brood: those closest to the exterior having a higher bacterial coverage. Bacterial biomass increase also accompanies an increase of mineral precipitations and thus clearly relates to the degree of exposure to vent fluids. In terms of diversity, most bacterial lineages were found in all samples and were also those found in the cephalothorax of adults. However, significant variation occurs in the relative abundance of these lineages, most of this variation being explained by body surface (egg vs. pleopod), vent field, and developmental stage. The occurrence of symbiont-related lineages of Epsilonbacteraeota, Gammaproteobacteria, Zetaproteobacteria, and Mollicutes provide a basis for dis

Symbiosis between Bathymodiolus and Gammaproteobacteria allows these deep-sea mussels to live in toxic environments such as hydrothermal vents and cold seeps. The quantity of endosymbionts within the gill-bacteriocytes appears to vary according to the hosts environment; however, the mechanisms of endosymbiont population size regulation remain obscure. We investigated the possibility of a control of endosymbiont density by apoptosis, a programmed cell death, in three mussel species. Fluorometric TUNEL and active Caspase-3-targeting antibodies were used to visualize and quantify apoptotic cells in mussel gills. To control for potential artefacts due to depressurization upon specimen recovery from the deep-sea, the apoptotic rates between mussels recovered unpressurised, versus mussels recovered in a pressure-maintaining device, were compared in two species from hydro thermal vents on the Mid-Atlantic Ridge: Bathymodiolus azoricus and B. puteoserpentis. Results show that pressurized recovery had no significant effect on the apoptotic rate in the gill filaments. Apoptotic levels were highest in the ciliated zone and in the circulating hemocytes, compared to the bacteriocyte zone. Apoptotic gill-cells in B. aff. boomerang from cold seeps off the Gulf of Guinea show similar distribution patterns. Deep-sea symbiotic mussels have much higher rates of apoptosis in their gills than the coastal mussel Mytilus edulis, which lacks chemolithoautotrophic symbionts. We discuss how apoptosis might be one of the mechanisms that contribute to the adaptation of deep-sea mussels to toxic environments and/or to symbiosis.

The hydrothermal vent mussel Bathymodiolus puteoserpentis hosts gill-associated sulfur- and methane-oxidizing bacteria which sustain host nutrition and allow it to reach high densities at various sites along the northern Mid-Atlantic Ridge. Previous studies have demonstrated that in similar dual symbioses, relative abundances of each bacterial type could change following variations in symbiont substrate availabilities. In this study, pressurized recovery and incubations in pressure vessels were used to test whether B. puteoserpentis symbionts displayed similar behavior in the presence of symbiont substrates. The relative abundances of both types of symbionts were analyzed using fluorescence in situ hybridization (FISH) and group-specific gene copy numbers were assessed using qPCR. Specimens sampled using isobaric and non-isobaric recovery contained similar relative proportions (in surface coverage) of sulfur- and methane-oxidizing bacteria indicating that recovery type did not have impact on measured relative areas. Similarly, pressurized incubations with different substrates did lead to significant differences in the relative surface coverage of the two types of bacteria, although slight variations were measured with qPCR, suggesting changes in relative abundances of gene copy numbers but not in relative areas covered. Total gill surface areas and total bacterial numbers in specimens were estimated for the first time. Symbiont bearing-mussels display exchange surfaces about 20-fold higher than those found in similar-sized coastal mussels, and mean bacterial numbers of 2.5*10(12) per specimen were estimated. This emphasizes that symbiotic mussels are a major reservoir of bacteria in vent ecosystems.

