Transcriptomic changes in relation to early-life events in the gilthead sea bream (Sparus aurata).

BACKGROUND: Teleosts are exposed to a broad range of external stimuli, which may be either of acute or chronic nature. The larval phase of certain fish species offer a unique opportunity to study the interactions between genes and environmental factors during early life. The present study investigates the effects of early-life events, applied at different time points of early ontogeny (first feeding, flexion and development of all fins; Phase 1) as well as on the subsequent juvenile stage after the application of an additional acute stressor (Phase 2) in the gilthead sea bream (Sparus aurata), a commercially important European aquaculture species. Animal performance, the cortisol response and gene expression patterns during early development as well as on the subsequent phases (juveniles) after the application of additional acute stressors were investigated. RESULTS: Significant differences on fish performance were found only for juveniles exposed to early-life events at the phase of the formation of all fins. On the transcriptome level distinct expression patterns were obtained for larvae as well as for juveniles with the most divergent expression pattern found to be again at the phase of the development of all fins, which showed to have also an impact later on in the acute stress response of juveniles. CONCLUSIONS: The present study showed that applying an early-life protocol, characterized by the unpredictable, variable and moderate intensity of the applied stimuli provides a relative realistic model to evaluate the impact of daily aquaculture practices on fish performance. In addition, the power of investigating global gene expression patterns is shown, providing significant insights regarding the response of early-life events during development and as juveniles after the application of extra acute stressors.

Strong population genetic structure and contrasting demographic histories for the small-spotted catshark (Scyliorhinus canicula) in the Mediterranean Sea.

Coastal and demersal chondrichthyans, such as the small-spotted catshark, are expected to exhibit genetic differentiation in areas of complex geomorphology like the Mediterranean Basin because of their limited dispersal ability. To test this hypothesis, we used a fragment of the mitochondrial cytochrome c oxidase subunit I gene and 12 nuclear microsatellite loci in order to investigate the genetic structure and historical demography of this species, and to identify potential barriers to gene flow. Samples were collected from the Balearic Islands, the Algerian Basin, the Ionian Sea, the Corinthian Gulf and various locations across the Aegean Sea. Additional sequences from the Atlantic and the Levantine Basin retrieved from GenBank were included in the mitochondrial DNA analysis. Both mitochondrial and nuclear microsatellite DNA data revealed a strong genetic subdivision, mainly between the western and eastern Mediterranean, whereas the Levantine Basin shared haplotypes with both areas. The geographic isolation of the Mediterranean basins seems to enforce the population genetic differentiation of the species, with the deep sea acting as a strong barrier to its dispersal. Contrasting historical demographic patterns were also observed in different parts of the species' distribution, most notably a population growth trend in the western Mediterranean/Atlantic area and a slight decreasing one in the Aegean Sea. The different effects of the Pleistocene glacial periods on the habitat availability may explain the contrasting demographic patterns observed. The current findings suggest that the small-spotted catshark exhibits several genetic stocks in the Mediterranean, although further study is needed.

Pseudomonas, Pantoea and Cupriavidus isolates induce calcium carbonate precipitation for biorestoration of ornamental stone.

AIMS: Bacterially induced calcium carbonate precipitation from various isolates was investigated aiming at developing an environmentally friendly technique for ornamental stone protection and restoration. METHODS AND RESULTS: Micro-organisms isolated from stone samples and identified using 16S rDNA and biochemical tests promoted calcium carbonate precipitation in solid and novel liquid growth media. Biomineral morphology was studied on marble samples with scanning electron microscopy. Most isolates demonstrated specimen weight increase, covering partially or even completely the marble surfaces mainly with vaterite. The conditions under which vaterite precipitated and its stability throughout the experimental runs are presented. CONCLUSIONS: A growth medium that facilitated bacterial growth of different species and promoted biomineralization was formulated. Most isolates induced biomineralization of CaCO3 . Micro-organisms may actually be a milestone in the investigation of vaterite formation facilitating our understanding of geomicrobiological interactions. Pseudomonas, Pantoea and Cupriavidus strains could be candidates for bioconsolidation of ornamental stone protection. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of biomineralization capacity of different bacterial species improves understanding of the bacterially induced mineralization processes and enriches the list of candidates for biorestoration applications. Knowledge of biomineral morphology assists in differentiating mineral from biologically induced precipitates.

