The multigenic family of the extracellular hemoglobin from the annelid polychaete Arenicola marina

The extracellular hemoglobin of the lugworm Arenicola marina which inhabits on the intertidal area, a sulfide-rich environment, comprises eight globin chains previously determined by mass spectrometry. We have cloned and sequenced five of the globin components. The deduced amino-acid sequences exhibit an extracellular signal peptide and two cysteine residues involved in an internal disulfide bond. The molecular weights calculated from the globin primary structures obtained from complete cDNA sequences are in good agreement with the mass spectrometry values obtained with the native hemoglobin. Phylogenetic analysis has allowed assigning the five A. marina sequences to the different globin sub-families. Two of the globins were found to be A2 globin chains lacking the cysteine residues proposed to be involved in the binding of hydrogen sulfide by such hemoglobin. We discuss the unusual absence of these cysteines in the light of their invariant occurrence in the A2 subfamily of hemoglobins from annelids inhabiting sulfide-rich environments.     

Purification and characterization of proteases from the polychaete annelid Sabellaria alveolata (L.)

Eleven proteases have been purified to electrophoretic homogeneity from crude digestive fluid of polychaete annelids, Sabellaria alveolata. Purification steps were Sephadex G-100 gel filtration, benzamidine-cellulose and SBTI-Sepharose (SBTI = soybean trypsin inhibitor) affinity chromatography, CM-Sepharose and DEAE-Sepharose ion-exchange chromatography. Nine proteases have been purified in sufficient quantities for characterization. All are active at basic pH and are probably serine proteases, since they are inhibited by phenylmethylsulfonyl fluoride, specific chloromethyl ketone amino acids derivatives, but not by EDTA and p-chloromercuribenzoate. They do not hydrolyse exopeptidase substrates. From their properties, they can be divided into five classes. 1. A trypsin-like protease, which hydrolyses only trypsin substrates and is inhibited by N-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl), leupeptin and antipain. It differs from bovine trypsin by its very acidic isoelectric point (below 3.3) and its higher Mr (35 000). 2. A chymotrypsin-like protease which hydrolyses only chymotrypsin substrates and is inhibited by TosPheCH2Cl, Z-PheCH2Cl, chymostatin but only slightly by leupeptin and antipain. Its isoelectric point is below 3.3 and its Mr 31 000. 3. Two minor chymotrypsin-like proteases with slightly broader specificity, since they hydrolyse trypsin substrates significantly and are much more inhibited by leupeptin. They have acidic isoelectric points (3.3 and 3.5) and slightly lower Mr (27 000). 4. Four proteases hydrolyse trypsin and chymotrypsin substrates equally well. Their chymotryptic character is, however, predominant since they are inhibited by TosPheCH2Cl and Z-PheCH2Cl but not TosLysCH2Cl. They have similar Mr (27 000) but isoelectric points ranging from 4.0 to above 9.1. 5. The last one is very similar but has lower esterolytic activities. These proteases of broad specificity do not resemble any known serine protease since they differ from subtilisins by their sensitivity to TosPheCH2Cl.     

Morphology and molecular phylogeny of Haplozoon praxillellae n. sp. (Dinoflagellata): a novel intestinal parasite of the maldanid polychaete Praxillella pacifica Berkeley

The genus Haplozoon comprises a group of endoparasites infecting the intestines of polychaete worms. Comparative studies using light microscopy, scanning and transmission electron microscopy, and small subunit rDNA have shown that these organisms are very unusual dinoflagellates. To date, there is only one species known from the Pacific Ocean, namely Haplozoon axiothellae Siebert. In this study, we describe Haplozoon praxillellae n. sp. from the intestine of the Pacific maldanid polychaete Praxillella pacifica Berkeley. The parasites are relatively small, oblong and about 35-125mum in length, consisting of the trophocyte (anterior-most compartment), rectangular gonocytes and bulbous sporocytes. The trophocyte bears an attachment apparatus with a prominent 'suction disc' and numerous stylets. We were able to detect spherical vesicles near the ventral surface of each gonocyte. The whole organism is covered with thecal barbs of different shape and size, except for the caudal end of the posterior-most sporocyte, which is instead covered with hexagonal or pentagonal alveoli. A continuous membrane encloses the whole pseudocolony. Molecular phylogenetic data, host specificity and morphological differences clearly distinguish H. praxillellae n. sp. from H. axiothellae.     

