Low-diversity bacterial microbiota in Southern Ocean representatives of lanternfish genera Electrona, Protomyctophum and Gymnoscopelus (family Myctophidae).

Myctophids are among the most abundant mesopelagic teleost fishes worldwide. They are dominant in the Southern Ocean, an extreme environment where they are important both as consumers of zooplankton as well as food items for larger predators. Various studies have investigated myctophids diet, but no data is yet available regarding their associated microbiota, despite that the significance of bacterial communities to fish health and adaptation is increasingly acknowledged. In order to document microbiota in key fish groups from the Southern Ocean, the bacterial communities associated with the gut, fin, gills and light organs of members of six species within the three myctophid genera Electrona, Protomyctophum and Gymnoscopelus were characterized using a 16S rRNA-based metabarcoding approach. Gut communities display limited diversity of mostly fish-specific lineages likely involved in food processing. Fin and skin communities display diversity levels and compositions resembling more those found in surrounding seawater. Community compositions are similar between genera Electrona and Protomyctophum, that differ from those found in Gymnoscopelus and in water. Low abundances of potentially light-emitting bacteria in light organs support the hypothesis of host production of light. This first description of myctophid-associated microbiota, and among the first on fish from the Southern Ocean, emphasizes the need to extend microbiome research beyond economically-important species, and start addressing ecologically-relevant species.

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.

Cryptic frenulates are the dominant chemosymbiotrophic fauna at Arctic and high latitude Atlantic cold seeps.

We provide the first detailed identification of Barents Sea cold seep frenulate hosts and their symbionts. Mitochondrial COI sequence analysis, in combination with detailed morphological investigations through both light and electron microscopy was used for identifying frenulate hosts, and comparing them to Oligobrachia haakonmosbiensis and Oligobrachia webbi, two morphologically similar species known from the Norwegian Sea. Specimens from sites previously assumed to host O. haakonmosbiensis were included in our molecular analysis, which allowed us to provide new insight on the debate regarding species identity of these Oligobrachia worms. Our results indicate that high Arctic seeps are inhabited by a species that though closely related to Oligobrachia haakonmosbiensis, is nonetheless distinct. We refer to this group as the Oligobrachia sp. CPL-clade, based on the colloquial names of the sites they are currently known to inhabit. Since members of the Oligobrachia sp. CPL-clade cannot be distinguished from O. haakonmosbiensis or O. webbi based on morphology, we suggest that a complex of cryptic Oligobrachia species inhabit seeps in the Norwegian Sea and the Arctic. The symbionts of the Oligobrachia sp. CPL-clade were also found to be closely related to O. haakonmosbiensis symbionts, but genetically distinct. Fluorescent in situ hybridization and transmission electron micrographs revealed extremely dense populations of bacteria within the trophosome of members of the Oligobrachia sp. CPL-clade, which is unusual for frenulates. Bacterial genes for sulfur oxidation were detected and small rod shaped bacteria (round in cross section), typical of siboglinid-associated sulfur-oxidizing bacteria, were seen on electron micrographs of trophosome bacteriocytes, suggesting that sulfide constitutes the main energy source. We hypothesize that specific, local geochemical conditions, in particular, high sulfide fluxes and concentrations could account for the unusually high symbiont densities in members of the Oligrobrachia sp. CPL-clade.

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.

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 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.

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.

Diversity of symbioses between chemosynthetic bacteria and metazoans at the Guiness cold seep site (Gulf of Guinea, West Africa).

Fauna from deep-sea cold seeps worldwide is dominated by chemosymbiotic metazoans. Recently, investigation of new sites in the Gulf of Guinea yielded numerous new species for which symbiosis was strongly suspected. In this study, symbioses are characterized in five seep-specialist metazoans recently collected from the Guiness site located at ≈ 600 m depth. Four bivalve and one annelid species belonging to families previously documented to harbor chemosynthetic bacteria were investigated using bacterial marker gene sequencing, fluorescence in situ hybridization, and stable isotope analyses. Results support that all five species display chemosynthetic, sulfur-oxidizing γ-proteobacteria. Bacteria are abundant in the gills of bivalves, and in the trophosome of the siboglinid annelid. As observed for their relatives occurring at deeper sites, chemoautotrophy is a major source of carbon for animal nutrition. Although symbionts found in each host species are related to symbionts found in other metazoans from the same families, several incongruencies are observed among phylogenetic trees obtained from the different bacterial genes, suggesting a certain level of heterogeneity in symbiont strains present. Results provide new insights into the diversity, biogeography, and role of symbiotic bacteria in metazoans from the Gulf of Guinea, at a site located at an intermediate depth between the continental shelf and the deep sea.

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.

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.

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.

Evidence for multiple reversals of asymmetric mutational constraints during the evolution of the mitochondrial genome of metazoa, and consequences for phylogenetic inferences.

Mitochondrial DNA (mtDNA) sequences are comonly used for inferring phylogenetic relationships. However, the strand-specific bias in the nucleotide composition of the mtDNA, which is thought to reflect assymetric mutational constraints, combined with the important compositional heterogeneity among taxa, are known to be highly problematic for phylogenetic analyses. Here, nucleotide composition was compared across 49 species of Metazoa (34 arthropods, 2 annelids, 2 molluscs, and 11 deuterosomes), and analyzed for a mtDNA fragment including six protein-coding genes, i.e., atp6, atp8, cox1, cox2, cox3, and nad2. The analyses show that most metazoan species present a clear strand assymetry, where one strand is biased in favor of A and C, whereas the other strand has reverse bias, i.e. in favor of T and G. the origin of this strand bias can be related to assymetric mutational constraints involving deaminations of A and C nucleotides during the replication and/or transcription processes. The analyses reveal that six unrelated genera are characterized by a reversal of the usual strand bias, i.e., Argiope (Araneae), Euscorpius (Scorpiones), Tigrioupus (Maxillopoda), Branchiostoma (Cephalochordata) Florometra (Echinodermata), and Katharina (Mollusca). It is proposed that assymetric mutational constraints have been independantly reversed in these six genera, through an inversion of the control region, i.e., the region that contains most regulatory elements for replication and transcription of the mtDNA. We show that reversals of assymetric mutational constraints have dramatic consequences on the phylogenetic analyses, as taxa characterized by reverse strand bias tend to group together due to long-branch attraction artifacts. We propose a new method for limiting this specific problem in tree reconstruction under the Bayesian approach. We apply our method to deal with the question of phylogenetic relationships of the major lineages of Arthropoda, This new approach provides a better congruence with nuclear analyses based on mtDNA sequences, our data suggest that Chelicerata, Crustacea, Myriapoda, Pancrustacea, and Paradoxopoda are monophyletic.