Quantitative trait loci affecting morphology traits in gilthead seabream (Sparus aurata L.).

We report a quantitative trait loci (QTL) mapping study on 18 morphometric characters in gilthead seabream based on a total of 74 informative microsatellite markers genotyped in 409 offspring coming from 10 paternal half-sib families. Statistical analysis was carried out using a linear regression approach, and various suggestive and significant morphology QTL were detected in three (9, 21 and 25) of nine linkage groups examined. Fitting body weight as a covariate reduced the significance of some QTL but revealed three new QTL in other linkage groups (LG6 and LG10). Current results combined with those obtained from previous studies underline highly significant loci affecting overall growth and morphology in S. aurata.

Quantitative trait loci for body growth and sex determination in the hermaphrodite teleost fish Sparus aurata L.

Gilthead sea bream (Sparus aurata L.) is an important marine fish in Mediterranean aquaculture. Sex determination by age and/or body weight is a critical life-history trait, the genetic basis for which is largely unknown in this sequential hermaphrodite species. Herein, we performed a partial genome scan to map quantitative trait loci (QTL) affecting body weight and sex using 74 informative microsatellite markers from 10 paternal half-sib families to construct nine linkage groups (LG). In total, four growth-related QTL (two chromosome-wide and two genome-wide) and six QTL related to sex determination (three pairs in three different LGs) were detected (two chromosome-wide and one genome-wide). The proportion of phenotypic variation explained by the body-weight QTL ranged from 9.3% to 17.2%, showing their potential for use in marker-assisted selection. The results obtained offer solid ground to investigate the structure and function of the genomic regions involved in the mechanisms of sex reversal.

Toxopsis calypsus gen. nov., sp. nov. (Cyanobacteria, Nostocales) from cave 'Francthi', Peloponnese, Greece: a morphological and molecular evaluation.

Representatives of a new cyanobacterial genus, Toxopsis Lamprinou & Pantazidou gen. nov., were found in fresh material from Cave 'Francthi' (Peloponnese, Greece) and isolated in cultures. Ecological data relating to the environmental parameters of the sampling sites are provided, such as the photosynthetically active radiation (PAR), temperature and relative humidity. Morphological characteristics and the life cycle of the type species Toxopsis calypsus Lamprinou & Pantazidou sp. nov. were studied using light microscopy and scanning and transmission microscopy. Molecular analysis based on the 16S rRNA gene sequence was also conducted. Toxopsis calypsus sp. nov. is a false-branched nostocalean cyanobacterium with both isopolar and heteropolar filaments bearing mono-pored and bi-pored heterocysts, and also hormogonia and akinetes. Isopolar filaments adhere by the centre to the substrate and are found mainly in fresh material and in young cultures; heteropolar filaments bearing a basic mono-pore heterocyst are dominant in aged (more than one-year-old) cultures. According to the revised taxonomic classification system of Komárek & Anagnostidis (1989) [Komárek, J. & Anagnostidis, K. (1989). Algol Stud, 56, 247-345] based mainly on morphological data, the new genus described here shares morphological characters with both nostocalean families Scytonemataceae and Microchaetaceae, showing similarities in particular to Scytonematopsis contorta [Vaccarino, M. A. & Johansen, J. R. (2011). Fottea 11, 149-161], Microchaetaceae. Molecular data from the 16S rRNA sequence determined in this paper showed that Toxopsis calypsus sp. nov. is more related to the family Microchaetaceae, and the five phylotypes analysed by PCR showed that the closest nostocalean relatives are Tolypothrix distorta SAG 93.79 (GenBank accession no. GQ287651) and Coleodesmium sp. ANT.L52B.5 (AY493596) with 95-96% and 96% similarity, respectively. In contrast, the five phylotypes showed a distant similarity to Scytonematopsis contorta (<91%). The phenotypic and genetic traits strongly supported the classification of the five phylotypes as a new taxon for which the name Toxopsis calypsus Lamprinou & Pantazidou gen. nov., sp. nov. is proposed.

Exploring neutral and adaptive processes in expanding populations of gilthead sea bream, Sparus aurata L., in the North-East Atlantic.

