Discovery and characterization of the α-amylases cDNAs from Enchytraeus albidus shed light on the evolution of "Enchytraeus-Eisenia type" Amy homologs in Annelida.

Although enchytraeids have gained popularity in scientific research, fundamental questions regarding their feeding ecology and biology remain largely unexplored. This study investigates α-amylases, major digestive enzymes responsible for hydrolyzing starch and similar polysaccharides into sugars, in Enchytraeus albidus. Genetic data related to α-amylases is currently lacking for the family Enchytraeidae but also for the entire Annelida. To detect and identify coding sequences of the expressed α-amylase genes in COI-monohaplotype culture (PL-A strain) of E. albidus, we used classical "gene fishing" and transcriptomic approaches. We also compared coding sequence variants of α-amylase retrieved from transcriptomic data related to freeze-tolerant strains. Our results reveal that E. albidus possesses two distinct α-amylase genes (Amy I and Amy II) that are homologs to earthworm Eisenia fetida Ef-Amy genes. Different strains of E. albidus possess distinctive alleles of α-amylases with unique SNP patterns specific to a particular strain. Unlike Amy II, Amy I seems to be a highly polymorphic and multicopy gene. The domain architecture of the putative Amy proteins was found the same as for classical animal α-amylases with ABC-domains. A characteristic feature of Amy II is the lack of GHGA motif in the flexible loop region, similarly to many insect amylases. We identified "Enchytraeus-Eisenia type" α-amylase homologs in other clitellates and polychaetes, indicating the ancestral origin of Amy I/II proteins in Annelida. This study provides the first insight into the endogenous non-proteolytic digestive enzyme genes in potworms, discusses the evolution of Amy α-amylases in Annelida, and explores phylogenetic implications.

Soluble esterases as biomarkers of neurotoxic compounds in the widespread serpulid Ficopomatus enigmaticus (Fauvel, 1923).

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.

Effects of short-term and long-term exposure to ocean acidification on carbonic anhydrase activity and morphometric characteristics in the invasive polychaete Branchiomma boholense (Annelida: Sabellidae): A case-study from a CO2 vent system.

The aim of this study was to test the effects of short- and long-term exposure to high pCO2 on the invasive polychaete Branchiomma boholense (Grube, 1878), (Sabellidae), through the implementation of a transplant experiment at the CO2 vents of the Castello Aragonese at the island of Ischia (Italy). Analysis of carbonic anhydrase (CA) activity, protein tissue content and morphometric characteristics were performed on transplanted individuals (short-term exposure) as well as on specimens resident to both normal and low pH/high pCO2 environments (long-term exposure). Results obtained on transplanted worms showed no significant differences in CA activity between individuals exposed to control and acidified conditions, while a decrease in weight was observed under short-term acclimatization to both control and low pH, although at low pH the decrease was more pronounced (∼20%). As regard individuals living under chronic exposure to high pCO2, the morphometric results revealed a significantly lower (70%) wet weight of specimens from the vents with respect to animals living in high pH/low pCO2 areas. Moreover, individuals living in the Castello vents showed doubled values of enzymatic activity and a significantly higher (50%) protein tissue content compared to specimens native from normal pH/low pCO2. The results of this study demonstrated that B. boholense is inclined to maintain a great homeostatic capacity when exposed to low pH, although likely at the energetic expense of other physiological processes such as growth, especially under chronic exposure to high pCO2.

Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities.