The AbyssBox project aims to provide the first permanent public exhibition of live deep-sea hydrothermal fauna maintained at in situ pressure. AbyssBox is a pressurized aquarium designed to function permanently. Here we present details of the project after the public exhibition functioned for more than three years at Océanopolis aquarium in Brest, France. We also describe the AbyssBox pressure aquarium, and provide data and observations on vent shrimp (Mirocaris fortunata) and crabs (Segonzacia mesatlantica) that were sampled from 1700 m depth at the Lucky Strike vent field (Mid-Atlantic Ridge) during different cruises. While mortalities exceeded 50% during the first days following sampling, the remaining animals appeared to acclimate fairly well. Some crabs have now been kept for more than 2 years, and some shrimp have spent more than 3 years in captivity. Primarily designed for a public exhibition, the AbyssBox is already used for scientific purposes, since it provides one of the most effective tools for long-term rearing of deep-sea fauna. AbyssBox is a first step towards maintaining a variety of deep-sea fauna year-round at in situ pressure, which will serve both scientific and public interests.

The shrimp Rimicaris exoculata dominates several hydrothermal vent ecosystems of the Mid- Atlantic Ridge and is thought to be a primary consumer harbouring a chemoautotrophic bacterial community in its gill chamber. The aim of the present study was to test current hypotheses concerning the epibiont’s chemoautotrophy, and the mutualistic character of this association. Invivo experiments were carried out in a pressurised aquarium with isotope-labelled inorganic carbon (NaH13CO3 and NaH14CO3) in the presence of two different electron donors (Na2S2O3 and Fe2þ) and with radiolabelled organic compounds (14C-acetate and 3H-lysine) chosen as potential bacterial substrates and/or metabolic by-products in experiments mimicking transfer of small biomolecules from epibionts to host. The bacterial epibionts were found to assimilate inorganic carbon by chemoautotrophy, but many of them (thick filaments of epsilonproteobacteria) appeared versatile and able to switch between electron donors, including organic compounds (heterotrophic acetate and lysine uptake). At least some of them (thin filamentous gammaproteobacteria) also seem capable of internal energy storage that could supply chemosynthetic metabolism for hours under conditions of electron donor deprivation. As direct nutritional transfer from bacteria to host was detected, the association appears as true mutualism. Import of soluble bacterial products occurs by permeation across the gill chamber integument, rather than via the digestive tract. This first demonstration of such capabilities in a decapod crustacean supports the previously discarded hypothesis of transtegumental absorption of dissolved organic matter or carbon as a common nutritional pathway.

The thermal limit for metazoan life, expected to be around 50 degrees 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 degrees C, repeated in situ temperature measurements depict an animal thriving at temperatures of 60 degrees 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 degrees 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 degrees 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 degrees C in its natural environment. The thermal optimum is nevertheless beyond 42 degrees C, which confirms that the Pompeii worm ranks among the most thermotolerant metazoans.

The caridean shrimp Rimicaris exoculata dominates the fauna at several Mid-Atlantic Ridge hydrothermal vent sites. This shrimp has an enlarged gill chamber, harboring a dense ectosymbiotic community of chemoautotrophic bacteria associated with mineral oxide deposits. Until now, their acquisition is not fully understood. At three hydrothermal vent sites, we analyzed the epibionts diversity at different moult stages and also in the first stages of the shrimp life (eggs, hatched eggs (with larvae) and juveniles). Hatched eggs associated with young larvae were collected for the first time directly from gravid females at the Logachev vent site during the Serpentine cruise. An approach using 16S rRNA clone libraries, scanning and transmission electron microscopy, and fluorescent in situ hybridization was used. Molecular results and microscope observations indicated a switch in the composition of the bacterial community between early R. exoculata life cycle stage (egg libraries dominated by the Gammaproteobacteria) and later stages (juvenile/adult libraries dominated by the Epsilonproteobacteria). We hypothesized that the epibiotic phylotype composition could vary according to the life stage of the shrimp. Our results confirmed the occurrence of a symbiosis with Gammaproteobacteria and Epsilonproteobacteria, but more complex than previously assumed. We revealed the presence of active type-I methanotrophic bacteria colonizing the cephalothorax of shrimps from the Rainbow site. They were also present on the eggs from the Logachev site. This could be the first 'epibiotic' association between methanotrophic bacteria and hydrothermal vent crustacean. We discuss possible transmission pathways for epibionts linked to the shrimp life cycle. The ISME Journal (2012) 6, 597-609; doi: 10.1038/ismej.2011.133; published online 13 October 2011