Recent studies in empirical population genetics have highlighted the importance of taking into account both neutral and adaptive genetic variation in characterizing microevolutionary dynamics. Here, we explore the genetic population structure and the footprints of selection in four populations of the warm-temperate coastal fish, the gilthead sea bream (Sparus aurata), whose recent northward expansion has been linked to climate change. Samples were collected at four Atlantic locations, including Spain, Portugal, France and the South of Ireland, and genetically assayed using a suite of species-specific markers, including 15 putatively neutral microsatellites and 23 expressed sequence tag-linked markers, as well as a portion of the mitochondrial DNA (mtDNA) control region. Two of the putatively neutral markers, Bld-10 and Ad-10, bore signatures of strong directional selection, particularly in the newly established Irish population, although the potential 'surfing effect' of rare alleles at the edge of the expansion front was also considered. Analyses after the removal of these loci suggest low but significant population structure likely affected by some degree of gene flow counteracting random genetic drift. No signal of historic divergence was detected at mtDNA. BLAST searches conducted with all 38 markers used failed to identify specific genomic regions associated to adaptive functions. However, the availability of genomic resources for this commercially valuable species is rapidly increasing, bringing us closer to the understanding of the interplay between selective and neutral evolutionary forces, shaping population divergence of an expanding species in a heterogeneous milieu.

Isolation and characterization of 18 new polymorphic microsatellite loci for the swordfish, Xiphias gladius.

Eighteen microsatellite loci (13 di- and 5 tri-repeats) were isolated from swordfish and characterized in two populations from the Atlantic Ocean and Mediterranean Sea. The number of alleles per locus ranged from two to 29 and the observed heterozygosity from 0.302 to 0.953. All but one locus conformed to Hardy-Weinberg expectations and there was no evidence for linkage disequilibrium between loci.

A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L.

European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) sustains a regional fishery and is commonly farmed in the Mediterranean basin, but has not undergone much long-term genetic improvement. An updated genetic linkage map of the European sea bass was constructed using 190 microsatellites, 176 amplified fragment length polymorphisms and two single nucleotide polymorphisms. From the 45 new microsatellite markers (including 31 type I markers) reported in this study, 28 were mapped. A total of 368 markers were assembled into 35 linkage groups. Among these markers, 28 represented type I (coding) markers, including those located within the peptide Y, SOX10, PXN1, ERA and TCRB genes (linkage groups 1, 7, 16, 17 and 27 respectively). The sex-averaged map spanned 1373.1 centimorgans (cM) of the genome. The female map measured 1380.0 cM, whereas the male map measured 1046.9 cM, leading to a female-to-male (F:M) recombination rate ratio of 1.32:1. The intermarker spacing of the second-generation linkage map of the European sea bass was 3.67 cM, which is smaller than that of the first-generation linkage map (5.03 cM). Comparative mapping of microsatellite flanking regions was performed with five model teleosts and this revealed a high percentage (33.6%) of evolutionarily conserved regions with the three-spined stickleback.

Linking the genomes of nonmodel teleosts through comparative genomics.

Recently the genomes of two more teleost species have been released: the medaka (Oryzias latipes), and the three-spined stickleback (Gasterosteus aculateus). The rapid developments in genomics of fish species paved the way to new and valuable research in comparative genetics and genomics. With the accumulation of information in model species, the genetic and genomic characterization of nonmodel, but economically important species, is now feasible. Furthermore, comparison of low coverage gene maps of aquacultured fish species against fully sequenced fish species will enhance the efficiency of candidate genes identification projected for quantitative trait loci (QTL) scans for traits of commercial interest. This study shows the syntenic relationship between the genomes of six different teleost species, including three fully sequenced model species: Tetraodon nigroviridis, Oryzias latipes, Gasterosteus aculateus, and three marine species of commercial and evolutionary interest: Sparus aurata, Dicentrarchus labrax, Oreochromis spp. All three commercial fish species belong to the order Perciformes, which is the richest in number of species (approximately 10,000) but poor in terms of available genomic information and tools. Syntenic relationships were established by using 800 EST and microsatellites sequences successfully mapped on the RH map of seabream. Comparison to the stickleback genome produced most positive BLAT hits (58%) followed by medaka (32%) and Tetraodon (30%). Thus, stickleback was used as the major stepping stone to compare seabass and tilapia to seabream. In addition to the significance for the aquaculture industry, this approach can encompass important ecological and evolutionary implications.

New polymorphic microsatellite loci for the Macedonian crested newt, Triturus macedonicus, and cross-priming testing in four other crested newt species.

Eleven microsatellites have been characterized for Triturus macedonicus. Nine loci showed different variation patterns in a sample of 40 individuals from a single breeding pond in Zagori province (Greece), with an average number of 4.2 alleles per locus and an expected heterozygosity ranging from 0.1199 to 0.8079. Distinct cross-priming amplification rates were recovered on four additional crested newt species. Two monomorphic T. macedonicus loci were polymorphic in other Triturus species. The microsatellites developed herein could be a useful intraspecific genetic tool to undertake fine-scale population genetic analyses as well as in the study of contact zones between crested newt species.

The shaping of mitochondrial DNA phylogeographic patterns of the brown hare (Lepus europaeus) under the combined influence of Late Pleistocene climatic fluctuations and anthropogenic translocations.

The phylogeographic structure of the brown hare (Lepus europaeus) was studied by analysing mtDNA control region sequences of 98 individuals from continental and insular Greece, Bulgaria, Cyprus and northern Israel, together with 44 published sequences from Italy and central Europe. We found two distinct clades separated by an average nucleotide divergence of 6.6%, which may correspond to a Balkan and to an Asia Minor refugium. The estimated time of separation of the two clades was dated back to 105,000- 490,000 years ago. These two clades coexist in the area of northeastern Greece and Bulgaria, most likely as a result of a post-glacial northward expansion. Within the southern Balkan refugium, network analyses showed geographical structuring, which supports the hypothesis of several isolated Late Pleistocene populations. The central European and Italian populations appear to have originated from a non-detected northern Balkan population that was genetically closely related to some northern Greek populations, as a result of postglacial expansion, translocations or a combination of both. Moreover, several cases of ancient and recent translocations by humans were detected, especially for some island populations, while the eastern Aegean islands off the Asia Minor coast were most likely colonized naturally through Late Pleistocene land bridge connection. The genetic analysis presented here provides a framework for designing proper conservation and management guidelines for this species.

Development and linkage relationships for new microsatellite markers of the sea bass (Dicentrarchus labrax L.).

Twenty-eight polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with oligonucleotide probes. Analysis for these markers and 11 recently described microsatellites of D. labrax found linkage between 26 loci and revealed eight linkage groups.

Comparison of genetic variability and parentage in different ploidy classes of the Japanese oyster Crassostrea gigas.

Chemical treatments with cytochalasin B were used to induce triploidy in the progeny of a mass fertilization of 3 male and 7 female Crassostrea gigas parents. Triploids were produced either by retention of the first (meiosis I (MI) triploids) or the second (meiosis II (MII) triploids) polar bodies. These animals, together with their diploid siblings, were divided for two experiments. One set was used to compare physiological performance, and the other set deployed to compare growth in two different natural environments. For both experiments, genetic variability in different ploidy classes was estimated using three microsatellite loci and eight allozyme loci. The microsatellite loci were highly polymorphic, allowing independent confirmation of ploidy status and the unambiguous identification of parentage for each oyster. Significant differences in parentage were found between ploidy classes, despite the fact they originated from the same mass fertilization. This indicates that the assumptions of a common genetic background among random samples of animals taken from the same mass fertilization may not be generally valid. Knowledge of parentage also allowed the more accurate scoring of allozyme loci. As expected, triploids were found to be significantly more polymorphic than diploids. However, MI triploids were not significantly more polymorphic than MII triploids. MII triploid genotypes were used to estimate recombination rates between loci and their centromeres. These rates varied between 0.29 and 0.71, indicating only moderate chiasma interference.

Separate effects of triploidy, parentage and genomic diversity upon feeding behaviour, metabolic efficiency and net energy balance in the Pacific oyster Crassostrea gigas.

Triploid oysters were induced using cytochalasin B upon retention of either the first (meiosis I triploids) or the second (meiosis II triploids) polar body in embryos from a single cohort derived from mixed parentage. Allozyme and microsatellite assays enabled the confirmation of both parentage and triploidy status in each oyster. Comparison of meiosis I triploids, meiosis II triploids and diploid siblings established that improved physiological performance in triploids was associated with increased allelic variation, rather than with the quantitative dosage effects of ploidy status. An unidentified maternal influence also interacted with genotype. Among full sibs, allelic variation measured as multi-locus enzyme heterozygosity accounted for up to 42% of the variance in physiological performance; significant positive influences were identified upon feeding rate, absorption efficiency, net energy balance and growth efficiency (= net energy balance divided by energy absorbed). Whilst allelic variation was greater in both meiosis I and meiosis II triploids than in diploid siblings, both allelic variation and net energy balance were highest in triploids induced at meiosis I. This suggests that it may be preferable to induce triploidy by blocking meiosis I, rather than meiosis II as has traditionally been undertaken during commercial breeding programmes.

Cortical atrophy detected by computed tomography in gasoline station attendants.

BACKGROUND: To evaluate the contribution of leaded gasoline in the presence of abnormal calcifications or cortical atrophy seen in computed tomographies (CT) of the head of occupationally exposed professionals working in the centre of Athens. METHODS: One hundred and twenty-two head CTs from gas station employees and traffic-exposed professionals (taxi and bus drivers) were analyzed for evidence of cortical atrophy or abnormal calcifications. Blood lead level (BLL) of these lead occupationally exposed groups was compared with 37 non-exposed subjects. RESULTS: All three occupationally exposed-to-lead groups had similar blood lead levels compared to the non-exposed group and within the currently accepted norms for lead. No abnormal calcifications were found. Cortical atrophy was more frequently seen in the gas station employees group using univariate and multivariate analysis. In the logistic regression model gas station employment had a stronger impact in developing cortical atrophy [odds ratio of 6.43 (1.46-28.3, 95% CI)] than BLL [odds ratio of 1.4 (1.01-2.05, 95% CI)]. CONCLUSIONS: These results show that employment in gasoline stations may be associated with detectable cortical atrophy in imaging studies and suggest the contribution of a leaded gasoline to its development.

Percutaneous drainage of intra-abdominal abscesses using large lumen tubes under computed tomographic control.

OBJECTIVE: To describe our experience of percutaneous drainage of intra-abdominal abscesses with large-bore catheters under computed tomographic control. DESIGN: Retrospective study. SETTING: Teaching hospital, Greece. SUBJECTS: 185 Patients treated for abdominal abscesses during the period 1989-94. INTERVENTIONS: Needle aspiration (n = 27), drainage through conventional pigtail catheters (n = 22), and drainage through large-bore (8-16F) Argyle drains (n = 136). MAIN OUTCOME MEASURES: Morbidity. RESULTS: The overall success rate was 166/185 (92%). Of the 136 patients for whom the large-bore drains were used, 9 (7%) developed major complications (bowel fistula, n = 5; and pneumothorax and haemorrhage, n = 2 each) and 10 (7%) developed minor complications (obstruction of the tube, n = 4; dislocation of the tube, n = 3; bleeding from the wound, n = 2; and haematoma of the liver, n = 1). There were no deaths. CONCLUSION: Large-bore Argyle drains are efficient and safe for the percutaneous drainage of certain types of abdominal abscesses.

MRI of nasopharyngeal carcinoma metastatic to the cerebellopontine angle.

In patients with nasopharyngeal carcinoma, intracranial spread may occur via direct extension from the base of the skull or via perineural spread. Perineural spread usually affects branches of the trigeminal nerve. We describe two patients with recurrent nasopharyngeal carcinoma, who presented with a solitary mass in the cerebellopontine angle without associated bony destruction. MRI findings mimicked those of acoustic schwannoma. The imaging findings and possible pathways of spread are discussed.

Evaluation of hydatid disease of the heart with magnetic resonance imaging.

Two patients with cardiac involvement of hydatid disease are presented: one with hydatid cyst of the interventricular septum and pulmonary arteries and the other with multiple pulmonary cysts associated with intracardiac and pericardial cysts. The ability of magnetic resonance imaging (MRI) to provide a global view of cardiac anatomy in any plane with high contrast between flowing blood and soft tissue ensures it an important role in the diagnosis and preoperative assessment of hydatid disease of the heart.