Short-term variability of fish condition and growth in estuarine and shallow coastal areas.

Short-term variability in condition factor: relative condition factor Kn; biochemical condition: RNA:DNA and protein content; and instantaneous growth rates were determined in estuarine and coastal fish. Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps were sampled in the Tejo estuary, while Trachurus trachurus was sampled in an adjacent shallow coastal area. Variation of condition indices was more frequent at the week scale (sampling periods with fortnight intervals) than at the daily scale (consecutive days in each sampling period) in all species. Water temperature was correlated with biochemical indices, while salinity showed no effect, evidencing the influence of environmental short-term variation (temperature) on biochemical condition in natural populations. Yet, decreasing individual variability in fish condition was observed along the sampled weeks, resulting in a more homogeneous condition of populations, particularly for T. trachurus likely due to a more stable coastal environment. Biochemical indices proved to be sensitive to short-term environmental variability, despite species-specific responses.

Biomarker responses to environmental contamination in estuaries: A comparative multi-taxa approach.

Estuaries are highly productive ecosystems subjected to numerous anthropogenic pressures with consequent environmental quality degradation. In this study, multiple biomarker responses [superoxide dismutase (SOD), catalase (CAT), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities, as well as lipid peroxidation (LPO) and DNA damage (DNAd)] were determined in two fish (Dicentrarchus labrax and Pomatoschistus microps) and four macroinvertebrate species (Carcinus maenas, Crangon crangon, Hediste diversicolor and Scrobicularia plana) from the Ria de Aveiro and Tejo estuaries over distinct months. Two sites per estuarine system were selected based on anthropogenic pressures and magnitude of environmental contamination. Antioxidant enzyme activities in fish species suggested a ubiquitous response to oxidative stress, while biotransformation and effect biomarkers exhibited higher spatial and temporal variation. In invertebrate species, biotransformation enzyme activity was clearly less variable than in fish evidencing lower xenobiotic transformation capability. Overall, largest biomarker responses were found in the most contaminated sites (Tejo), yet species-specific patterns were evident. These should be factored in multi-taxa approaches, considering that the differential functional traits of species, such as habitat use, life-stage, feeding or physiology can influence exposure routes and biomarker responses. The Integrated Biomarker Response index highlighted patterns in biomarker responses which were not immediately evident when analyzing biomarkers individually. Overall, results provided insights into the complexity of species responses to contamination in naturally varying estuarine environments. Ultimately, multi-taxa and multi-biomarker approaches provide a comprehensive and complementary view of ecosystem health, encompassing diverse forms of biological integration and exposure routes, and allow the validation of results among markers and species.

Global patterns and predictors of fish species richness in estuaries.

1. Knowledge of global patterns of biodiversity and regulating variables is indispensable to develop predictive models. 2. The present study used predictive modelling approaches to investigate hypotheses that explain the variation in fish species richness between estuaries over a worldwide spatial extent. Ultimately, such models will allow assessment of future changes in ecosystem structure and function as a result of environmental changes. 3. A comprehensive worldwide data base was compiled of the fish assemblage composition and environmental characteristics of estuaries. Generalized Linear Models were used to quantify how variation in species richness among estuaries is related to historical events, energy dynamics and ecosystem characteristics, while controlling for sampling effects. 4. At the global extent, species richness differed among marine biogeographic realms and continents and increased with mean sea surface temperature, terrestrial net primary productivity and the stability of connectivity with a marine ecosystem (open vs. temporarily open estuaries). At a smaller extent (within a marine biogeographic realm or continent), other characteristics were also important in predicting variation in species richness, with species richness increasing with estuary area and continental shelf width. 5. The results suggest that species richness in an estuary is defined by predictors that are spatially hierarchical. Over the largest spatial extents, species richness is influenced by the broader distributions and habitat use patterns of marine and freshwater species that can colonize estuaries, which are in turn governed by history contingency, energy dynamics and productivity variables. Species richness is also influenced by more regional and local parameters that can further affect the process of community colonization in an estuary including the connectivity of the estuary with the adjacent marine habitat, and, over smaller spatial extents, the size of these habitats. In summary, patterns of species richness in estuaries across large spatial extents seem to reflect from global to local processes acting on community colonization. The importance of considering spatial extent, sampling effects and of combining history and contemporary environmental characteristics when exploring biodiversity is highlighted.

Assessing food web dynamics and relative importance of organic matter sources for fish species in two Portuguese estuaries: a stable isotope approach.

Stable carbon and nitrogen isotopes (δ¹³C, δ¹5N) were used to analyse food web dynamics of two of the main estuaries of the Portuguese coast: Tejo and Mira. The ultimate sources of organic matter supporting production of some of the most abundant and commercially important fish species were determined; and seasonal, inter- and intra- estuarine differences in the trophic relations among producers and consumers were identified. Stable isotope analysis was performed in different producers, primary consumers (main prey items for fish) and fish species (Solea solea, Solea senegalensis, Pomatoschistus microps, Dicentrarchus labrax, Liza ramada, Diplodus vulgaris and Atherina presbyter) of two areas in each estuary, in July and October 2009. Model calculations showed that the main prey for the fish species in the Tejo estuary used mostly salt marsh-derived organic matter as nutritional sources, with no marked differences between the sampled months. Trophic levels of fish species from the same estuary differed at multiple scales: inter-species, seasonally and spatially (both between and within estuaries). Significant differences in isotopic composition of fish species were more pronounced spatially (between the two sampled areas in the estuary) than seasonally (between sampled months). Trophic relationships in both estuaries demonstrated that organic matter is transferred to higher trophic positions mainly through benthic pathways. This shows the flexibility of these species to share resources and to exploit temporary peaks in prey populations. The present results showed that extensive disturbance in intertidal habitats from both estuaries may potentially change the balance of organic matter in the base of these complex food webs.

