The molecular mechanisms underlying oocyte maturation in the annelid polychaetes Arenicola marina and Arenicola defodiens were investigated. In both species, a hitherto unidentified hormone triggers synchronous and rapid transition from prophase to metaphase, a maturation process which can be easily reproduced in vitro. Activation of a roscovitine- and olomoucine-sensitive M-phase-specific histone, H1 kinase, occurs during oocyte maturation. Using affinity chromatography on immobilized p9CKShs1, we purified CDK1 and cyclin B from oocyte extracts prepared from both phases and both species. In prophase, CDK1 is present both as an inactive, but Thr161-phosphorylated monomer, and as an inactive (Tyr15-phosphorylated) heterodimer with cyclin B. Prophase to metaphase transition is associated with complete tyrosine dephosphorylation of the cyclin B-associated CDK1, with phosphorylation of cyclin B, and with dramatic activation of the kinase activity of the CDK1/cyclin B complex. We propose that Arenicola oocytes may provide an ideal model system to investigate the acquisition of the ability of oocytes to be fertilized that occurs as oocyte shift from prophase to metaphase, an important physiological event, probably regulated by active CDK1/cyclin B.

Equipe Ecophysiologie, UPMC–CNRS UMR 7127–INSUE, Station Biologique, BP 74, Roscoff cedex 29682, France Laboratoire de Spectrometrie de Masse Bio-Organique, ULP–CNRS UMR 7509, ECPM, 25 rue Becquerel, Strasbourg Cedex 67087, France Laboratoire de Chimie Analytique et Minerale, ULP–CNRS UMR 7512 ECPM 25, rue Becquerel, Strasbourg Cedex 67087, France Institut fur Zoophysiologie, Heinrich-Heine-Universitat, Dusseldorf D-40225, Germany IFREMER DRO/EP, BP 70, Plouzane F-29280, France Equipe Adaptations aux Milieux Extremes, UPMC-CNRS UMR 7622, 7 Quai St-Bernard, Paris 75005, France

Segonzacia mesatlantica (Crustacea; Decapoda; Brachyura) is the only endemic crab species known from the Mid-Atlantic Ridge (MAR) hydrothermal vents. Known from all explored sites in the Atlantic, its wide distribution makes this species a model to study physiological adaptation, and specifically respiratory strategies. Native haemocyanin (Hc) comprises four non-covalent associations in equilibrium formed by monomers, hexamers, dodecamers and octadecamers made up of approximately 75 kDa polypeptide chains. Four different amino acid chains are observed with a molecular mass ranging from 75,234 to 75,972 Da. Experiments carried-out under pressure suggested that the percentage of monomer increased in the haemolymph under hypoxic condition. We have also observed a shift of the proportion of the two dodecamer series, suggesting a rapid modification of the Hc phenotype between hypoxic and hyperoxic conditions. Native Hc possesses a high oxygen affinity ( P50 = 2.2 Torr at 15 degrees C and pH 7.5), a large Bohr effect (Deltalog P50 / DeltapH approximately -2.7) and a slightly reverse temperature effect (DeltaH = +17.19 kJ mol(-1). The composition of Segonzacia haemolymph is similar to that of other littoral species except for the large enrichment in free copper and zinc. As for other species from hydrothermal vent sites, Segonzacia haemolymph possesses a higher buffer capacity than littoral species. Moreover, species from the hydrothermal vent decapods from Pacific hydrothermal vent that encounter higher CO2 content in their environment have a higher buffer capacity than Atlantic vent species. The results presented are discussed in relation with the physico-chemical characteristics of the hydrothermal vent environment.

