Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization

<p>Abstract</p> <p>Background</p> <p>Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm <it>Riftia pachyptila </it>and its symbiotic sulfide-oxidizing γ-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH) in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water) and in the trophosome (the organ housing the symbiotic bacteria) using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species.</p> <p>Results</p> <p>We produced four cDNA libraries: i) body wall-subtracted branchial plume library (BR-BW), ii) and its reverse library, branchial plume-subtracted body wall library (BW-BR), iii) body wall-subtracted trophosome library (TR-BW), iv) and its reverse library, trophosome-subtracted body wall library (BW-TR). For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively). Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr), cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr), myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue.</p> <p>Conclusion</p> <p>Quantitative PCR analyses were congruent with our libraries results thereby confirming the existence of tissue-specific transcripts identified by SSH. We focused our study on the transcripts we identified as the most interesting ones based on the BLAST results. Some of the keys to understanding metabolite exchanges may remain in the sequences we could not identify (hypothetical proteins and no similarity found). These sequences will have to be better studied by a longer -or complete- sequencing to check their identity, and then by verifying the expression level of the transcripts in different parts of the worm.</p

Insights into metazoan evolution from Alvinella pompejana cDNAs.

International audienceBACKGROUND: Alvinella pompejana is a representative of Annelids, a key phylum for evo-devo studies that is still poorly studied at the sequence level. A. pompejana inhabits deep-sea hydrothermal vents and is currently known as one of the most thermotolerant Eukaryotes in marine environments, withstanding the largest known chemical and thermal ranges (from 5 to 105°C). This tube-dwelling worm forms dense colonies on the surface of hydrothermal chimneys and can withstand long periods of hypo/anoxia and long phases of exposure to hydrogen sulphides. A. pompejana specifically inhabits chimney walls of hydrothermal vents on the East Pacific Rise. To survive, Alvinella has developed numerous adaptations at the physiological and molecular levels, such as an increase in the thermostability of proteins and protein complexes. It represents an outstanding model organism for studying adaptation to harsh physicochemical conditions and for isolating stable macromolecules resistant to high temperatures. RESULTS: We have constructed four full length enriched cDNA libraries to investigate the biology and evolution of this intriguing animal. Analysis of more than 75,000 high quality reads led to the identification of 15,858 transcripts and 9,221 putative protein sequences. Our annotation reveals a good coverage of most animal pathways and networks with a prevalence of transcripts involved in oxidative stress resistance, detoxification, anti-bacterial defence, and heat shock protection. Alvinella proteins seem to show a slow evolutionary rate and a higher similarity with proteins from Vertebrates compared to proteins from Arthropods or Nematodes. Their composition shows enrichment in positively charged amino acids that might contribute to their thermostability. The gene content of Alvinella reveals that an important pool of genes previously considered to be specific to Deuterostomes were in fact already present in the last common ancestor of the Bilaterian animals, but have been secondarily lost in model invertebrates. This pool is enriched in glycoproteins that play a key role in intercellular communication, hormonal regulation and immunity. CONCLUSIONS: Our study starts to unravel the gene content and sequence evolution of a deep-sea annelid, revealing key features in eukaryote adaptation to extreme environmental conditions and highlighting the proximity of Annelids and Vertebrates

Adaptations respiratoires des annélides polychètes de milieux abyssaux hypoxiques