Invertebrates, unlike vertebrates which have adaptive immune system, rely heavily on the innate immune system for the defense against pathogenic bacteria. Lysozymes, along with other immune effectors, are regarded as an important group in this defense. An invertebrate-type (i-type) lysozyme, designated Urechis unicinctus invertebrate-type lysozyme, Uu-ilys, has been isolated from nephridia of Urechis unicinctus using a series of high performance liquid chromatography (HPLC), and ultrasensitive radial diffusion assay (URDA) as a bioassay system. Analyses of the primary structure and cDNA cloning revealed that Uu-ilys was approximately 14 kDa and composed of 122 amino acids (AAs) of which the precursor had a total of 160 AAs containing a signal peptide of 18 AAs and a pro-sequence of 20 AAs encoded by the nucleotide sequence of 714 bp that comprises a 5' untranslated region (UTR) of 42 bp, an open reading frame (ORF) of 483 bp, and a 3' UTR of 189 bp. Multiple sequence alignment showed Uu-ilys has high homology to i-type lysozymes from several annelids. Relatively high transcriptional expression levels of Uu-ilys was detected in nephridia, anal vesicle, and intestine. The native Uu-ilys exhibited comparable lysozyme enzymatic and antibacterial activities to hen egg white lysozyme. Collectively, these data suggest that Uu-ilys, the isolated antibacterial protein, plays a role in the immune defense mechanism of U. unicinctus. Recombinant Uu-ilys (rUu-ilys) produced in a bacterial expression system showed significantly decreased lysozyme lytic activity from that of the native while its potency on radial diffusion assay detecting antibacterial activity was retained, which may indicate the non-enzymatic antibacterial capacity of Uu-ilys.

The NF-κB pathway participates in the response to sulfide stress in Urechis unicinctus.

The NF-κB pathway is known to be involved in regulating apoptosis, inflammation and immunity in organisms. In this study, we first identified full-length cDNA sequences of two key molecules in the NF-κB pathway, namely, NEMO and p65, and characterized their responses in the hindgut of Urechis unicinctus (Echiura, Urechidae) exposed to sulfide. The full-length of cDNA was 2491 bp for U. unicinctus NEMO (UuNEMO) and 1971 bp for U. unicinctus p65 (Uup65), and both polyclonal antibodies were prepared using UuNEMO or Uup65 expressed prokaryotically with the sequence of their whole open reading frame. Immunoprecipitation and Western blotting showed that the NF-κB pathway was activated in U. unicinctus exposed to sulfide, in which the content of UuNEMO ubiquitination and nuclear Uup65 increased significantly (p < 0.05) in hindgut tissue of U. unicinctus exposed to sulfide. Furthermore, the mRNA level of UuBcl-xL, a downstream anti-apoptosis gene of the NF-κB pathway, increased significantly (p < 0.05) from 48 h to 72 h and the mRNA level of UuBax, a Bcl-xL antagonist gene, decreased significantly (p < 0.05) at 48 h in the hindgut of U. unicinctus exposed to 50 µM sulfide. During the 150 µM sulfide exposure, the level of UuBcl-xL showed no obvious change, whereas the UuBax mRNA level increased significantly (p < 0.05) at 72 h post-exposure to 150 µM sulfide. We suggested that the activated NF-κB pathway up-regulates UuBcl-xL expression, and evokes an anti-apoptotic response to resist sulfide damage at 50 µM in U. unicinctus. Meanwhile, a Bax-mediated pro-apoptotic response occurs when U. unicinctus is exposed to 150 µM sulfide.

Expression characteristics of sulfur dioxygenase and its function adaption to sulfide in echiuran worm Urechis unicinctus.

Animals living in coastal burrows are periodically exposed to the sulfide, a mixture of H2S, HS(-) and S(2-), during low tide. Mitochondrial sulfide oxidation is an important strategy that allows organisms to avoid injury from sulfide exposure, and sulfur dioxygenase (SDO) plays an essential role. In this study, we characterized the SDO expression and the total SDO-specific activity (T-SDO SA) in different organs of Urechis unicinctus, which inhabitU-shaped burrows in intertidal and subtidal mudflats. The SDO expressions at both mRNA and protein levels were highest in the anal sac, followed by the midgut, and were extraordinarily low in the body wall and hindgut; SDO was located mainly in the epithelial cells of all organs by immunohistochemistry. Moreover, the T-SDO SA was different in the detected organs, but with no significant differences and SDO SAs were strong positive correlation with GSH contents. Furthermore, we investigated the responses of the SDO in the midgut and hindgut of U. unicinctus during sulfide exposure. The SDO contents increased significantly at 48h and 72h, respectively, in both the midgut and hindgut when the worms were exposed to 50 and 150µM sulfide. However, the T-SDO SA was no significantly different in the midgut except that at 72h for 150µM sulfide treatment, meanwhile in the hindgut, the T-SDO SA increased significantly after 24h exposure for 50 and 150µM sulfide treatments. We concluded that the hindgut plays more important role than the midgut in sulfide tolerance for U. unicinctus.