Since electrospray ionization mass spectrometry (ESI-MS) has demonstrated capabilities for observing intact, weak interactions, there has been increasing interest in studying by this method noncovalently bound complexes. In this communication, we report for the first time the structure obtained by a commercial ESI quadrupole time-of-flight spectrometer on a native hemocyanin of deep-sea crab Bythograea thermydron with a molecular mass of 1.3 MDa. ESI-MS analysis of the native hemocyanin revealed the formation of a 18-mer noncovalent assembly with a measured molecular mass of 1354940 +/- 480 Da. ESI-MS data also revealed that this huge structure is an equilibrium with several assemblages, dodecamer (measured molecular weight = 902570 +/- 110 Da), hexamer (measured molecular weight = 450310 +/- 260 Da), and monomeric structures (measured molecular weight = 74999 +/- 85 Da).

Les sources hydrothermales sont caracterisees par des conditions environnementales spatio-temporelles hyper-variables. Les crustaces decapodes hydrothermaux des dorsales Pacifique et Atlantique, bien que presentant une repartition differentielle sur les cheminees, sont soumis a des parametres physico-chimiques relativement similaires. Ils rencontrent des variations de pH importants, de fortes concentrations en H2S et en CO2 et de faibles teneurs en O2. Les especes du Pacifique rencontrent des conditions plus marquees que les especes de l'Atlantique. Les branchies des brachyoures hydrothermaux apparaissent similaires a celles des crustaces cotiers. Des structures ressemblant a des chemorecepteurs ont ete observees sur le vaisseur efferent des branchies. Ces structures pourraient intervenir dans la perception par l'animal de son environnement chimique. La composition ionique de l'hemolymphe de ces crustaces est peu differente de celle des crustaces littoraux, a l'exception de fortes teneurs en lactate, en proteines et en cuivre. Les hemocyanines (Hc) fonctionnelles ne representent pas la majorite des proteines totales, ce qui pourrait etre du a la presence de forte concentration en apoHc qui jouerait un role dans la detoxification des metaux. La structure quaternaire des Hc de crustaces hydrothermaux semble essentiellement correlee avec la position taxonomique des especes, sans presenter de particularites majeures liees a l'environnement hydrothermal. Ces Hc presentent de fortes affinites pour l'O2, un effet Bohr marque, un effet lactate faible a nul et un effet temperature faible, voire inverse. Un facteur diminuant l'affinite Hc-O2 a ete mis en evidence chez Cyanagraea praedator. Le pouvoir tampon de l'hemolymphe des crustaces du Pacifique, vivant dans un milieu plus hypercapnique, semble superieur a celui des especes de l'Atlantique. La crevette Rimicaris exoculata presente face a une hypercapnie une reponse rapide et complete comparee a celle des especes littorales.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.

Carcinus maenas and Necora puber were exposed to air for 72 h and 18 h, respectively, at 18 °C. Changes in the free amino acid (FAA) content of their muscle, hepatopancreas and haemolymph were recorded during air-exposure and subsequent reimmersion. Muscle and hepatopancreas urate contents and haemolymph serum protein levels were also measured during emersion. In air-exposed C. maenas, the muscle FAA pool increased significantly within the first 24 h of emersion. This increase was due to an increase in the non-essential amino acid (NEAA) pool only; the essential amino acid (EAA) pool did not change. In haemolymph, the EAA pool decreased during the first 24 h of emersion, whereas the FAA and NEAA pools did not change. However, in this compartment, glutamine levels increased throughout the air-exposure period. No significant changes in FAA, NEAA and EAA contents of the hepatopancreas were observed during the 72 h emersion. In air-exposed N. puber, the FAA pools of muscle and hepatopancreas did not change, although changes in the levels of some amino acids were observed during the 18 h emersion period. In this species, large increases in both the NEAA and EAA pools in the haemolymph were recorded. High levels of urate were observed in the muscle and hepatopancreas of immersed N. puber, but no significant changes occurred during emersion. In contrast, immersed C. maenas exhibited low levels of urate in both compartments, and hepatopancreas urate levels increased slightly during emersion. Haemolymph protein content did not change in air-exposed N. puber, whereas it increased in the haemolymph of 72 h emersed C. maenas. The origin of newly formed NEAAs and their role in ammonia detoxification, particularly in C. maenas, which is able to regulate its internal ammonia levels during such a prolonged emersion, are discussed.

The functional properties of the haemocyanin ofVampyroteuthis infernalis (Cephalopoda: Vampyromorpha), measured at 5 °C, are reported and discussed in relation to hypoxia. The oxygen affinity of this haemocyanin (P50=0.47−0.55 kPa) is higher than any previously measured for a cephalopod. The high cooperativity (n50=2.20−2.23) and Bohr coefficient (−0.22) suggest a true transport function for this haemocyanin. This high-affinity haemocyanin, in conjunction with moderate gill diffusion capacity, provides a sufficient oxygen gradient from the environment to the blood to support the low routine oxygen consumption rate of V. infernalis

Deep-sea hydrothermal vents, first discovered in 1977, are characterized by variable and extreme conditions, in particular by a low level of oxygen, a low pH, a high temperature, a high sulphide content, and a high level of carbon dioxide. Decapod crustaceans, which are found in all the hydrothermal vents yet known at the East Pacific Rise (e.g. Bythograea thermydron Williams, 1980, Cyanagraea praedator de Saint-Laurent, 1984, Alvinocaris lusca Williams & Chace Jr., 1982) and at the Mid-Atlantic Ridge (e.g. Rimicaris exoculata, Williams & Rona 1986, Chorocaris chacei (Williams & Rona, 1986), Segonzacia mesatlantica Williams, 1988), had to develop adaptation mechanisms, in particular respiratory adaptations to survive in this environment. The respiratory pigment of decapod crustaceans is haemocyanin that binds reversibly oxygen to a pair of copper atoms. It is a large extracellular protein, with a blue colour, which has a typical hexameric structure (Markl & Decker, 1992) and a large potential for functional plasticity (Truchot & Lallier, 1992).

Chez les Crustaces decapodes euryhalins, les branchies posterieures se differencient des branchies anterieures par leur ultrastructure et leur fonction; des etudes histochimiques et cytochimiques ont egalement revele des differences quant a la distribution et la densite des granules de glycogene presents dans les branchies anterieures et posterieures. La presente etude a pour but de quantifier les reserves en glycogene et en glucose des branchies anterieures et posterieures du crabe Carcinus maenas en intermue. La teneur moyenne en glycogene dans les branchies anterieures est de 56,54 ± 8,84 μg. mg -1 de poids sec (PS) et dans les branchies posterieures, de 115,2 ± 14,59 μg. mg -1 PS. L'estimation des reserves totales en glycogene montre que celles-ci sont quatre fois plus elevees dans les branchies posterieures que dans les branchies anterieures. En revanche, les teneurs en glucose des deux types de branchies sont faibles et non significativement differentes: 5,25 ± 1,18 μg. mg -1 PS dans les branchies anterieures et 4,29 ± 0,9 μg. mg -1 PS dans les branchies posterieures. Une eventuelle relation entre ces reserves energetiques et la fonction des deux types de branchies est discutee.

In euryhaline decapod Crustaceans, the ultrastructure and function of posterior gills are known to be different from those of anterior gills. Histochemical and cytochemical studies have also shown that both distribution and density of glycogen granules in anterior and posterior gills are not similar. This study was aimed at quantifying and comparing glycogen and glucose reserves of both anterior and posterior gills of the euryhaline crab Carcinus maenas at the intermoult stage. The mean glycogen content was 56.54 +/- 8.84 mu g. mg(-1) tissue dry weight (DW) and 115.20 +/- 14.59 mu g. mg(-1) DW in anterior and posterior gills, respectively. The glycogen amount stored in the posterior gills of a crab was four times as high as that stored in the anterior gills. Glucose content was low and similar in all gills: 5.25 +/- 1.18 mu g. mg(-1) DW and 4.29 +/- 0.90 mu g. mg(-1) DW in anterior and posterior gills, respectively. A possible relationship between these energetic stores and the function of each type of gills is discussed.