The animals living near hydrothermal vents and cold-seeps experience environmental conditions which are challenging for respiration (little oxygen, presence of large amounts of sulfide and carbon dioxide ). We used polychaetes from these two environments: Alvinella pompejana, from the hot part of the hydrothermal vent ecosystem, Branchipolynoe spp., commensal with the mussels found in the cold part of hydrothermal vents and an orbiniid from the Gulf of Mexico cold-seeps. We investigated the anatomical and physiological adaptations facilitating gas exchange in these three species. The anatomical adaptations are an increase of the gill surface area and a decrease of the diffusion distance at the level of these gas exchange organs. Branchipolynoe is unusual in having gills, an anatomical feature lacking in littoral species of the same family. These gills are perfused by the coelomic fluid instead of the blood as in the other species. In the orbiniid, most of the gas exchanges take place in the anterior part of the body. The three species possess hemoglobin, another feature distinguishing Branchipolynoe from the littoral polynoids. While the two other species exhibit typical annelid hemoglobins, the coelomic hemoglobins of Branchipolynoe are very unusual: they are dimers and trimers of tetradomain globins. The body fluids of A. pompejana and Branchipolynoe are able to buffer the effects of the variations of carbon dioxide concentration. All the the species studied possess hemoglobins with very high oxygen affinities, allowing a good extraction of the oxygen from their hypoxic environment. In A. pompejana, there is an internal gas transfer system allowing the worm to store oxygen when the conditions are good and to release it into the vascular system when its environmental concentration is too low.Les animaux vivant au niveau des sources hydrothermales et des suintements froids se trouvent dans des conditions contraignantes pour la respiration (peu d'oxygène, présence d'importantes quantités de sulfures et de dioxyde de carbone). Nous nous sommes intéressés aux annélides polychètes de ces milieux, prenant trois modèles : Alvinella pompejana, du pôle chaud hydrothermal, Branchipolynoe spp., commensal des moules du pôle froid des sources hydrothermales et un Orbiniidé provenant des suintements froids du Golfe du Mexique. Nous avons recherché les adaptations anatomiques et physiologiques facilitant les échanges gazeux chez ces trois espèces. Du point de vue anatomique, ces adaptations sont une augmentation des surfaces branchiales et une réduction des distances de diffusion au niveau de branchies. Branchipolynoe présente la particularité de posséder des branchies, structure anatomique absente chez les espèces littorales de la même famille. Ces branchies sont perfusées par du liquide coelomique et non du sang comme c'est le cas pour les deux autres espèces. Chez l'Orbiniidé, les échanges gazeux se situent principalement au niveau des branchies situées dans la partie antérieure du ver. Les trois espèces possèdent de l'hémoglobine, encore une caractéristique distinguant Branchipolynoe des Polynoidés littoraux. Si les deux autres espèces possèdent des hémoglobines "classiques" pour les annélides, les hémoglobines de Branchipolynoe sont tout à fait originales : elles correspondent à des dimères ou des trimères de globines tétradomaines. Les fluides corporels d' A. pompejana et de Branchipolynoe sont capables de limiter les effets des variations de concentration en dioxyde de carbone. Toutes les espèces étudiées possèdent des hémoglobines à très forte affinité pour l'oxygène qui permettent une bonne extraction de l'oxygène du milieu environnant hypoxique. Chez A. pompejana, il y a un système de transfert gazeux interne qui permet de stocker de l'oxygène quand les conditions sont favorables et de le relarguer dans la circulation quand il manque dans le milieu environnant

Cardiac response of the hydrothermal vent crab Segonzacia mesatlantica to variable temperature and oxygen levels

International audienceSegonzacia mesatlantica inhabits different hydrothermal vent sites of the Mid-Atlantic Ridge where it experiences chronic environmental hypoxia, and highly variable temperatures. Experimental animals in aquaria at in situ pressure were exposed to varying oxygen concentrations and temperature, and their cardiac response was studied. S. mesatlantica is well adapted to these challenging conditions and capable of regulating its oxygen uptake down to very low concentrations (7.3–14.2 µmol l−1). In S. mesatlantica, this capacity most likely relies on an increased ventilation rate, while the heart rate remains stable down to this critical oxygen tension. When not exposed to temperature increase, hypoxia corresponds to metabolic hypoxia and the response likely only involves ventilation modulation, as in shallow-water relatives. For S. mesatlantica however, an environmental temperature increase is usually correlated with more pronounced hypoxia. Although the response to hypoxia is similar at 10 and 20 °C, temperature itself has a strong effect on the heart rate and EKG signal amplitude. As in shallow water species, the heart rate increases with temperature. Our study revealed that the range of thermal tolerance for S. mesatlantica ranges from 6 through 21 °C for specimens from the shallow site Menez Gwen (800 m), and from 3 through 19 °C for specimens from the deeper sites explored (2700–3000 m)

&lt;i&gt;Lamellibrachia anaximandri&lt;/i&gt; n. sp., un nouveau ver vestimentifère (Annelida) de Méditerranée, avec des remarques sur les vers frénulés du même habitat

