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De Novo Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Mesenchytraeus (Annelida, Enchytraeidae).

The Changbai Mountain range is one of the key glacial refugia in Northeast Asia. Mesenchytraeus exhibits high species diversity, strong endemism, and widespread cryptic species in this region, for which mitogenomes provide useful molecular markers for exploring cryptic species complexes. This makes Mesenchytraeus an ideal model for studying mitogenome evolution among closely related lineages; however, no mitogenome data have been reported for this genus to date. In this study, we performed de novo assembly, annotation, and comparative analysis of the mitogenomes of 13 Mesenchytraeus species (14 individuals) from Changbai Mountain. All mitogenomes are typical circular molecules containing 37 genes, but putative control regions are rearranged and consistently located between ATP6 and trnR. All species exhibit annelid-specific strand nucleotide biases, characterized by negative GC skew and near-zero AT skew. Codon usage analysis reveals that codon families with wobble U are significantly biased toward mtDNA codons, whereas those with wobble C or G are biased toward non-mtDNA codons, suggesting a conserved mitochondrial codon usage pattern in annelids. All tRNAs form typical cloverleaf secondary structures except trnS2, which lacks the D-stem and the dihydrouridine (DHU) arm in some species. The putative control regions commonly contain complex palindromic repeats, hairpins, and repetitive elements, and may harbor dual replication origins. Phylogenetic analyses support the monophyly of Mesenchytraeus and reveal significant molecular divergence among morphologically cryptic species. This study provides the first mitogenome dataset for Mesenchytraeus and offers new insights into the evolution and replication mechanisms of mitogenomes in Clitellata and broader Annelida.

Mesenchytraeus↗

Structure of the germinal vesicle during oogenesis in leech Glossiphonia heteroclita (Annelida, Hirudinea, Rhynchobdellida).

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.

Animals↗

Occurrence and coexistence in Nereis diversicolor O.F. Müller (Annelida Polychaeta) of substances immunologically related to vertebrate neuropeptides.

Numerous immunochemical and immunohistochemical studies have shown a wide distribution of several families of neuropeptides in invertebrates as well as vertebrates. There are relatively few data available for Annelida: Polychaeta. Therefore, we undertook an immunohistochemical investigation in the marine worm Nereis. Among the vertebrate type antibodies tested, those against met-enkephalin, LH-RH, vasopressin, oxytocin and ACTH had negative or only very slight effects. Slight to moderate reactions were obtained for VIP, SRIF, CRF, GRF, and leu-enkephalin. Moderate to very strong responses were found with anti-CCK/gastrin, -substance P, and -beta-MSH sera. Immunopositive reactions were usually observed in the entire CNS (except, until now, in neurosecretory cells, type II, in nuclei 20, and in nerve fibres located in the infracerebral neurohemal area). The immunoreactivity was, however, more or less abundant according to different CNS regions. For example, it appeared that the immunostaining for CRF is more important in the VNC while the leu-enkephalin family is more abundant in the brain (particularly in fuchsinophilic neurosecretory cells, type I, in nuclei 20). Moreover, several vertebrate type peptides (such as CRF/GRF and CCK/gastrin) may coexist in a single neurone. Several antisera may elicit a positive reaction in some specific area (for example, substance P in the nuchal organ; SRIF in oocytes; CCK/gastrin in the gastrointestinal tract). Nothing is known about the role of the different substances immunologically detected in Nereis. It is suggested that CCK/gastrin-, beta-MSH- and substance P-like materials transmit external stimuli to neurosecretory centres located in the caudal part of the brain.

Animals↗

Analysis and reconstruction of unusual obliquely striated fibres in Lumbriculids (Annelida, Oligochaeta).