Arginine kinase from Myzostoma cirriferum, a basal member of annelids.

We assembled a phosphagen kinase gene from the Expressed Sequence Tags database of Myzostoma cirriferum, a basal member of annelids. The assembled gene sequence was synthesized using an overlap extension polymerase chain reaction method and was expressed in Escherichia coli. The recombinant enzyme (355 residues) exhibited monomeric behavior on a gel filtration column and showed strong activity only for l-arginine. Thus, the enzyme was identified as arginine kinase (AK). The two-substrate kinetic parameters were obtained and compared with other AKs. Phylogenetic analysis of amino acid sequences of phosphagen kinases indicated that the Myzostoma AK gene lineage differed from that of the polychaete Sabellastarte spectabilis AK, which is a dimer of creatine kinase (CK) origin. It is likely that the Myzostoma AK gene lineage was lost at an early stage of annelid evolution and that Sabellastarte AK evolved secondarily from the CK gene. This work contributes to our understanding of the evolution of phosphagen kinases of annelids with marked diversity.

Molecular characterization of an acetylcholinesterase from the hemichordate Saccoglossus kowalevskii.

Our goal is to understand the evolution of the structure and function of cholinesterases (ChEs) in the deuterostome lineage and in particular to understand the role of paralogous enzymes such as the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of the vertebrates. We have, in the past, characterized ChEs in two acraniate deuterostomes: amphioxus (a cephalochordate) and Ciona intestinalis (a urochordate). Here we present results on an AChE from a basal deuterostome, a model hemichordate, the acorn worm Saccoglossus kowalevskii. Of the eight genes coding for putative ChE-like proteins possessing Trp84, a characteristic of the choline-binding catalytic subsite of ChEs, we cloned a full length cDNA with a coding sequence typical of an acraniate AChE possessing a C-terminal exon coding for a typical T-peptide. We then used in vitro expression of the cDNA in COS-7 cells to characterize the AChE kinetically, pharmacologically, and biochemically. The cDNA codes for an AChE (AChE1), which is found in monomeric (G1), dimeric (G2), and tetrameric (G4) forms; and interacts with poly-L-proline, PRiMA, and ColQ, characteristic of an AChE possessing a T-peptide. The expression of the AChE is temperature dependent, with greater expression at 30 °C. We discuss the implications of these data for the evolution of the ChEs in the deuterostomes.

Nurse eggs form through an active process of apoptosis in the spionid Polydora cornuta (Annelida).

The production of nurse eggs is fundamental to poecilogony in some species of spionid annelids. In species such as Polydora cornuta, nurse-egg production varies among females and ingestion of nurse eggs varies among young, resulting in a form of poecilogony with divergent phenotypes for females (e.g., fecundity and per-offspring investment) as well as for larvae (e.g., trophic mode, size, and stage at hatching). We tested the hypothesis that nurse eggs of P. cornuta form through an active developmental process and specifically, through apoptosis. Results of a TUNEL assay indicate nuclear fragmentation occurs in a process that is characteristic of apoptosis. Cellular indicators of apoptosis in nurse eggs include activation of caspase-3, a positive Annexin V reaction indicating exposure of phosphatidylserine on the outer cell membrane, and invagination of the membrane to form yolk vesicles. These results indicate that formation of nurse eggs in this population of P. cornuta occurs through an active, adaptive process. Furthermore, while apoptosis also occurs in some cells of P. cornuta embryos, it was not detected until later in development. This suggests that nurse eggs originate through heterochrony in a developmental process (apoptosis) that is common to all young of P. cornuta.

Response of sulfide:quinone oxidoreductase to sulfide exposure in the echiuran worm Urechis unicinctus.

