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Molecular phylogeny of coleoid cephalopods (Mollusca: Cephalopoda) using a multigene approach; the effect of data partitioning on resolving phylogenies in a Bayesian framework.

The resolution of higher level phylogeny of the coleoid cephalopods (octopuses, squids, and cuttlefishes) has been hindered by homoplasy among morphological characters in conjunction with a very poor fossil record. Initial molecular studies, based primarily on small fragments of single mitochondrial genes, have produced little resolution of the deep relationships amongst coleoid cephalopod families. The present study investigated this issue using 3415 base pairs (bp) from three nuclear genes (octopine dehydrogenase, pax-6, and rhodopsin) and three mitochondrial genes (12S rDNA, 16S rDNA, and cytochrome oxidase I) from a total of 35 species (including representatives of each of the higher level taxa). Bayesian analyses were conducted on mitochondrial and nuclear genes separately and also all six genes together. Separate analyses were conducted with the data partitioned by gene, codon/rDNA, gene+codon/rDNA or not partitioned at all. In the majority of analyses partitioning the data by gene+codon was the appropriate model with partitioning by codon the second most selected model. In some instances the topology varied according to the model used. Relatively high posterior probabilities and high levels of congruence were present between the topologies resulting from the analysis of all Octopodiform (octopuses and vampire "squid") taxa for all six genes, and independently for the datasets of mitochondrial and nuclear genes. In contrast, the highest levels of resolution within the Decapodiformes (squids and cuttlefishes) resulted from analysis of nuclear genes alone. Different higher level Decapodiform topologies were obtained through the analysis of only the 1st+2nd codon positions of nuclear genes and of all three codon positions. It is notable that there is strong evidence of saturation among the 3rd codon positions within the Decapodiformes and this may contribute spurious signal. The results suggest that the Decapodiformes may have radiated earlier and/or had faster rates of evolution than the Octopodiformes. The following taxonomic conclusions are drawn from our analyses: (1) the order Octopoda and suborders Cirrata, Incirrata, and Oegopsida are monophyletic groups; (2) the family Spirulidae (Ram's horn squids) are the sister taxon to the family Sepiidae (cuttlefishes); (3) the family Octopodidae, as currently defined, is paraphyletic; (4) the superfamily Argonautoidea are basal within the suborder Incirrata; and (5) the benthic octopus genera Benthoctopus and Enteroctopus are sister taxa.

Animals↗

Long-term conservation of six duplicated structural genes in cephalopod mitochondrial genomes.

The complete nucleotide sequences of the mitochondrial (mt) genomes of three cephalopods, Octopus vulgaris (Octopodiformes, Octopoda, Incirrata), Todarodes pacificus (Decapodiformes, Oegopsida, Ommastrephidae), and Watasenia scintillans (Decapodiformes, Oegopsida, Enoploteuthidae), were determined. These three mt genomes encode the standard set of metazoan mt genes. However, W. scintillans and T. pacificus mt genomes share duplications of the longest noncoding region, three cytochrome oxidase subunit genes and two ATP synthase subunit genes, and the tRNA(Asp) gene. Southern hybridization analysis of the W. scintillans mt genome shows that this single genome carries both duplicated regions. The near-identical sequence of the duplicates suggests that there are certain concerted evolutionary mechanisms, at least in cephalopod mitochondria. Molecular phylogenetic analyses of mt protein genes are suggestive, although not statistically significantly so, of a monophyletic relationship between W. scintillans and T. pacificus.

Animals↗

Actin gene family evolution and the phylogeny of coleoid cephalopods (Mollusca: Cephalopoda).

Phylogenetic analysis conducted on a 784-bp fragment of 82 actin gene sequences of 44 coleoid cephalopod taxa, along with results obtained from genomic Southern blot analysis, confirmed the presence of at least three distinct actin loci in coleoids. Actin isoforms were characteri zed through phylogenetic analysis of representative cephalopod sequences from each of the three isoforms, along with translated actin cDNA sequences from a diverse array of metazoan taxa downloaded from GenBank. One of the three isoforms found in cephalopods was closely related to actin sequences expressed in the muscular tissues of other molluscs. A second isoform was most similar to cytoplasmic-specific actin amino acid sequences. The muscle type actins of molluscs were found to be distinct from those of arthropods, suggesting at least two independent derivations of muscle actins in the protostome lineage, although statistical support for this conclusion was lacking. Parsimony and maximum-likelihood analyses of two of the isoforms from which >30 orthologous coleoid sequences had been obtained (one of the cytoplasmic actins and the muscle actin) supported the monophyly of several higher-level coleoid taxa. These included the superorders Octopodiformes and Decapodiformes, the order Octopoda, the octopod suborder Incirrata, and the teuthoid suborder Myopsida. The monophyly of several taxonomic groups within the Decapodiformes was not supported, including the orders Teuthoidea and Sepioidea and the teuthoid suborder Oegopsida. Parametric bootstrap analysis conducted on the simulated cytoplasmic actin data set provided statistical support to reject the monophyly of the Sepioidea. Although parametric bootstrap analysis of the muscle actin isoform did not reject sepioid monophyly at the 5% level, the results (rejection at P: = 0.068) were certainly suggestive of sepioid nonmonophyly.