Une nouvelle espèce de vestimentifère lamellibrachiidé, Lamellibrachia anaximandri n. sp. a été trouvée en Méditerranée orientale à proximité de suintements froids riches en méthane et de sources de sulfures dissous dans les sédiments superficiels. Elle vit à une profondeur d’environ 1100 à 2100 m, sur certains volcans de boue de la chaîne d’Anaximandre au sud de la Turquie, sur la ride méditerranéenne au sud de la Crète, et dans l’estuaire profond du Nil. Elle a d’autre part été trouvée dans du papier putréfié à l’intérieur d’un bateau coulé, torpillé en 1915 et gisant par 2800 m de fond au sud-est de la Crète. Quelques pogonophores frenulés sont aussi présents au niveau des volcans de boue (dont une espèce de Siboglinum ressemblant à S. carpinei ainsi que des tubes d’autres genres non identifiés). Le nouveau Lamellibrachia est la première espèce de vestimentifère décrite de Méditerranée. Il diffère de L. luymesi de la population du Golfe du Mexique par le très faible développement des annulations de son tube et par un plus petit nombre de paires de lamelles branchiales de son panache branchial. Le séquençage des gènes COI et mt16S confirme la différence spécifique entre cette nouvelle espèce et L. luymesi, ainsi qu’une différence entre ces deux espèces et les quatre espèces de Lamellibrachia décrites de l’océan Pacifique. Les plus grands individus de L. anaximandri n. sp. sont peut-être très vieux, mais la présence de nombreux jeunes individus sur certains sites montre que les conditions actuelles sont favorables à son recrutement et à sa croissance initiale. Le développement du panache branchial dans une série de jeunes stades révèle que les gaines lamellaires enveloppantes, qui sont caractéristiques du genre Lamellibrachia, commencent à se former seulement après le déploiement de plusieurs paires de lamelles branchiales. L’examen du trophosome de l’adulte en microscopie électronique à transmission montre des bactéries Gram-négatives sans membranes internes empilées, indiquant que les symbiontes sont probablement sulfo-oxydants.A new species of lamellibrachiid vestimentiferan, Lamellibrachia anaximandri n. sp., has been found in the Eastern Mediterranean, close to cold seeps of fluid carrying dissolved methane and sources of sulfide in superficial sediments. It occurs at about 1100 to 2100 m depth, on some of the mud volcanoes on the Anaximander Mountains, south of Turkey, on the Mediterranean Ridge, south of Crete, and on the Nile deep-sea fan. In addition, it has been obtained from rotting paper inside a sunken ship, torpedoed in 1915 and lying at 2800 m depth, southeast of Crete. Some frenulate pogonophores also occur on the mud volcanoes (including a species of Siboglinum resembling S. carpinei and tubes of other unidentified genera). The new Lamellibrachia is the first vestimentiferan species to be described from the Mediterranean. It differs from L. luymesi taken from the Gulf of Mexico population in the very weak development of collars on its tube and in having a smaller number of pairs of branchial lamellae in the branchial plume. Sequencing of the COI and the mt16S genes confirms a difference at the species level between the new species and L. luymesi, and a difference between these two species and four described species of Lamellibrachia from the Pacific Ocean. The largest individuals of L. anaximandri n. sp. may be many years old, but there are numerous young individuals at some sites, showing that favourable conditions are available for settlement and early growth. The development of the branchial plume in a series of young stages reveals that the sheath lamellae, which are characteristic of the genus Lamellibrachia, begin to form only after the establishment of several pairs of branchial lamellae. Examination of the adult trophosome by transmission electron microscopy shows Gram-negative bacteria without internal stacked membranes, indicating that the symbionts are most probably sulfide oxidizing.</p

Oxygen consumption rates in deep-sea hydrothermal vent scale worms: Effect of life-style, oxygen concentration, and temperature sensitivity

