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The complete mitochondrial genome of the orbiniid polychaete Orbinia latreillii (Annelida, Orbiniidae)--A novel gene order for Annelida and implications for annelid phylogeny.

Relationships of annelid subtaxa are controversially discussed and additional markers are necessarily needed to get further insights into their evolution. Due to their high content of information, mitochondrial genomes have been proven very useful in phylogenetic analyses. Whereas many complete mitochondrial genomes of arthropods are available, lophotrochozoan taxa are only scarcely represented and this is especially true for annelids. Here we present the complete mitochondrial genome of the orbiniid polychaete Orbinia latreillii. The circular genome is 15,558 bp in size and contains the same 37 genes as found in most other metazoans. As in the case for all studied annelids all genes are transcribed from the same strand. Compared with the known data from other annelids at least five gene translocations must be hypothesized for O. latreillii. A comparison of the available data shows that gene translocations within Annelida seem to be less frequent than in molluscs, but more frequent as previously assumed. Phylogenetic analyses of mitochondrial DNA sequence data and amino acid data support an inclusion of Sipuncula within Annelida and a closer relationship to orbiniids is recovered for this taxon.

Animals↗

Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: sequence and gene arrangement comparisons indicate that Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa.

We report a contiguous region of more than half (> 7,500 nt) of the mitochondrial genomes for Platynereis dumerii (Annelida: Polychaeta), Helobdella robusta (Annelida: Hirudinida), and Galathealinum brachiosum (Pogonophora: Perviata). The relative arrangements of all 22 genes identified for Helobdella and Galathealinum are identical to one another and to their arrangements in the mtDNA of the previously studied oligochaete annelid Lumbricus. In contrast, Platynereis differs from these taxa in the positions of several tRNA genes and in having two additional tRNA genes (trnC and trnM) and a large noncoding sequence in this region. Comparisons of relative gene arrangements and of the nucleotide and inferred amino acid sequences among these and other published taxa provide strong support for an annelid-mollusk clade that excludes arthropods, and for the inclusion of pogonophorans within Annelida, rather than giving them separate phylum status. Gene arrangement comparisons include the first use of a recently described method on previously unpublished data. Although a variety of alternative initiation codons are typically used by mitochondrial protein-encoding genes, ATG appears to be the initiator for all but one reported here. The large noncoding region (1,091 nt) identified in Platynereis has no significant sequence similarity to the noncoding region of Lumbricus, although each contains runs of TA dinucleotides and of homopolymers, which could potentially serve as signaling elements. There is strong bias for synonymous codon usage in Helobdella and especially in Galathealinum. In this latter taxon, 5 codons are completely unused, 13 are used three or fewer times, and G appears at third codon positions in only 26 of the 2,236 codons. Nucleotide composition bias appears to influence amino acid composition of the proteins.

Amino Acid Sequence↗

Close phylogenetic relationship between Vestimentifera (tube worms) and Annelida revealed by the amino acid sequence of elongation factor-1 alpha.

To clarify the phylogenetic position of Vestimentifera (tube worms), 346-bp fragments of the elongation factor-1 alpha (EF-1 alpha) gene (939-1286 according to the numbering of the human gene) of a vestimentiferan, Lamellibrachia sp., a sternaspid polychaete, Sternaspis scutata, an earthworm, Pheretima sp., and a gastropod, Alviniconcha hessleri, were sequenced. From the amino acid sequences of these EF-1 alpha, and those of two other vertebrates and two arthropods, phylogenetic relationships were deduced by the maximum likelihood (ML) method, by which the phylogenetic tree can be inferred without assuming constancy of the molecular evolutionary rate. For the ML tree and all of seven alternative trees, whose log-likelihoods could not be discriminated from that of the ML tree by the criterion of the standard error, the vestimentiferan, the polychaete, and the oligochaete formed a clade, excluding the arthropods and the gastropod as outgroups. This result is convincing evidence that Vestimentifera are protostomes that are closely related to Annelida. The ML tree suggests that Vestimentifera are more closely related to Polychaeta than to Oligochaeta, though the data were not sufficient to discriminate these three groups at a significant level. From recent evidence such as morphological characteristics and molecular information, it may safely be said that vestimentiferans should be included in the Annelida provided this phylum contains polychaetes and oligochaetes.