The longitudinal muscle fibres of Lumbriculids (Annelida, Oligochaeta) have been studied by means of electron microscopy. These fibres are obliquely striated (flattened circomyarian) and possess Z-elements and sarcotubules which frequently cross the centre of the fibre connecting the cell surface on the opposite sides. The myosin filaments have, in cross-section, a rectangular profile with the long side parallel to the fibre surface; they look wavy (S-shaped) on the XZ plane, thus crossing the whole thickness of the sarcomere. The three-dimensional interpretation of the contractile apparatus was facilitated by the comparison of the micrographs from ad hoc sections of the three spatial planes with corresponding calculated "sections', obtained with a dynamic computer model. The real periodicity of the different contractile structures has been determined and the wavy shape of the thick filaments on the XZ plane has been demonstrated by indirect proofs: computed images in which rectangular filaments are parallel to the fibre axis cannot be made to yield a situation comparable with real images. Furthermore, the distances (centre to centre) between thick filaments in fibres at different states of contraction are so distributed in each "sarcomere' as to be compatible only with the wavy filaments.

Actin Cytoskeleton↗

Organization and nucleotide sequence of the cluster of five histone genes in the polichaete worm Chaetopterus variopedatus: first record of a H1 histone gene in the phylum Annelida.

Histone genes were identified and their nucleotide sequences were determined in the polychaete marine worm Chaetopterus variopedatus. The genes are organized in about 390 clusters of 7.3 kbp. Each cluster contains one copy of the five histone genes. The H1 histone gene present in the clusters is the first ever isolated in the phylum Annelida. The cluster has the unique peculiarity that all genes contain both the replication-dependent and the replication-independent 3' mRNA termination signals. Despite the differences in cluster organization and transcription polarity of the individual histone genes between C. variopedatus and Platynereis dumerilii, the other annelid in which histone genes have been studied, phylogenetic analysis of the encoded amino acid sequences clearly groups together those two organisms in a tree in which the other studied worms find closely related positions on the same evolutionary branch.

Amino Acid Sequence↗

Cloning and molecular characterization of the first innexin of the phylum annelida-expression of the gene during development.

A novel member of the innexin family (cv-inx) has been isolated from the annelid polychaete worm Chaetopterus variopedatus using a PCR approach on genomic DNA and sequence analysis on genomic DNA clones. The gene is present in a HindIII-HindIII segment of 2250 bp containing an uninterrupted open reading frame of 1196 bp encoding a protein of 399 amino acids. The predicted protein shows the typical structural features of innexins and consensus sites for phosphorylation. Analyses on genomic DNA demonstrate that cv-inx is a single copy gene with no introns in the coding region, exactly corresponding to the cDNA sequence. The gene expression is regulated during development as shown by Northern blots analyses of the RNA and by immunoreaction with antibodies against the protein at several embryonic stages. The finding of an innexin in the phylum Annelida, outside of the Ecdysozoa clade, and its peculiar gene structure suggest the necessity to reconsider the current hypothesis on the origin and evolution of gap junctional proteins.

Amino Acid Sequence↗

Localization of a visual Gq protein in the photoreceptors of a polychaete, Perinereis brevicirris (Annelida).

Heterotrimeric GTP-binding proteins (G proteins) play an important role in phototransduction. The presence of G-protein subclasses has been reported in photoreceptive membranes, e.g., the Gi subgroup (transducin) in vertebrate rods, and the Gq subgroup in the eyes of the Arthropoda and the Mollusca. We examined the immunoreactivity and distribution of a Gq homologue in the cerebral ocelli of Perinereis brevicirris (Polychaeta, Annelida) using an anti-GqC antibody raised against a conserved sequence at the C-terminal of the alpha-subunit of Gq (Gq-alpha). The anti-GqC antibody labeled a 48-kDa band on the Western blot of proteins from the Perinereis ocelli. The anti-GtC antibody, which is raised against the C-terminal sequence of bovine transducin alpha-subunit (Gt-alpha), did not cross-react to the ocellar proteins of Perinereis. The rhabdomeric layers of the anterior and posterior ocelli were strongly labeled by anti-GqC on light-microscopic immunohistology. Immunoelectron microscopy showed that the Gq molecules were specifically localized in the photoreceptive membrane of the rhabdomeric microvilli. These results suggest that the Gq protein plays a role in the phototransduction of the Perinereis ocelli.

Amino Acid Sequence↗

Bioavailability and toxicity of pentachlorophenol in contaminated soil evaluated on coelomocytes of Eisenia andrei (Annelida: Lumbricidae).

Pentachlorophenol (PCP) is widely distributed and highly persistent in soil, and represents a threat to the health of ecosystems. The present study aimed to assess the toxicity and bioavailability of PCP in soils as a function of different aging periods with the attempt to select a good toxicological assay for Eisenia andrei Bouché (Annelida: Lumbricidae). The experiments were performed on soil contaminated with PCP at 15 and 150ppm. After different aging periods (20, 60 and 120 days from spiking), bioavailability and toxicity were evaluated on E. andrei kept for 7 and 14 days in treated soils. The actual bioavailability decreased in relation to the aging for both PCP concentrations. No membrane damage was observed on coelomocytes collected by ethanol extrusion. Modifications in distribution of coelomocyte subpopulations were detected by flow cytometry on samples aged for 60 and 120 days at 150ppm PCP contamination. The reduction of lysosomal membrane stability, measured by neutral red retention time, was observed in all treatments. Worm mortality increased with aging in soils spiked with 150ppm of PCP. In conclusion, aging did not seem to reduce PCP cytotoxicity. This is the first report on in vivo toxicity of PCP evaluated on coelomocytes of E. andrei using different assays.

Animals↗

Clearance rates of Sabella spallanzanii and Branchiomma luctuosum (Annelida: Polychaeta) on a pure culture of Vibrio alginolyticus.

The influence exerted by filter-feeding activity on bacterial density by two sabellid species from the Mediterranean Sea (Ionian Sea, Italy), Branchiomma luctuosum Grube and Sabella spallanzanii Gmelin (Annelida: Polychaeta) was investigated. Clearance rates and retention efficiencies were estimated utilizing the species Vibrio alginolyticus selected on account of previous field studies and its importance in fish culture pathogenicity. The Cmax was 43.2+/-2.63 L h(-1) g(-1) DW for B. luctuosum and 12.4+/-2.22 L h(-1) g(-1) DW for S. spallanzanii. The Retention efficiency was 98% corresponding to a removed bacterial biomass of 44.8+7.88 microgC L(-1) g(-1) DW for B. luctuosum and 70% corresponding to a bacterial biomass of 23.8+2.95 microgC L(-1) g(-1) DW for S. spallanzanii. Maximum retention was recorded after 20 min for the first species and after 30 min for the second one. Present laboratory experiments represent a contribution to the knowledge of the filtration activity of the two polychaetes, characterizing the filtration process on bacterioplankton. Both species resulted extremely efficient in removing V. alginolyticus from seawater in experimental tanks, thus confirming the previous data from the field studies and suggesting their employment as biofilters of microbially contaminated waters in intensive aquaculture.

Animals↗

Calcium phosphate granules in muscle cells of Nephtys (Annelida, Polychaeta)--a novel skeleton?

Analyses of various marine polychaete worms (phylum Annelida) reveal that members of the family Nephtyidae are exceptional in containing very high concentrations of calcium and phosphorus. We report here that in the genus Nephtys, the two elements form rounded granules packing the sarcoplasmic core of obliquely striated muscle cells. Intracellular calcium granules rich in phosphorus are found throughout the animal kingdom and occur in a wide range of tissues. Although some examples of granules in muscle tissue have been recorded (as in Syllis proventriculus), as far as we are aware, none appears in the abundance found in Nephtys.

Animals↗

Physical mapping of rDNA genes, (TTAGGG)n telomeric sequence and other karyological features in two earthworms of the family Lumbricidae (Annelida: Oligochaeta).

A cytogenetical study was carried out on the chromosomes and nuclear DNA amounts of the terrestrial earthworms Octodrilus complanatus and Eisenia foetida (Annelida: Oligochaeta: Lumbricidae). Chromosomes were studied using Giemsa staining, banding methods and fluorescent in situ hybridization (FISH) with two repetitive DNA probes [rDNA and (TTAGGG)n]. rDNA FISH and silver staining consistently identified one chromosome pair per spread in both species. The telomeric sequence (TTAGGG)n hybridized with termini of all the chromosomes in both earthworms. Flow cytometry DNA assays showed that O. complanatus and E. foetida had different nuclear DNA contents (2C value=1.72 and=1.40 pg, respectively) but very similar base composition in their genomes.

Animals↗

Novel galactose-binding proteins in Annelida. Characterization of 29-kDa tandem repeat-type lectins from the earthworm Lumbricus terrestris.

Novel type lectins were found in the phylum Annelida, i.e. in the earthworm, tubifex, leech, and lugworm. The lectins (29-31 kDa) were extracted from the worms without the use of detergent and purified by affinity chromatography on asialofetuin-agarose. On the basis of the partial primary structures of the earthworm Lumbricus terrestris 29-kDa lectin (EW29), degenerate primers were synthesized for use in the reverse transcriptase-polymerase chain reaction. An amplified 155-base pair fragment was used to screen a cDNA library. Four types of full-length clones were obtained, all of which encoded 260 amino acids, but which were found to differ at 29 nucleotide positions. Since three of them resulted in non-silent substitutions, EW29 mRNA was considered to be a mixture of at least three distinct polynucleotides encoding the following proteins: Ala44-Gln197-Ile213 (clone 5), Gly44-Gln197-Val213 (clone 7), and Ala44-His197-Ile213 (clones 8 and 9; different at the nucleotide level, but encoding an identical polypeptide). Genomic polymerase chain reaction using DNA from a single worm revealed that the single worm already had four sets of cDNAs. The EW29 protein showed two features. First, the lectin was composed of two homologous domains (14,500 Da) showing 27% identity with each other. When each of the domains was separately expressed in Escherichia coli, the C-terminal domain was found to bind to asialofetuin-agarose as strongly as the whole protein, whereas the N-terminal domain did not bind and only retardation was observed. EW29 was found to exist as a monomer under non-denaturing conditions. It had significant hemagglutinating activity, which was inhibited by a wide range of galactose-containing saccharides. Second, EW29 contained multiple short conserved motifs, "Gly-X-X-X-Gln-X-Trp." Similar motifs have been found in many carbohydrate-recognizing proteins from an extensive variety of organisms, e.g. plant lectin ricin B-chain and Clostridium botulinum 33-kDa hemagglutinin. Therefore, these carbohydrate-recognition proteins appear to form a protein superfamily.

Amino Acid Sequence↗

Phylogeny of Eunicida (Annelida) and exploring data congruence using a partition addition bootstrap alteration (PABA) approach.

Even though relationships within Annelida are poorly understood, Eunicida is one of only a few major annelid lineages well supported by morphology. The seven recognized eunicid families possess sclerotized jaws that include mandibles and a maxillary apparatus. The maxillary apparatuses vary in shape and number of elements, and three main types are recognized in extant taxa: ctenognath, labidognath, and prionognath. Ctenognath jaws are usually considered to represent the plesiomorphic state of Eunicida, whereas taxa with labidognath and prionognath are thought to form a derived monophyletic assemblage. However, this hypothesis has never been tested in a statistical framework even though it holds considerable importance for understanding annelid phylogeny and possibly lophotrochozoan evolution because Eunicida has the best annelid fossil record. Therefore, we used maximum likelihood and Bayesian inference approaches to reconstruct Eunicida phylogeny using sequence data from nuclear 18S and 28S rDNA genes and mitochondrial 16S rDNA and cytochrome c oxidase subunit I genes. Additionally, we conducted three different tests to investigate suitability of combining data sets. Incongruence length difference (ILD) and Shimodaira-Hasegawa (SH) test comparisons of resultant trees under different data partitions have been widely used previously but do not give a good indication as to which nodes may be causing the conflict. Thus, we developed a partition addition bootstrap alteration (PABA) approach that evaluates congruence or conflict for any given node by determining how bootstrap scores are altered when different data partitions are added. PABA shows the contribution of each partition to the phylogeny obtained in the combined analysis. Generally, the ILD test performed worse than the other approaches in detecting incongruence. Both PABA and the SH approach indicated the 28S and COI data sets add conflicting signal, but PABA is more informative for elucidating which data partition may be misleading at a given node. All our analyses indicate that the monophyly of the labidognath/prionognath taxa and even a labidognath clade (i.e., a "Eunicidae"/Onuphidae/Lumbrineridae clade) is significantly rejected. We show that the definition of both the labidognath and ctenognath jaw type does not address adequately the variation within Eunicida and thus misleads our current evolutionary understanding. Based on the presented results a symmetric maxillary apparatus with a carrier and four to six maxillae is most likely the plesiomorphic condition for Eunicida. [COI; conflicting data; fossil record; ILD; Jaw Evolution; molecular phylogeny; rDNA; SH test.].

Animals↗

rDNA (18S-28S and 5S) colocalization and linkage between ribosomal genes and (TTAGGG)(n) telomeric sequence in the earthworm, Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), revealed by single- and double-color FISH.

Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes-5S rDNA, 18S-26S rDNA, and (TTAGGG)(n). Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S-26S rDNA as probes. Simultaneous (18S-26S)-5S and (18S-26S)-(TTAGGG)(n) FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.

Animals↗

Feeding behavior of the cirratulid Cirriformia filigera (Delle Chiaje, 1825) (Annelida: Polychaeta).

Observations of the feeding behavior of Cirriformia filigera (Delle Chiaje, 1825) (Annelida: Polychaeta) from the intertidal zone of São Francisco and Engenho D'água beaches (São Sebastião, State of São Paulo) were made in the laboratory. This species, like other cirratulids, is a deposit feeder, feeding mainly on sediment surface with the aid of its grooved and ciliated palps, which are used to capture food particles. The worm lies just beneath the substrate surface in a J-shaped tube. When feeding, it extends up to 4 palps over the sediment surface, capturing food particles which pass down the groove of each palp directly to the mouth. Only fine sand grains are ingested. The worm frequently extends 4 branchial filaments into the overlying water for aeration. When it moves with the prostomium sideways, it collects and transports sand grains that pass backwards along its ventral region until reaching the middle part of its body. Next, the parapodia and palps move the sand grains to the dorsal posterior end of the animal, covering this area with sand. Some sand grains are also ingested as the worm moves.

Animals↗

Sabellaria tottoriensis n. sp. (Annelida: Polychaeta: Sabellariidae) from shallow water off Tottori, the Sea of Japan.

A new species of the genus Sabellaria Lamarck, 1812 (Annelida: Polychaeta: Sabellariidae), is described from shallow water off Tottori, the Sea of Japan. Sabellaria tottoriensis n. sp., is gregarious with tubes constructed of sand and shell debris. The new species is distinguished by the character combination of 1 or 2 pairs of nuchal spines, two forms (long and short) of opercular paleae in the middle row, with the slender blades of long ones recurved outward. Detailed morphological features of the species are described and compared with other Japanese and worldwide congeners.

Animals↗

Some Nereididae (Annelida: Polychaeta) from the Pacific Coast of Costa Rica.

Fifteen species of Nereididae (Annelida: Polychaeta) belonging to nine genera are reported from the subtidal and intertidal of the Pacific Coast of Costa Rica. A new species, Nereis costaricaensis, is described. Laeonereis brunnea Hartmann-Schröder 1959 is removed from synonomy with L. culveri (Webster 1880) while Perinereis seridentata (Hartmann-Schröder 1959) is moved from the genus Neanthes based upon the presence of bar-shaped paragnaths on area VI of the proboscis. The presence of Neanthes micromma Harper 1979 is an extension of the range of this species from the Gulf of California and Neanthes roosevelti Hartman 1939 is the first record of this species since its description from the Galapagos. A taxonomic key to these nereidid species is included.

Animals↗

Capitellidae (Annelida: Polychaeta) from the Pacific Coast of Costa Rica.

Eleven species of Capitellidae (Annelida: Polychaeta) belonging to eight genera are reported from the subtidal and intertidal of the Pacific Coast of Costa Rica. Three species, Mediomastus ambiseta, M. californiensis and Notomastus hemipodus, are found in highest abundances and are widespread in subtidal and intertidal environments. A taxonomic key to these species is included.

Animals↗