Deep-sea hydrothermal vents are a challenging environment inhabited by very specialized species. To reap the benefits of the local primary production, species need to cope with a number of constraints among which low oxygen is probably the most basic. This hypoxia is further complicated by the highly variable temperature these species experience. We studied the response of deep-sea hydrothermal species of scale worms (Annelida, Polynoidae) to varying levels of oxygen and showed that they were capable of compensating a decrease of environmental oxygen concentration (= oxyregulators), down to values of about 30 μmol l−1. This contrasts with shallow-water temperate species, for which oxygen consumption is directly proportional to its concentration (= oxyconformers). We measured oxygen consumption rates in 11 species from hydrothermal vents, as well as 2 species from the general deep-sea, and compared them to three shallow-water species. Life-style (free-living vs. commensal) and habitat of origin (shallow-water, deep-sea, and hydrothermal vent) did not affect oxygen consumption rates. In agreement with thermodynamic expectations, as temperature increases, oxygen consumption increases as well for all species. The sensitivity of oxygen consumption to temperature variation in the shallow-water species is however smaller than that from the deep-sea hydrothermal vent species. This unexpected result could correspond to a pronounced increase of activity (avoidance behaviour) in the vent species, which was not observed for the shallow-water species

Oxygen consumption rates in deep-sea hydrothermal vent scale worms: effect of lifestyle, oxygen concentration, and temperature sensitivity

International audienceDeep-sea hydrothermal vents are a challenging environment inhabited by very specialized species. To reap the benefits of the local primary production, species need to cope with a number of constraints among which low oxygen is probably the most basic. This hypoxia is further complicated by the highly variable temperature these species experience. We studied the response of deep-sea hydrothermal species of scale worms (Annelida, Polynoidae) to varying levels of oxygen and showed that they were capable of compensating a decrease of environmental oxygen concentration (= oxyregulators), down to values of about 30 µmol.l-1. This contrasts with shallow-water temperate species, for which oxygen consumption is directly proportional to its concentration (= oxyconformers). We measured oxygen consumption rates in 11 species from hydrothermal vents, as well as 2 species from the general deep-sea, and compared them to three shallow-water species. Lifestyle (free-living vs. commensal) and habitat of origin (shallow-water, deep-sea, and hydrothermal vent) did not affect oxygen consumption rates. In agreement with thermodynamic expectations, as temperature increases, oxygen consumption increases as well for all species. The sensitivity of oxygen consumption to temperature variation in the shallow-water species is however smaller than that from the deep-sea hydrothermal vent species. This unexpected result could correspond to a pronounced increase of activity (avoidance behavior) in the vent species, which was not observed for the shallow-water species

Les endosymbioses chimioautotrophes : des modèles contemporains de la symbiogenèse ?

L'oxygène semble bien être un des facteurs clés pour comprendre l'évolution de la vie sur Terre. Quasiment absent de l'atmosphère durant plus de 2 milliards d'années, son accumulation ultérieure est corrélée à l'activité photosynthétique des cyanobactéries et à l'apparition des procaryotes aérobies, puis à celle des eucaryotes, tous primitivement aérobies, et enfin, à celle des organismes complexes pluricellulaires. Néanmoins il subsiste aujourd'hui à la surface de notre planète, et particulièrement au fond des océans, de nombreux environnements réduits (sources hydrothermales profondes, zones de suintements froids, apports massifs de matière organique,...) au sein desquels une grande diversité de Procaryotes anaérobies ou micro-aérobies continue de prospérer, y compris certains chimioautotrophes, fixant le carbone inorganique grâce à l'énergie chimique comme celle de l'oxydation des sulfures par exemple. Quelques métazoaires parviennent à coloniser ces milieux, les plus abondants formant des endosymbioses avec ces procaryotes. Les exemples les mieux étudiés (les bivalves du genre Bathymodiolus, l'annélide vestimentifère Riftia pachyptila, ou encore les bivalves du genre Calyptogena) mettent en évidence de nombreuses différences dans le degré de dépendance entre l'hôte et ses symbiotes et dans le mode de transmission de ceux-ci en particulier. Ce processus évolutif de la symbiose n'est pas sans rappeler les endosymbioses primaires qui sont à l'origine des organites des eucaryotes hétérotrophes (acquisition de la mitochondrie) et des eucaryotes phototrophes (acquisition des chloroplastes), et l'étude de ces modèles contemporains pourrait éclairer cette symbiogenèse d'un jour nouveau, voire révéler l'existence d'une nouvelle lignée, les Eucaryotes thiotrophes