Actins↗

Neuropeptidergic control of the optic gland of Octopus vulgaris: FMRF-amide and GnRH immunoreactivity.

In cephalopods, the endocrine optic glands on the optic tract control the maturation of the gonads. The glands are innervated by the optic gland nerve, which originates in the central nervous system. To explore the involvement of neuropeptides in the nervous control of the optic gland of Octopus vulgaris, the presence and distribution of Phe-Met-Arg-Phe-NH2 (FMRF-amide)-like and gonadotropin releasing homone (GnRH)-like peptides were examined in the central nervous system and optic gland by immunohistochemistry. For GnRH immunodetection, antibodies against four different forms of GnRH were used: cGnRH-I, cGnRH-II, sGnRH, and mGnRH. The optic gland nerve provides direct and indirect signals coming from the centres of integration of chemical, visual, and olfactive stimuli to modulate the glandular activity. In these centres, the subpedunculate area, the olfactory and optic lobes, and FMRF-amide-like and GnRH-like immunoreactivities were detected. The subpedunculate area seems to be the source of the FMRF-amide-like peptide, whereas the posterior olfactory lobule is the source of the GnRH-like peptide. The immunoreactive fibres for both neuropeptides leave their sources and directly enter the optic gland nerve. FMRF-amide- and GnRH-immunoreactive nerve endings are seen on the glandular cells. The evidence of a possible neuropeptidergic control of optic gland activity reinforces the analogies and the functional parallels in the octopus, insect, crustacean, and vertebrate hormonal systems.

Animals↗

Studies on the structure of sperm heads of Eledone cirrhosa by means of CLSM linked to bioimage-oriented devices.

We have used a confocal laser scanning optical microscope imaging device and a bioimage-oriented workstation equipped for augmented reality to study the helical sperm head of the octopus Eledone cirrhosa. This approach allows us to study different complex organisational motifs due to the spatial arrangement of linear helical structures. We consider this helical specimen an enlarged copy of one of the most important biostructures governing cell functioning such as chromatin-DNA. Moreover, this very same sample is made of highly compacted chromatin that can be studied at higher resolution, i.e., by means of scanning force microscopy. Fluorescence optical sectioning has been used to enter the spatial organisation. Three-dimensional images of single, twisted, and folded fibers are shown.

Animals↗

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.

Animals↗

Progesterone receptor in the reproductive system of the female of Octopus vulgaris: characterization and immunolocalization.

In this study for the first time we have characterized a progesterone receptor in the reproductive system of the female of Octopus vulgaris. Scatchard analysis revealed that one binding component with high affinity and low capacity for the ligand was present only in the nuclear extract. Competition experiments showed that the progesterone receptor was strictly specific for progesterone. DNA-cellulose binding and DEAE-Sephacel both confirmed the presence of one 3H-progesterone binding component which eluted at a salt concentration of 0.14 +/- 0.05 M NaCl and 0.15 +/- 0.05 M NaCl respectively. By using monoclonal antibodies against chicken progesterone receptor (subunits A and B), we have localized on Western Blot one band of about 70 kDa. Immunoreactivity for progesterone binding molecules has been localized in the nuclei of the follicle cells of the ovary, of the proximal portion of the oviduct and of the outer region of the nidimental gland. These data, taken together, provide evidence that in Octopus vulgaris the progesterone receptor has biochemical and immunohistochemical characteristics resembling those of progesterone receptor in vertebrates.

Animals↗

Sex steroid hormone fluctuations and morphological changes of the reproductive system of the female of Octopus vulgaris throughout the annual cycle.

Sex steroids (17beta-estradiol and progesterone) and morphological variations of the reproductive system of the female of Octopus vulgaris from the Bay of Naples were followed over a period of 2 years. The increase in the ovary weight was independent of body weight as demonstrated by the gonado-somatic index (GSI). Both 17beta-estradiol and progesterone have been detected in the ovary of O. vulgaris, and their concentrations changed in correlation with the ovarian development. No 17beta-estradiol or progesterone was found in the hemolymph. 3beta-Hydroxysteroid dehydrogenase activity has been detected in the ovary, indicating that in the female of O. vulgaris the reproductive system is a source of sex steroid hormones. According to the morphological changes of the ovary, the ovarian cycle can be divided into the following phases: previtellogenesis; early vitellogenesis, full vitellogenesis and late vitellogenesis. The morphological changes of the oviducts and oviducal glands throughout the reproductive cycle were in accordance with their role in the transport and secretion of gelatinous coat covering the eggs, as well as in sperm storage and sperm reactivation during fertilization. J. Exp. Zool. 289:33-47, 2001.