Sulfide is a natural, widely distributed, poisonous substance, and sulfide:quinone oxidoreductase (SQR) is responsible for the initial oxidation of sulfide in mitochondria. In this study, we examined the response of SQR to sulfide exposure (25, 50, and 150 µM) at mRNA, protein, and enzyme activity levels in the body wall and hindgut of the echiuran worm Urechis unicinctus, a benthic organism living in marine sediments. The results revealed SQR mRNA expression during sulfide exposure in the body wall and hindgut increased in a time- and concentration-dependent manner that increased significantly at 12 h and continuously increased with time. At the protein level, SQR expression in the two tissues showed a time-dependent relationship that increased significantly at 12 h in 50 µM sulfide and 6 h in 150 µM, and then continued to increase with time while no significant increase appeared after 25 µM sulfide exposure. SQR enzyme activity in both tissues increased significantly in a time-dependent manner after 50 µM sulfide exposure. We concluded that SQR expression could be induced by sulfide exposure and that the two tissues studied have dissimilar sulfide metabolic patterns. A U. unicinctus sulfide-induced detoxification mechanism was also discussed.

The structure of lombricine kinase: implications for phosphagen kinase conformational changes.

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

Sulfide:quinone oxidoreductase from echiuran worm Urechis unicinctus.

Sulfide is a natural, widely distributed, poisonous substance, and sulfide:quinone oxidoreductase (SQR) has been identified to be responsible for the initial oxidation of sulfide in mitochondria. In this study, full-length SQR cDNA was cloned from the echiuran worm Urechis unicinctus, a benthic organism living in marine sediments. The protein consisted of 451 amino acids with a theoretical pI of 8.98 and molecular weight of 50.5 kDa. Subsequently, the SQR mRNA expression in different tissues was assessed by real-time reverse transcription and polymerase chain reaction and showed that the highest expression was in midgut, followed by anal sacs and coelomic fluid cells, and then body wall and hindgut. Furthermore, activated SQR was obtained by dilution refolding of recombinant SQR expression in E. coli, and the refolded product showed optimal activity at 37 °C and pH 8.5 and K (m) for ubiquinone and sulfide at 15.6 µM and 40.3 µM, respectively. EDTA and GSH had an activating effect on refolded SQR, while Zn(2+) caused decreased activity. Western blot showed that SQR in vivo was located in mitochondria and was ∼ 10 kDa heavier than the recombinant protein. In addition, SQR, detected by immunohistochemistry, was mainly located in the epithelium of all tissues examined. Ultrastructural observations of these tissues' epithelium by transmission electron microscopy provided indirect cytological evidence for its mitochondrial location. Interesting aspects of the U. unicinctus SQR amino acid sequence, its catalytic mechanism, and the different roles of these tissues in sulfide metabolic adaptation are also discussed.

Evolution of the diverse array of phosphagen systems present in annelids.

Annelids as a group express a variety of phosphagen kinases including creatine kinase (CK), glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and a unique arginine kinase (AK) restricted to annelids. In prior work, we have determined and compared the intron/exon organization of the annelid genes for cytoplasmic GK, LK, AK, and mitochondrial TK and LK (MiTK and MiLK, respectively), and found that these annelid genes, irrespective of cytoplasmic or mitochondrial, have the same 8-intron/9-exon organization strikingly similar to mitochondrial CK (MiCK) genes. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids. To gain a greater understanding of the evolutionary processes leading to the diversity of annelid phosphagen kinases, we determined for the first time the intron/exon organization of a cytoplasmic CK gene from a polychaete as well as that of another polychaete MiCK gene. These gene structures, coupled with a phylogenetic analyses of annelid enzymes and assessment of the fidelity of substrate specificity of some these phosphagen kinases, provide insight into the pattern of radiation of the annelid enzymes. Annelid phosphagen kinases appeared to have diverged in the following order (earliest first): (1) cytoplasmic AK, LK and TK, (2) GK, and (3) mitochondrial MiLK and MiTK. Interestingly, phylogenetic analyses showed that the above phosphagen kinases appear to be basal to all CK isoforms (mitochondrial, cytoplasmic and flagellar CKs). This somewhat paradoxical placement of CKs most likely reflects a higher rate of evolution and radiation of the annelid-specific LK, TK and GK genes than the CK isoform genes.

Cloning and characterization of Arenicola marina peroxiredoxin 6, an annelid two-cysteine peroxiredoxin highly homologous to mammalian one-cysteine peroxiredoxins.