Cell lineage and fate map of the primary somatoblast of the polychaete annelid Capitella teleta

Like most polychaete annelids, Capitella teleta (formerly Capitella sp. I) exhibits a highly stereotypic program of early development known as spiral cleavage. Animals with spiral cleavage have diverse body plans, and homologous embryonic cells can be readily identified among distantly related animals. Spiralian embryos are particularly amenable to studies of fate-mapping, and larval fates of identified cells are conserved among diverse taxa. One cell of particular importance in spiralian development is 2d, or the primary somatoblast, which generates ectoderm of the body posterior to the prototroch. We are interested in the evolution of the primary somatoblast, and thus far, the 2d sublineage has only been analyzed in a few species. In Capitella teleta, 2d generates ectoderm of the segmented trunk and post-segmental pygidium. In this study, development of the 2d lineage was characterized in detail through intracellular injections of DiI, and time-lapse as well as confocal microscopy to analyze cleavage patterns and the fates of larval cells. Analysis of cleavage patterns reveals that the first bilateral division in the 2d sublineage occurs with the division of 2d112, the same 2d daughter cell that first divides bilaterally in the polychaete Platynereis dumerilii. Larval fates of blastomeres 2d1, 2d2, 2d11, 2d12, 2d112, 2d1121, and 2d1122 were determined. All cells show stereotypic descendant clones that are consistent with segregation within sublineages. In the first few divisions of the 2d sublineage, larval-specific structures (neurotroch and telotroch) and pygidial ectoderm are segregated from segmental ectoderm and ventral nerve cord. The daughters of the first bilateral division, 2d1121 and 2d1122, generate the right and left halves of the segmental ectoderm and ventral nerve cord respectively, although the clones are consistently asymmetric across the dorsal midline. The pattern of cleavage divisions and the fates of the 2d daughters in Capitella teleta are compared to those in other spiralians with special attention to annelids.     

A novel polymer produced by a bacterium isolated from a deep-sea hydrothermal vent polychaete annelid

AIMS: The objective of the present work was to describe an aerobic, mesophilic and heterotrophic marine bacterium, designated HYD657, able to produce an exopolysaccharide (EPS). It was isolated from a East Pacific Rise deep-sea hydrothermal vent polychaete annelid. METHODS AND RESULTS: This micro-organism, on the basis of the phenotypical features and genotypic investigations, can be clearly assigned to the Alteromonas macleodii species and the name A. macleodii subsp. fijiensis biovar deepsane is proposed. Optimal growth occurs between 30 and 35 degrees C, at pH between 6.5 and 7.5 and at ionic strengths between 20 and 40 g x l(-1) NaCl. The G + C content of DNA was 46.5%. This bacterium excreted, under laboratory conditions, an EPS consisting of glucose, galactose, rhamnose, fucose and mannose as neutral sugars along with glucuronic and galacturonic acids and a diacidic hexose identified as a 3-0-(1 carboxyethyl)-D-glucuronic acid. Its average molecular mass was 1.6 x 10(6) Da. CONCLUSIONS: The bacterium HYD657, for which the name A. macleodii subsp. fijiensis biovar deepsane is proposed, produces an unusual EPS in specific medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to its interesting biological activities, applications have been found in cosmetics. Its probable contribution to the filamentous microbial mat in the Alvinella pompejana microenvironment can be also mentioned.     

A fine structural study of the calcareous opercular plate and associated cells a polychaete annelid

The structure of the organic material and inorganic elements of the opercular plate and associated cells in the serpulid annelid, Pomatoceros lamarckii Quatrefages, have been described by transmission and scanning electron microscopy. After decalcification the organic material of the opercular plate was found to consist of three major structurally different components, an outer, thin, electron-dense layer, parallel rows of rectangular profiles partitioned into large units by cross-walls, and layers of orthogonally arranged fibres. The inorganic aragonite components were found, in contrast, to consist of two structurally different elements namely, highly ordered crystals with a prismatic-like morphology and smaller needle-like crystallites. Two morphologically distinct cell types, columnar opercular rim and cuboidal opercular plate cells, are responsible for the formation of the opercular plate. Both possess membrane-bound bodies containing filamentous material. However, in addition, membrane-bound bodies, containing calcium carbonate crystals, are found in some cells. Such bodies are seen to be closely related to the Golgi system. Based on the cytoarchitecture of the cells, the mechanisms involved in the formation and calcification of the opercular plate are discussed.     

Three nephromixial patterns in polychaete species currently assigned to the genus Pista (Annelida,Terebellidae)

A comparative morphological study of nephromixial systems in three Californian terebellid polchaetes currently assigned to the genus Pista shows that P. fimbriata has all attributes of the generic type, but that P. pacifica and P. elongata differ markedly. The features of typical Pista include (among others): two pairs of usually unequal branchiae, long-handled anterior uncini (hooks) of crested avicular (bird-head-like) from, muddy, unornamented tubes, one pair of anterior excretory nephromixia (ENMX), and two pairs of separate thoracic reproductive nephromixia (RNMX) with genital papillae on segments VI and VII. A review indicates that P. fimbriata shares these typical features with practically all adequately described Pista species. However, P. pacifica and P. elongata possess three pairs of branchiae, long-handled uncini of distinctive crochet-like form, and membranous tubes with apertural hoods. Both have two pairs of ENMX, the first supplied by one pair of ciliated renal funnels, the second by two pairs of such funnels. But they differ in their RNMX: P. pacifica has three pairs of complex RNMX, those on each side united by a common duct. P. elongata has 11–13 pairs of simple RNMX united by common ducts. Although these species do not fit into Pista, no genus has been found to accommodate them. Generic placement is complicated by the fact that no instances of intra-generic nephromixial variation have been reported in the Terebellidae, although inter-generic variation is well known. If they are congeneric, this would be the first example of intrageneric RNMX variation in Terebellidae. But if assigned to separate genera or subgenera on the basis of their RNMX, their similarity of anterior uncini might be attributable to parallel or convergent adaptation to life in comparable tubes. More evidence, including molecular analysis, is needed for phylogenetic studies of Terebellidae. © 1992 Wiley-Liss, Inc.     

Purification and properties of the arginase from Jerusalem artichoke tubers

Arginase [l-arginine amidinohydrolase] in Jerusalem artichoke tubers occurs in a particulate fraction from which it was released in active form by detergent treatment. The particulate enzyme was purified 450-fold with ca 3% yield. The enzyme has a MW of ca 140 000 and pI of 5.3. The enzyme required Mn²⁺ for activity and was unstable when Mn²⁺ was removed. In tissue extracts the K_m for arginine was ca 1OmM, but when purified the K_m (arginine) was 145 mM. The artichoke arginase was shown to be more substrate specific than other plant and animal arginases which have been described, and to be very sensitive to competitive inhibition by indospicine, ornithine and citrulline.     

First record of an actinosporean (Myxozoa) in a marine polychaete annelid

The marine polychaete Nereis (Hediste) diversicolor (Annelida) from shallow water in the Oresund, Denmark, was found to be infected with an actinosporean stage of a myxozoan parasite. The body length of the pyriform actinospore is 12-16 microm and its maximum width is 10-12 microm. The spore is triangular in apical view, with the 3 spherical polar capsules distally. The spore is without caudal processes. Eight spores develop in each pansporocyst. Free spores and pansporocysts were found in the musculature and parapodia but not in the intestine. The myxosporean stage in fish is unknown. This is the first record of an actinosporean stage in a marine polychaete, but because marine oligochaetes are rare, compared with polychaetes, the latter are believed to play an important role as invertebrate (alternate) hosts in marine myxozoan life cycles.     

Monoamine-containing structures in the intestines of bivalve molluscs and a polychaete annelid revealed by fluorescence histochemistry

Summary Monoamine-containing elements in the intestines of Bivalvia and Polychaeta species have been found by use of histochemical fluorescence methods according to Falck and Furness. Catecholamine-containing perikarya and fibers are seen within the epithelium and subepithelial layers of the midgut of the bivalves Mytilus edulis, Mya arenaria, Arctica islandica, as well as the polychaete Harmothoe imbricata. In addition, intraepithelial cell bodies and fibers containing serotonin-like substance are present in Mytilus edulis. Results obtained with the Furness method, applied earlier to vertebrates, correlate with those obtained with the Falck method.     

Characterization of a novel spirochete associated with the hydrothermal vent polychaete annelid, Alvinella pompejana

A highly integrated, morphologically diverse bacterial community is associated with the dorsal surface of Alvinella pompejana, a polychaetous annelid that inhabits active high-temperature deep-sea hydrothermal vent sites along the East Pacific Rise (EPR). Analysis of a previously prepared bacterial 16S ribosomal DNA (rDNA) library identified a spirochete most closely related to an endosymbiont of the oligochete Olavius loisae. This spirochete phylotype (spirochete A) comprised only 2.2% of the 16S rDNA clone library but appeared to be much more dominant when the same sample was analyzed by denaturing gradient gel electrophoresis (DGGE) and the terminal restriction fragment length polymorphism procedure (12 to 18%). PCR amplification of the community with spirochete-specific primers used in conjunction with DGGE analysis identified two spirochete phylotypes. The first spirochete was identical to spirochete A but was present in only one A. pompejana specimen. The second spirochete (spirochete B) was 84.5% similar to spirochete A and, more interestingly, was present in the epibiont communities of all of the A. pompejana specimens sampled throughout the geographic range of the worm (13 degrees N to 32 degrees S along the EPR). The sequence variation of the spirochete B phylotype was less than 3% for the range of A. pompejana specimens tested, suggesting that a single spirochete species was present in the A. pompejana epibiotic community. Additional analysis of the environments surrounding the worm revealed that spirochetes are a ubiquitous component of high-temperature vents and may play an important role in this unique ecosystem.