3-Hydroxysteroid Dehydrogenases↗

Embryonic and paralarval development of the central nervous system of the loliginid squid Sepioteuthis lessoniana.

The embryonic development of the central nervous system (CNS) in the oval squid Sepioteuthis lessoniana is described. It has three distinct phases: (1) The ganglionic accumulation phase: Ganglionic cell clusters develop by ingression, migration, and accumulation of neuroblasts. (2) The lobe differentiation phase: Ganglia differentiate into lobes. The phase is identified by the beginning of an axogenesis. During this phase, neuropils are first formed in the suboesophageal mass, then in the basal lobe system, and finally in the inferior frontal lobes and the superior frontal-vertical lobe systems. (3) The neuropil increment phase: After the shape of the lobes reached its typical form, neuropil growth occurs, specifically in the vertical lobe. The paralarval central nervous system (CNS) is characterized by neuronal gigantism of the giant fibers and some suboesophageal commissures and connectives. The neuropil formation in the CNS of S. lessoniana occurs somewhat earlier than in Octopus vulgaris, although the principal developmental plan is quite conservative among the other coleoids investigated. Some phylogenetic aspects are discussed based on the similarities in the morphologic organization of their brains.

Animals↗

Expression and distribution of octopus gonadotropin-releasing hormone in the central nervous system and peripheral organs of the octopus (Octopus vulgaris) by in situ hybridization and immunohistochemistry.

We recently purified a peptide with structural features similar to vertebrate gonadotropin-releasing hormone (GnRH) from the brain of Octopus vulgaris, cloned a cDNA encoding the precursor protein, and named it oct-GnRH. In the current study, we investigated the expression and distribution of oct-GnRH throughout the central nervous system (CNS) and peripheral organs of Octopus by in situ hybridization on the basis of the cDNA sequence and by immunohistochemistry using a specific antiserum against oct-GnRH. Oct-GnRH mRNA-expressing cell bodies were located in 10 of 19 lobes in the supraesophageal and subesophageal parts of the CNS. Several oct-GnRH-like immunoreactive fibers were seen in all the neuropils of the CNS lobes. The sites of oct-GnRH mRNA expression and the mature peptide distribution were consistent with each other as judged by in situ hybridization and immunohistochemistry. In addition, many immunoreactive fibers were distributed in peripheral organs such as the heart, the oviduct, and the oviducal gland. Modulatory effects of oct-GnRH on the contractions of the heart and the oviduct were demonstrated. The results suggested that, in the context of reproduction, oct-GnRH is a key peptide in the subpedunculate lobe and/or posterior olfactory lobe-optic gland-gonadal axis, an octopus analogue of the hypothalamo-hypophysial-gonadal axis. It may also act as a modulatory factor in controlling higher brain functions such as feeding, memory, movement, maturation, and autonomic functions

Amino Acid Sequence↗

Excess DNA in the nuclei of the subesophageal region of octopus brain.

Cytophotometric analyses of Feulgen-stained nuclei present in homogenates of vertical and subesophageal lobes of octopus brain have shown that the latter region contains larger nuclei with up to several times the amount of DNA present in vertical nuclei. No obvious relationship was found between DNA content and nuclear size. Except for a rather small minority, nuclei of the vertical lobe have a uniform size and the expected diploid amount of DNA. These parameters are not substantially dependent on body weight. In contrast, the DNA content of subesophageal nuclei increases progressively with body weight. The amount of DNA found in subesophageal nuclei does not seem to be a simple multiple of the diploid or haploid value.

Animals↗

Afferent synaptic connections between hair cells and the somata of intramacular neurons in the gravity receptor system of the statocyst of Octopus vulgaris.

In the sensory epithelium (macula) of the gravity receptor system of the statocyst of Octopus vulgaris, there are two types of afferent neurons, distinguished according to their position in the epithelium. The somata of one type lie accumulated in a ring peripheral to the hair cell layer of the epithelium; these are designated as perimacular neurons. The somata of the other type lie among the hair cells, below the level of their nuclei; these are designated as intramacular neurons. Axons of afferent neurons which touch the hair cells are postsynaptic to some of the hair cells touched. As the somata of the intramacular neurons also touch the hair cells, they were investigated by serial electron microscopic reconstruction to determine if afferent synaptic contacts between hair cells and these somata occur. An average of about 600 intramacular neurons was counted in two maculae. Afferent synapses were seen to occur between hair cells and the somata of 76% of the intramacular neurons investigated. The postsynaptic processes of the intramacular neurons' somata were of two morphological types; one with a finger-like and one with a flat postsynaptic process (average of one synapse of each type per soma). The soma of an intramacular neurons can be postsynaptic to more than one hair cell simultaneously (average of 1--2 hair cells per soma). In addition to being presynaptic to only one neuron's soma, a hair cell could be simultaneously presynaptic to the axons of one or more afferent neurons. The morphological findings are discussed as to their possible physiological consequences.