Peroxiredoxins (PRDXs) are a superfamily of thiol-dependent peroxidases found in all phyla. PRDXs are mechanistically divided into three subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs. To reduce peroxides, the N-terminal peroxidatic Cys of PRDXs is first oxidized into sulfenic acid. This intermediate is reduced by forming a disulfide bond either with a resolving Cys of another monomeric entity (typical 2-Cys) or of the same molecule (atypical 2-Cys). In 1-Cys PRDXs, the resolving Cys is missing and the sulfenic acid of the peroxidatic Cys is reduced by a heterologous thiol-containing reductant. In search of a homolog of human 1-Cys PRDX6 in Arenicola marina, an annelid worm living in intertidal sediments, we have cloned and characterized a PRDX exhibiting high sequence homology with its mammalian counterpart. However, A. marina PRDX6 possesses five Cys among which two Cys function as peroxidatic and resolving Cys of typical 2-Cys PRDXs. Thus, A. marina PRDX6 belongs to a transient group exhibiting sequence homologies with mammalian 1-Cys PRDX6 but must be mechanistically classified into typical 2-Cys PRDXs. Moreover, PRDX6 is highly expressed in tissues directly exposed to the external environment, suggesting that this PRDX may be of particular importance for protection against exogenous oxidative attacks.

Biochemical and enzymatic properties of a novel marine fibrinolytic enzyme from Urechis unicinctus.

A novel potent protease, Urechis unicinctus fibrinolytic enzyme (UFE), was first discovered by our laboratory. In this study, we further investigated the enzymatic properties and dynamic parameters of UFE. As a low molecular weight protein, UFE appeared to be very stable to heat and pH. When the temperature was <50 degrees C, the remnant enzyme activity remained almost unchanged, but when the temperature was raised to 60 degrees C the remnant enzyme activity began to decrease rapidly. UFE was quite stable in a pH range of 3.0-12.0, especially at slightly alkaline pH values. Mn(2+), Cu(2+), and Fe(2+) ions were activators of UFE, whereas Fe(3+) and Ag(+) ions were inhibitors. Fe(2+) ion along with Fe(3+) ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8.0 and 50 degrees C, respectively. When using casein as substrate and a substrate concentration <0.1% casein (w/v), the reaction velocity was increased with substrate concentration. Also when using casein as substrate, the determined K(m) and V(max) of UFE were 0.5298 mg/mL and 3.0845 mol of L-tyrosine equivalent, respectively. Our systematic research results are significant when UFE is applied for medical and industrial purposes.

Toxicological responses in Laeonereis acuta (annelida, polychaeta) after arsenic exposure.

Several environmental pollutants, including metals, can induce oxidative stress. So, the objective of this study was to evaluate the effects of arsenic (As(III), as As(2)O(3)) on the antioxidant responses in the polychaete Laeonereis acuta. Worms were exposed to two environmentally relevant concentrations of As, including the highest previously allowed by Brazilian legislation (50 microg As/l). A control group was kept in saline water (10 per thousand) without added metal. It was observed that: (1) a peak concentration of lipid peroxide was registered after 2 days of exposure to 50 microg As/l (61+/-3.2 nmol CHP/g wet weight) compared to the control group (43+/-4.5 nmol CHP/g wet weight), together with a lowering of the activity of the antioxidant enzyme catalase (-47 and -48%, at 50 or 500 microg As/l respectively) and a higher superoxide dismutase activity (+305% at 50 microg As/l with respect to the control group); (2) a lower conjugation capacity through glutathione-S-transferase activity was observed after 7 days of exposure to 50 microg As/l (-48% compared to the control group); (3) a significant increase in As concentration was verified after 1 week of exposure to both As concentrations (50 and 500 microg/l); (4) worms exposed to As showed a limited accumulation of related methylated As species and the levels of non-toxic As species like arsenobetaine (AsB) and arsenocholine (AsC) remained unchanged during the exposure period when compared with the controls. Overall, it can be concluded that As interfered in the antioxidant defense system of L. acuta, even at low concentrations (50 microg/l) that Brazilian legislation previously considered safe. The fact that worms exposed to As showed high levels of methylated As species indicates the methylation capability of L. acuta, although the high levels of inorganic As suggest that not all the administered As(III) (as As(2)O(3)) is completely removed or biotransformed after 7 days of exposure.

Marine invertebrate cytochrome P450: emerging insights from vertebrate and insects analogies.