Amino Acid Sequence↗

On the status of the study of invertebrate neurons in tissue culture--phyla Mollusca and Annelida.

The utilization of tissue culture in neurobiological studies is discussed for all phyla phylogenetically preceding Phylum Arthropoda. Only two phyla, Mollusca and Annelida, are represented in such studies. The members of Phylum Mollusca which have been so investigated are Aplysia, Helisoma and Lymnaea. The mollusc Aplysia has been used to investigate several processes, including neurosecretion, synaptic transmission and synaptogenesis. Helisoma was employed to study factors regulating neurite growth and the specificity of synapse formation; mechanisms of neurite growth were investigated in the snail Lymnaea. The only member of Phylum Annelida involved in appropriate studies has been the leech Hirudo. This organism was used to investigate axonal regeneration and synaptic mechanisms.

Animals↗

Mitochondrial genomes of Clymenella torquata (Maldanidae) and Riftia pachyptila (Siboglinidae): evidence for conserved gene order in annelida.

Mitochondrial genomes are useful tools for inferring evolutionary history. However, many taxa are poorly represented by available data. Thus, to further understand the phylogenetic potential of complete mitochondrial genome sequence data in Annelida (segmented worms), we examined the complete mitochondrial sequence for Clymenella torquata (Maldanidae) and an estimated 80% of the sequence of Riftia pachyptila (Siboglinidae). These genomes have remarkably similar gene orders to previously published annelid genomes, suggesting that gene order is conserved across annelids. This result is interesting, given the high variation seen in the closely related Mollusca and Brachiopoda. Phylogenetic analyses of DNA sequence, amino acid sequence, and gene order all support the recent hypothesis that Sipuncula and Annelida are closely related. Our findings suggest that gene order data is of limited utility in annelids but that sequence data holds promise. Additionally, these genomes show AT bias (approximately 66%) and codon usage biases but have a typical gene complement for bilaterian mitochondrial genomes.

Animals↗

The nucleotide sequences of 5S rRNAs from two Annelida species, Perinereis brevicirris and Sabellastarte japonica, and an Echiura species, Urechis unicinctus.

The nucleotide sequences of 5S rRNAs from two Annelida species, Perinereis brevicirris and Sabellastarte japonica, and an Echiura species, Urechis unicinctus have been determined. Their sequences are all 120 nucleotides long. The sequence similarity percents are 88% (Perinereis/Sabellastarte), 90% (Sabellastarte/Urechis) and 92% (Perinereis/Urechis), indicating that the Echiura is indistinguishable from the Annelida by their 5S rRNAs. The 5S rRNA sequences from the Annelida/Echiura are most related to those from the Nemertinea (87%), the Mollusca (87%) and the Rotifera (88%).

Animals↗

Immunohistochemical localization of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta).

The present immunohistochemical study provides the first evidence of the presence of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta) a lower invertebrate. The entire epidermis was labelled for calmodulin which is in agreement with its ubiquitous occurrence. Immunopositivity for calbindin D28K was limited to mucous cells, while that for S-100 protein was present only in neuroendocrine-like small granular cells. Finally, labelling for parvalbumin was specifically present in the subcutaneous nerve plexus. S-100 protein is considered to be a marker of neuroendocrine cells, at least in lower invertebrates such as Annelida. Although calbindin D28K is considered to be a marker of these cells in vertebrates, the same function cannot be attributed in Lumbricus terrestris. However, we can conclude that S-100 protein, as a regulatory protein, is phylogenetically older than calbindin D28K. We assume that the latter has an autoregulatory function in secretory processes. In agreement with previous data, we suggest that small granular cells exert a paracrine action in osmoregulatory and secretory processes.

Animals↗

Ultrastructure and localization of a visual Gq protein in hypertrophied epitoke ocelli of Perinereis brevicirris (Polychaeta, Annelida).

Functional ultrastructural changes in the rhabdomeric photoreceptors of the cerebral ocelli are described for normal and sexually mature (epitoke) Perinereis brevicirris (Polychaeta, Annelida). With sexual maturation, the cerebral ocelli hypertrophied, increasing in volume to 5.5 times that of ocelli in the normal state, and the thickness of the retinal layer increased up to 10 times. Perinereis ocelli have a pigmented retinal layer consisting of at least two cell types: photoreceptor cell (PR) and pigmented supporting cells (PS). In epitoke ocelli, PR bear well-developed rhabdomeric microvilli, multilamellar bodies, and numerous cytoplasmic membranous structures, including vesicles, smooth endoplasmic reticulum, and secondary lysosomes. Localization of a visual Gq protein in the ocelli was studied with anti-GqC antibody. The antibody strongly labeled not only microvilli and multilamellar bodies throughout the retinal layer, but also secondary lysosomes and vesicles in the cytoplasm of the PR in the epitoke ocelli, although labeling was observed only in the microvilli and multilamellar bodies in normal ocelli. Reverse transcription/polymerase chain reaction analysis revealed that the amount of G protein alpha subunit mRNA in the epitoke head increased by roughly twice that of the normal head. Since Gq protein is essential for phototransduction in Perinereis ocelli, these results suggest that the sites are involved in photoreceptive membrane turnover, which occurs much more extensively in epitoke ocelli. Thus, epitoke ocelli may represent a model system for studying rhabdomeric photoreceptive membrane turnover.

Animals↗

An ESR study of nitrosyl-Aplysia brasiliana myoglobin and nitrosyl annelidae Glossoscolex paulistus erythrocruorin.

The nitrosyl derivatives of Annelidae Glossoscolex paulistus hemoglobin (an earth worm erythrocruorin (Ec AGp)) and Aplysia brasiliana myoglobin (Mb Apb) are studied using ESR spectroscopy. These two proteins have a quite similar ESR spectra at 100 K, but a different temperature behaviour. The temperature dependence of the nitrosyl Mb Apb spectrum is in good agreement with the Boltzmann distribution. In the case of nitrosyl-Ec AGp, the results are explained by the existence of two types of spectrum in thermodynamic equilibrium, with delta H = 9.08 kJ/mol, delta S = 47.15 J/mol and T1/2 = 193 K. There is a great similarity of the nitrosyl-Ec AGp spectra with those reported for elephant myoglobin, suggesting the presence of the same heme environment with a glutamine residue in the distal site. The pH dependence of the spectrum of nitrosyl-Mb Apb shows that the affinity of nitrosyl binding is higher at high pH (7.3) than at low pH (4.6). The ESR parameters are the same for these two pH values.

Animals↗

Diverse methyl-end desaturases enable LC-PUFA biosynthesis across Annelida.

Recent studies have demonstrated that annelids possess front-end desaturases and elongases involved in the biosynthesis of physiologically important long-chain polyunsaturated fatty acids (LC-PUFA). However, methyl-end desaturases (ωx desaturases), enzymes that play a central role in the de novo biosynthesis of polyunsaturated fatty acids and their subsequent conversion into LC-PUFA, have so far been reported in only a limited number of polychaete species. To advance our understanding of ωx desaturases across the phylum Annelida, this study performed a comprehensive molecular and functional characterisation of these enzymes in the major annelid taxa Polychaeta, Clitellata and Sipuncula, encompassing species from diverse taxonomic groups and ecological niches. A total of 110 ωx desaturase sequences were retrieved from available genomes and transcriptomes. The number of ωx desaturase genes varied among species, ranging from zero to four copies. Phylogenetic analyses revealed that annelid ωx desaturases are classified into two major clades, designated Cluster A and Cluster B. Analyses of histidine-box motifs and exon-intron organisation revealed conserved patterns within each clade. Functional characterisation of ωx desaturases from Eisenia fetida (Clitellata) and Sipunculus nudus (Sipuncula), together with previously published data from polychaetes, demonstrated that annelids generally possess two ωx desaturases, one with Δ12 desaturase activity and another with ω3 desaturase activity. Collectively, these results demonstrate that annelids possess a phylogenetically and functionally diverse ωx desaturase repertoire that underpins their LC-PUFA biosynthetic capacity and may reflect adaptation to different ecological and nutritional environments.

Animals↗

The structure of the ovary and oogenesis in the earthworm, Dendrobaena veneta (Annelida, Clitellata).

The structure of the ovary and the type of oogenesis were determined in the earthworm Dendrobaena veneta (Oligochaeta, Haplotaxida, Lumbricidae) with histological, electron-microscopic and immunocytochemical methods. In this species the ovary is of the alimentary, nutrimentary type because it contains oocytes and the nurse cells (trophocytes). The ovarian stroma is built by somatic cells, the processes of which are connected to each other via numerous desmosomes. The somatic cells and their processes envelop the germ cells tightly and play a supportive role. Oogonia, oocytes and trophocytes are arranged in distinct zones in the ovary. Trophocytes form chains of cells, which are interconnected by intercellular bridges. Numerous microtubules are located within the latter. The oocytes are distally arranged in the ovary. Vitellogenesis involves both auto- and heterosyntheses. The results obtained were compared with the reports on oogenesis in other representatives of Annelida.

Animals↗

Phylogeny and evolution of reproductive modes in Autolytinae (Syllidae, Annelida).

The phylogeny of 31 autolytine taxa (Syllidae, Polychaeta, and Annelida) was estimated based on 16S rDNA and 18S rDNA sequences. Outgroups included 12 non-autolytine syllids and four other annelids from related groups. The phylogeny was used to trace the evolution of the various reproductive strategies (i.e., epigamy, anterior and posterior scissiparity, and gemmiparity) within the group, and it will also serve as a basis for a forthcoming revision of autolytine taxonomy. The two genes were analysed both separately and in combination using parsimony, maximum likelihood, and Bayesian inference. Regardless of method used the combined analysis supported a division of Autolytinae into three major clades: one with epigamous Autolytus; a second comprising Autolytus and Myrianida with posterior scissiparity and gemmiparity; and a third containing Proceraea, Procerastea, and Virchowia with anterior scissiparity. The relationship between these three groups is uncertain. Ancestral reproductive states were reconstructed with parsimony and maximum likelihood, and the results unequivocally support epigamy as the plesiomorphic reproductive mode in Syllidae, and that schizogamy in Syllinae and Autolytinae are separate events. The evolution of reproductive traits is ambiguous within Autolytinae, and either of the different reproductive modes could represent the ancestral state.

Animals↗

Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG)n and (GATA)n repeats in the leech Haemopis sanguisuga (L.) (Annelida: Hirudinea).

In the present paper the chromosome complement (n = 13; 2n = 26) of the common leech Haemopis sanguisuga (L.) (Annelida: Hirudinea: Hirudinidae) was analyzed using banding techniques and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG)n and (GATA)n]. FISH with the rDNA probe consistently mapped major ribosomal clusters (18S-28S rDNA) in the pericentromeric region of one large metacentric chromosome pair; this region, which consisted of heterochromatin rich in GC base pairs, was preferentially stained by silver nitrate (Ag-NOR). The (TTAGGG)n telomeric probe was hybridized with the termini of nearly all chromosomes, whereas the (GATA)n probe did not label any chromosome areas.

Animals↗

The mitochondrial genome of the Sipunculid Phascolopsis gouldii supports its association with Annelida rather than Mollusca.

We determined the sequence of about half (7,470 nt) of the mitochondrial genome of the sipunculid Phascolopsis gouldii, the first representative of this phylum to be so studied. All of the 19 identified genes are transcribed from the same DNA strand. The arrangement of these genes is remarkably similar to that of the oligochaete annelid Lumbricus terrestris. Comparison of both the inferred amino acid sequences and the gene arrangements of a variety of diverse metazoan taxa reveals that the phylum Sipuncula is more closely related to Annelida than to Mollusca. This requires reinterpretation of the homology of several embryological features and of patterns of animal body plan evolution.

Amino Acid Sequence↗

Slanted centriole and transient anchoring apparatus during spermiogenesis of an Oligochaete (Annelida).

An electron microscopic analysis of Tubifex tubifex (Annelida, Oligochaeta) spermiogenesis has revealed the presence of a slanted basal body during the early stages: the centriole has the shape of a cylinder obliquely cut at one extremity. In these stages an anchoring apparatus is also present, similar to the one of primitive spermatozoa, and disappearing in the mature sperm. The slanted centriole in cross section gives images of "incomplete" centrioles similar to the ones reported in the literature and interpreted as images of centriologenesis. The transient anchoring apparatus is interpreted as a rudimentary organelle used during spermiogenesis to maintain the centriole in a correct position. This function in the mature spermatozoon is performed by the mitochondria.

Animals↗

Fruiting-inducing activity and antifungal properties of lipid components in members of Annelida.

Fruiting-inducing activity and antifungal properties of lipid components in the phylum Annelida were examined. Some amphoteric cerebrosides carrying a phosphocholine group showed fruiting-inducing activity on Schizophyllum commune, and one of them possessed activity comparable to that of Sch II, the most potent substance known. Furthermore, alkyl lysophosphatidylcholines were found to have an inhibitory effect on the growth of phytopathogenic fungi, Alternaria kikuchiana and Phomopsis mali. The relationship between structure and biological activities is discussed.

Alternaria↗

Holopelagic Poeobius meseres ("Poeobiidae," Annelida) is derived from benthic flabelligerid worms.

Phylogenetic relationships among and within the more than 70 recognized families of Annelida are poorly understood. In some cases, such as the monotypic Poeobiidae, derived morphology hinders the ability to find convincing synapomorphies that help elucidate evolutionary origins. In such cases, molecular data can be useful. Poeobiidae consists of the holopelagic polychaete Poeobius meseres, which is typically found in midwater depths off California. Morphologists have speculated that it is close to or within Flabelligeridae, but definitive evidence was lacking. Herein we use maximum likelihood phylogenetic reconstruction methods to examine the nuclear 18S rDNA (SSU) gene and the mitochondrial cytochrome b gene. Our results strongly support the hypothesis that P. meseres is a highly derived flabelligerid annelid closely related to Therochaeta. Thus, Poeobiidae is a junior synonym for Flabelligeridae. This result raises interesting questions about the evolution of the holopelagic P. meseres from a benthic ancestral flabelligerid.

Animals↗

Nereis virens (Annelida: Polychaeta) is not an adequate sentinel species to assess the genotoxic risk (comet assay) of PAH exposure to the environment.

Polychaetes, because of their bioturbation capacity, play an important role in the distribution of anthropogenic contaminants (including polycyclic aromatic hydrocarbons [PAHs]) throughout the sediments. In this work the use of Nereis virens (Annelida: Polychaeta) as a bioindicator to assess the genotoxic risk of PAH exposure for the environment was evaluated. For this purpose the alkaline single cell gel electrophoresis [comet] assay was applied on the coelomocytes of in vivo exposed Nereis virens. Benzo[a]pyrene (B[a]P) was chosen because it is classified by the IARC (International Agency for Research on Cancer) as "probably carcinogenic to humans" and because its mechanisms of action are well-known. Nereis virens was exposed to B[a]P in concentrations of 0.3, 0.6, 10, 20, 35 and 45 mg/ml by an intracoelomic injection of B[a]P (20 microliters) dissolved in dimethyl sulphoxide (DMSO). A solvent control with DMSO, a positive control with ethyl methane sulphonate (EMS) (12.1 mg/ml) and a negative control were included in each experiment. For each treatment four animals were analysed. After 1 hr treatment coelomocytes were harvested by puncturing the coelomic cavity with a sharpened Pasteur pipette, mixed with 0.5% low melting point agarose and sandwiched between two other gel layers. Ethidium bromide stained nuclei were analysed for tail length and tail moment. 12.1 mg/ml EMS, pure DMSO (98.9%) and B[a]P in all tested concentrations induced a statistically significant increase of DNA single strand breaks in the comet assay. The effect of B[a]P, however, was only at the highest concentration (45 mg/ml) significantly stronger than the effect of DMSO alone. Although a relatively large heterogeneity in the results could be observed, these experiments clearly showed that Nereis virens is not suited as a sentinel species for the assessment of the genotoxic risk of PAH exposure because this species seems to be very resistant to benzo[a]pyrene.

Animals↗