Afferent Pathways↗

Connections of the octopus optic lobe: an HRP study.

The major visual centers of the octopus central nervous system are the paired optic lobes. Bidirectional transport of horseradish peroxidase (HRP) was used to determine connections of the optic lobe. Cells afferent to the optic lobe were identified by retrograde HRP transport in the following lobes of the central ganglia: anterior basal, median basal, dorsal basal, interbasal, subvertical, precommissural, brachial, and magnocellular. Labeled cells were also observed within the contralateral optic lobe, various optic tract lobes bilaterally, and in photoreceptors of the ipsilateral retina. Additionally, individual fibers, in part originating from cells in the posterior subvertical lobe, were labeled within the central neuropil core of various vertical lobules. Differences in results between superficial and deep optic lobe medulla injections indicate that some afferent projections from central sources may terminate on cell populations at specific depths within the lobe. Efferent optic lobe fibers into the superior frontal and lateral basal lobes were labeled by anterograde transport. Other possible optic lobe efferent projections terminated in supraesophageal lobes and the magnocellular lobe. The many inputs to the optic lobe from higher motor and associative centers in the central ganglia emphasize that the medulla region of the optic lobe is an exceptionally complex integrative area.

Animals↗

Microscopic and biochemical characterization of lectin binding sites in the cephalopod retina.

Using light and electron microscope cytochemistry and lectin blotting techniques, we have shown that the lectins concanavalin A (Con A), Ricinus communis agglutinin (RCA), and peanut agglutinin (PNA) bind to specific glycoconjugants in the adult cephalopod retina. For light microscope lectin cytochemistry, aldehyde-fixed, frozen, or Araldite-embedded, etched sections of cephalopod retinas were incubated with FITC- or TRITC-conjugated lectins and examined by using epifluorescence microscopy. Con A labeled structures in the entire retina including the inner limiting membrane (ILM), rhabdomeric membranes, interphotoreceptor matrix (IPM), and structures in the photoreceptor inner segments. RCA labeling was similar to that of Con A except that there was a decrease in the staining of the rhabdom tips near the ILM. PNA labeled only the interphotoreceptor matrix between apposing rhabdomeres. The intensity of staining of the IPM by PNA also decreased or was absent toward the rhabdom tips. None of the lectins labeled the myeloid bodies located in the photoreceptor inner segments. Electron microscope (EM) lectin cytochemistry was performed on aldehyde-fixed, LR White-embedded tissue or on Araldite-embedded, periodate-etched sections by using gold-conjugated lectins. EM results confirmed the observations made by light microscopy. Lectin blots with a retinal extract or light-sensitive membrane fraction revealed a variety of protein bands labeled by all three lectins. Con A and RCA labeled opsin and its aggregates whereas PNA did not. None of the lectins labeled retinochrome. The labeling of the cephalopod IPM by PNA suggests a structural similarity between the IPM of vertebrates and invertebrates. In other studies, we have demonstrated the presence of a retinoid binding protein in the IPM of cephalopods.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Carbocyanine dye labeling reveals a new motor nucleus in octopus brain.

This work aims at a better understanding of the organization of the brain of Octopus vulgaris, emphasizing the touch and visual learning centers. We injected the carbocyanine dye, DiI, into the cerebrobrachial connectives and, separately, into the brachial nerves of living octopuses. In both experiments, retrogradely transported granules of DiI appeared in motor neurons in the superior buccal, posterior buccal and subvertical lobes and in a hitherto unsuspected motor nucleus of several hundred neurons in the posterior dorsal basal and median basal lobes. In addition we labeled afferent fibers by injecting DiI into the caudal (sensory) division of the cerebrobrachial connective on one side; the label spread throughout the superior buccal, posterior buccal and the lateral and median inferior frontal lobes mainly on the injected side. It extended through the cerebral tract into the subvertical lobe, into the superior frontal lobe through the interfrontal tract, through the posterior buccal commissure into the opposite posterior buccal lobe and into the median inferior frontal lobe. The work suggests a new function for the posterior dorsal and median basal lobes, which are shown for the first time to project through the inferior frontal lobe system into the brachial nerves. In addition it represents the first full report of the successful use of the carbocyanine dyes DiI and DiO for labeling nerve tissue in a live invertebrate animal.

Animals↗