Cytochrome P450 enzymes (P450s) are responsible for the oxidative metabolism of a plethora of endogenous and exogenous substrates. P450s and associated activities have been demonstrated in numerous marine invertebrates belonging to the phyla Cnidaria, Annelida (Polychaeta), Mollusca, Arthropoda (Crustacea) and Echinodermata. P450s of marine invertebrates and vertebrates show considerable sequence divergence and the few orthologs reveal the selective constraint on physiologically significant enzymes. P450s are present in virtually all tissues of marine invertebrates, although high levels usually are found in hepatic-like organs and steroidogenic tissues. High-throughput technologies result in the rapid acquisition of new marine invertebrate P450 sequences; however, the understanding of their function is poor. Based on analogy to vertebrates and insects, it is likely that P450s play a pivotal role in the physiology of marine invertebrates by catalyzing the biosynthesis of signal molecules including steroids such as 20-hydroxyecdysone (the molting hormone of crustaceans). The metabolism of many exogenous compounds including benzo(a)pyrene (BaP), pyrene, ethoxyresorufin, ethoxycoumarin and aniline is mediated by P450 enzymes in tissues of marine invertebrates. P450 gene expression, protein levels and P450 mediated metabolism of xenobiotics are induced by PAHs in some marine invertebrate species. Thus, regulation of P450 enzyme activity may play a central role in the adaptation of animals to environmental pollutants. Emphasis should be put on the elucidation of the function and regulation of the ever-increasing number of marine invertebrate P450s.

World-wide whale worms? A new species of Osedax from the shallow north Atlantic.

We describe a new species of the remarkable whalebone-eating siboglinid worm genus, Osedax, from a whale carcass in the shallow north Atlantic, west of Sweden. Previously only recorded from deep-sea (1500-3000 m) whale-falls in the northeast Pacific, this is the first species of Osedax known from a shelf-depth whale-fall, and the first from the Atlantic Ocean. The new species, Osedax mucofloris sp. n. is abundant on the bones of an experimentally implanted Minke whale carcass (Balaenoptera acutorostrata) at 125m depth in the shallow North Sea. O. mucofloris can be cultured on bones maintained in aquaria. The presence of O. mucofloris in the shallow North Sea and northeast Pacific suggests global distribution on whale-falls for the Osedax clade. Molecular evidence from mitochondrial cytochrome oxidase 1 (CO1) and 18S rRNA sequences suggests that O. mucofloris has high dispersal rates, and provides support for the idea of whale-falls acting as 'stepping-stones' for the global dispersal of siboglinid annelids over ecological and evolutionary time.

Biochemical and enzymological aspects of the symbiosis between the deep-sea tubeworm Riftia pachyptila and its bacterial endosymbiont.

Riftia pachyptila (Vestimentifera) is a giant tubeworm living around the volcanic deep-sea vents of the East Pacific Rise. This animal is devoid of a digestive tract and lives in an intimate symbiosis with a sulfur-oxidizing chemoautotrophic bacterium. This bacterial endosymbiont is localized in the cells of a richly vascularized organ of the worm: the trophosome. These organisms are adapted to their extreme environment and take advantage of the particular composition of the mixed volcanic and sea waters to extract and assimilate inorganic metabolites, especially carbon, nitrogen, oxygen and sulfur. The high molecular mass hemoglobin of the worm is the transporter for both oxygen and sulfide. This last compound is delivered to the bacterium which possesses the sulfur oxidizing respiratory system, which produces the metabolic energy for the two partners. CO2 is also delivered to the bacterium where it enters the Calvin-Benson cycle. Some of the resulting small carbonated organic molecules are thus provided to the worm for its own metabolism. As far as nitrogen assimilation is concerned, NH3 can be used by the two partners but nitrate can be used only by the bacterium. This very intimate symbiosis applies also to the organization of metabolic pathways such as those of pyrimidine nucleotides and arginine. In particular, the worm lacks the first three enzymes of the de novo pyrimidine biosynthetic pathways as well as some enzymes involved in the biosynthesis of polyamines. The bacterium lacks the enzymes of the pyrimidine salvage pathway. This symbiotic organization constitutes a very interesting system to study the molecular and metabolic basis of biological adaptation.

Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic invertebrate from deep-sea hydrothermal vents.

The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed.