Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.     

Interannual variation in the at‐sea behavior of California sea lions (Zalophus californianus)

To be successful, marine predators must alter their foraging behavior in response to changes in their environment. To understand the impact and severity of environmental change on a population it is necessary to first describe typical foraging patterns and identify the underlying variability that exists in foraging behavior. Therefore, we characterized the at‐sea behavior of adult female California sea lions (n = 32) over three years (2003, 2004, and 2005) using satellite transmitters and time‐depth recorders and examined how foraging behavior varied among years. In all years, sea lions traveled on average 84.7 ± 11.1 km from the rookery during foraging trips that were 3.2 ± 0.3 d. Sea lions spent 42.7% ± 1.9% of their time at sea diving and displayed short (2.2 ± 0.2 min), shallow dives (58.5 ± 8.5 m). Among individuals, there was significant variation in both dive behavior and movement patterns, which was found in all years. Among years, differences were found in trip durations, distances traveled, and some dive variables (e.g., dive duration and bottom time) as sea lions faced moderate variability in their foraging habitat (increased sea‐surface temperatures, decreased upwelling, and potential decreased prey abundance). The flexibility we found in the foraging behavior of California sea lions may be a mechanism to cope with environmental variability among years and could be linked to the continuing growth of sea lion populations.     

Morphological phylogenetics of the sea spiders (Arthropoda: Pycnogonida)

Pycnogonids or sea spiders are a group of marine arthropods whose relations to the chelicerates have been an issue of controversy. Higher-level phylogenetic relationships among the lineages of sea spiders are investigated using 36 morphological characters from 37 species from all extant families and a Devonian pycnogonid fossil. This is one of the first attempts to analyze the higher-level relationships of the Pycnogonida using cladistic techniques. Character homoplasy (implied weights) is taken into account to construct a polytomous, most-parsimonious tree in which two major clades within Pycnogonida are obtained. Clade A includes Ammotheidae paraphyletic with Colossendeidae, Austrodecidae and Rhynchothoracidae, and clade B is formed by Nymphonidae, Callipallenidae (apparently paraphyletic), Pycnogonidae and Phoxichilidiidae. The analysis of equally weighted data is presented and helps to identify those characters less consistent. The reduction of the chelifores, palps and ovigers — shown independently within each of the clades as parallel evolution events — challenges the assumption of a gradual mode of reduction within the group, according to analysis of unordered vs ordered characters. Most of the phylogenetic affinities proposed here are compatible with traditional classifications. However, traditional taxonomic characters need to be complemented by sets of anatomical, molecular and developmental data, among others, to produce more robust phylogenetic hypotheses on the higher- and lower-level relationships of the sea spiders.     

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer‐assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time‐consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm² vs 4.50 ± 0.04 μm² and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm² vs 3.18 ± 0.06 μm² and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.     

DNA barcoding of Brazilian sea turtles (Testudines)

Five out of the seven recognized species of sea turtles (Testudines) occur on the Brazilian coast. The Barcode Initiative is an effort to undertake a molecular inventory of Earth biodiversity. Cytochrome Oxidase c subunit I (COI) molecular tags for sea turtle species have not yet been described. In this study, COI sequences for the five species of sea turtles that occur in Brazil were generated. These presented widely divergent haplotypes. All observed values were on the same range as those already described for other animal groups: the overall mean distance was 8.2%, the mean distance between families (Dermochelyidae and Cheloniidae) 11.7%, the mean intraspecific divergence 0.34%, and the mean distance within Cheloniidae 6.4%, this being 19-fold higher than the mean divergence observed within species. We obtained species-specific COI barcode tags that can be used for identifying each of the marine turtle species studied.     

Ultrastructure of the mesoglea of the sea anemone Nematostella vectensis (Edwardsiidae)

Abstract. Cnidarians have extracellular matrix, or mesoglea, situated between an outer epidermis and an inner gastrodermis. In this article, we describe the ultrastructure of the mesoglea of polyps of Nematostella vectensis during development and regeneration. The column wall of recently metamorphosed polyps had basal laminae composed of a meshwork of thin filaments underlying each epithelium and a network of unstriated thick (20–25 nm in diameter) and thin fibrils (～5 nm) decorated with particulate matter. In juvenile polyps with eight tentacles, the system of thick fibrils was concentrated near the gastrodermis. In the column wall and mesenteries of the adult there were bundles of thick fibrils that ran parallel to the myonemes. In regenerating polyps 2 days after transection, the network of thin fibrils and particulate material as well as the basal lamina largely disappeared in the healing part of the oral, but not aboral, half. In the regenerating portion of the aboral half 1 and 2 days after transection, the bundles of thick fibrils were smaller and less organized, and the basal laminae were thicker than in the column wall of untransected polyps. In both regenerating halves, the general organization of the mesoglea of normal polyps was reattained by 5 days after transection. At all stages the mesoglea contained cellular processes that may belong to amebocytes; nucleated amebocytes with a range of shapes were present in the mesoglea of the column wall and mesenteries of adult polyps. Certain features of the mesoglea of members of N. vectensis and Hydra are similar, especially the ultrastructure of the basal laminae, but the fibrillar systems of these two model cnidarians are different. Temporal and spatial differences in the composition of the mesoglea of N. vectensis point to different roles for its components during development and regeneration.     

The coelomic elements of sea urchins (Strongylocentrotus)

Summary Coelomocytes of Strongylocentrotus purpuratus and Strongylocentrotus franciscanus include phagocytic leukocytes, flagellated vibratile cells, and two types of spherule-bearing cells; one of which contains echinochrome, a red naphthaquinone pigment. Vibratile cells are PAS positive and contain a strongly acidic mucopolysaccharide. Substances with similar staining and chemical properties appear in the coelomic fluid under certain stress conditions. It is supposed that these substances enter the fluid by diffusion from the vibratile cells. Tests indicate that both spherule-cell types, like the spherule cells of holothurians (sea cucumbers), may undergo a cycle during which they pass from a basophilic to an acidophilic state. Chemical properties of the spherule cells approximate those of vertebrate mast cells. The variability of staining and chemical reactions of spherule cells explains why published accounts of their properties have differed so widely. It is suggested that the properties of mucoid substances in invertebrate spherule-bearing cells may be as variable as is true of vertebrate mast cells.This investigation was supported by Public Health Service Research Grant No. 5358 (to E. A. Steinhaus) from the National Institute of Allergy and Infectious Diseases.     

The coelomic elements of sea urchins (Strongylocentrotus)

Summary The four coelomocyte classes of the red sea urchin,Strongylocentrotus franciscanus, described by light-microscope studies, are confirmed and the fine structure described. Material examined included fresh, non-aggregated cells; partially aggregated ones that had been heldin vitro up to four days; and aggregated cells heldin vitro for 40 days. Leukocytes from youngin-vitro preparations differed from most fresh leukocytes by having enlarged dense nucleoli and enlarged rough endoplasmic reticulum, which was often filled with secretion, and sometimes connected to the perinuclear cisterna. Leukocytes held 40 daysin vitro were mainly plasmodial. Unlike cells held a limited timein vitro, the 40-day leukocytes had nuclei much like those in fresh preparations.The three classes of spherule-bearing cells (vibratile cells, red spherule cells, and colorless spherule cells) differed greatly in ultrastructure, and varied in appearance according to the fixative and pH present during fixation. Vibratile-cell spherules were of biphasic construction, suggesting the condition of certain vertebrate mast cells. Red spherule cells occurred in two forms. The most common form in fresh preparations had despherulated,i.e., lacked material in the spherules; and the spherules of the second type were filled with either granular or homogeneous material. Colorless spherule cells had evenly and finely granular material in the spherules. Colorless spherule cells were uncommon or missing in material that had been heldin vitro. Certain unidentifiable spherule cells occurred in some preparations.Although samples are small, it is notable that in May and June, recognizable glycogen was present only in leukocytes that had been heldin vitro, not in any fresh cells. Glycogen occurred in fresh cells of all classes from samples taken in December and February (during or shortly before the normal spawning season ofS. franciscanus). Unlike the cells in fresh preparations made in May, June, and December, fresh leukocytes and vibratile cells taken in February often had extremely lobed nuclei and considerably developed rough endoplasmic reticulum.This investigation was supported by Public Health Service Research Grant No. 9296 (to P. T.Johnson) from the National Institute of Allergy and Infectious Diseases.     

Occurrence of plant hormone (cytokinin)-producing bacteria in the sea

Plant hormones, which are considered to be cytokinins, were detected in the culture media of marine bacteria using the Amaranthus bioassay method. The proportion of cytokinin-producing bacteria to total microbes tested was higher in sediments (45–55%) than in seawater (5–15%). The amount of cytokinin-like substances in the culture media was estimated as 0.05–0.30 μg of zeatin equivalents/l. Thin layer chromatography analysis using the soybean callus bioassay method suggested that the active substance produced by one of these bacteria was isopentenyladenine or its riboside. Taxonomic examination of the cytokinin-producing bacteria showed that the production of the hormone was not specific to any genus. A possible role of cytokinins produced by sediment bacteria on the development of red tide is discussed.     

Parallelisms in the evolution of sea cucumbers (Echinodermata: Holothuroidea)

The importance of taking into account parallelisms in the evolution of morphological characters is analyzed for the taxonomy of the class Holothuroidea. The establishment of the order Dactylochirotida and classification of the order Elasipodida serve as examples to illustrate insufficient appreciation of parallelisms in Holothuroidea. The following characters, evolving independently in different groups of sea cucumbers, are considered: a stout skeleton, reduction of the calcareous ring and the body wall sclerites; similarity of body shape; similarity in the shape of tentacles; reduction in the number of tentacles from 12 to 10 in different fam-ilies and subfamilies of the order Synaptida. Based on the analysis of morphological and molecular data, the family Deimatidae is transferred from the order Elasipodida to the order Aspidochirotida. It is hypothesized that the concave cup-shaped sclerites with three to five rays occurring in the family Laetmogonidae (order Elasipodida) are of paedomorphic origin and correspond to the early growth stages of the laetmogonid wheels; the concave cross-shaped sclerites of the families Elpidiidae and Psychropotidae may have originated from laetmogonid concave cup-shaped sclerites. Emended diagnosis of the order Elasipodida is proposed. The family Vaneyellidae previously synonymized by the author with the family Cucumariidae is reestablished, and its emended diagnosis is also proposed.     

Mechanics of the limbs of the walrus (Odobenus rosmarus) and the california sea lion (Zalophus californianus)

Moment arms and cross-sectional areas of muscles from the forelimbs and hind limbs of Odobenus and Zalophus were used to estimate relative torques associated with several locomotor movements. Odobenus uses predominantly the hind limbs for aquatic propulsion, while Zalophus only uses the forelimbs. Torques contributing to locomotor movements used during the power and recovery strokes were substantially greater for the hind limbs of Odobenus and for the forelimbs of Zalophus. Apparently, the lineages of these two genera shared an aquatic ancestor during the Miocene; therefore, they also shared a common method of aquatic propulsion. This common method of aquatic propulsion could have been either forelimb, hind limb, or both. Since the Miocene then, the method of aquatic propulsion and the mechanical characteristics of the limbs have diverged for at least one of these two lineages. A model is advanced to explain key mechanical differences between Odobenus and Zalophus that enable Odobenus to maneuver effectively in a head-down posture on the ocean floor.     

Tetranucleotide microsatellites from the loggerhead sea turtle (Caretta caretta)

We describe primers and polymerase chain reaction conditions to amplify 15 tetranucleotide microsatellite loci from the loggerhead sea turtle (Caretta caretta). The primers were tested on 30 individuals that nested along the Georgia, USA coast. The primer pairs developed in this study yielded an average of 13.9 alleles per locus (range of 10–21), an average observed heterozygosity of 0.91 (range 0.79–1.00), and an average polymorphic information content of 0.88 (range 0.84–0.92).