Topology of splicing and snRNP biogenesis in dinoflagellate nuclei.

BACKGROUND INFORMATION: Dinoflagellates are protists that are hypothesized to have experienced a secondary loss of histones. Amongst eukaryotes, they are unique in lacking these proteins. To date, information on the mechanisms involving remodelling, transcription and splicing of their chromatin is limited. Dinoflagellate genes lack TATA boxes and downstream polyadenylation sites and particular linear arrangements. They have an alpha-amanitin-sensitive RNA polymerase, specific transcription factors and regulators, and both transcriptional and post-transcriptional regulation of gene expression. Dinoflagellates produce either polycistronic or discrete mRNAs, and have conserved snRNAs (small nuclear RNAs), indicating that their genes are spliced. RESULTS: Five representative dinoflagellate species (Amphidinium carterae, Akashiwo sanguinea, Alexandrium lusitanicum, Alexandrium fundyense and Prorocentrum micans), which show diversity in their DNA content, nuclear organization and taxonomic position, were investigated. The nuclear distribution and ultrastructural organization of splicing and snRNP (small nuclear ribonucleoprotein) biogenesis were determined by fluorescent and electron microscopy immunolabelling with Y12 sera [recognizing the sDMA (symmetrical dimethylarginine) domain of Sm and other nuclear proteins], anti-p105-PANA [proliferation-associated nuclear antigen; a marker of IGs (interchromatin granules)] and anti-DNA antibodies. In parallel, ultrastructural analysis, including cytochemical staining for RNA, phosphorylated proteins and DNA, was carried out. Splicing factors were distributed in a diffuse perichromosomal layer containing perichromatin granules and fibrils that co-localized with the decondensed peripheral DNA loops, but not with the main chromosome body. Interchromosomal domains with IGs and Cajal-like bodies were also detected. CONCLUSIONS: Dinoflagellates are rather dissimilar to other eukaryotes in their genomes, their mechanisms of gene expression and their chromosome ultrastructure. However, they share common splicing nuclear domains and snRNP biogenesis with that of other eukaryotes.

Tratamento do tumor do córtex adrenal na infância / Treatment of childhood adrenocortical tumor

O tumor do córtex adrenal (TCA) na infância é raro. Entretanto, no Paraná sua incidência é 15 vezes maior do que a observada em outros países. A mutação germinativa R337H TP53, presente em mais de 95 por cento dos nossos pacientes, provavelmente está relacionada à maior incidência. Cento e vinte e cinco pacientes foram tratados no período de 1966 a 2003. A cirurgia é o único tratamento curativo. Em nossa experiência, tumores no estádio I, ausência de spillage durante a cirurgia e ausência de trombo são parâmetros relacionados à maior sobrevida. Dados preliminares mostram que a associação de etoposídeo, doxorrubicina, cisplatina e mitotano produziu remissão completa do tumor e/ou das metástases em alguns pacientes. Os efeitos colaterais destas drogas são comuns e pode ocorrer insuficiência adrenocortical. As doses de reposição de glicocorticóides e mineralocorticóides devem ser 2 a 3 vezes maiores que as doses fisiológicas.

Organization of the genome and gene expression in a nuclear environment lacking histones and nucleosomes: the amazing dinoflagellates.

Dinoflagellates are fascinating protists that have attracted researchers from different fields. The free-living species are major primary producers and the cause of harmful algal blooms sometimes associated with red tides. Dinoflagellates lack histones and nucleosomes and present a unique genome and chromosome organization, being considered the only living knockouts of histones. Their plastids contain genes organized in unigenic minicircles. Basic cell structure, biochemistry and molecular phylogeny place the dinoflagellates firmly among the eukaryotes. They have G1-S-G2-M cell cycles, repetitive sequences, ribosomal genes in tandem, nuclear matrix, snRNAs, and eukaryotic cytoplasm, whereas their nuclear DNA is different, from base composition to chromosome organization. They have a high G + C content, highly methylated and rare bases such as 5-hydroxymethyluracil (HOMeU), no TATA boxes, and form distinct interphasic dinochromosomes with a liquid crystalline organization of DNA, stabilized by metal cations and structural RNA. Without histones and with a protein:DNA mass ratio (1:10) lower than prokaryotes, they need a different way of packing their huge amounts of DNA into a functional chromatin. In spite of the high interest in the dinoflagellate system in genetics, molecular and cellular biology, their analysis until now has been very restricted. We review here the main achievements in the characterization of the genome, nucleus and chromosomes in this diversified phylum. The recent discovery of a eukaryotic structural and functional differentiation in the dinochromosomes and of the organization of gene expression in them, demonstrate that in spite of the secondary loss of histones, that produce a lack of nucleosomal and supranucleosomal chromatin organization, they keep a functional nuclear organization closer to eukaryotes than to prokaryotes.

[Treatment of childhood adrenocortical tumor]. / Tratamento do tumor do córtex adrenal na infância.

Adrenocortical tumors (ACT) in children are uncommon. However, the incidence of these tumors in Paraná is 15 times higher than that worldwide. A germline mutation, R337H TP53, present in more than 95% of our patients, is probably the reason for the higher incidence in our state. A hundred twenty-five patients were treated in the period of 1966 to 2003. Surgery is the only curative treatment. In our experience, disease stage I, absence of spillage during surgery and absence of intravenous thrombus are associated with better survival rates. Preliminary data with the combination of etoposide, doxorubicin, cisplatin, and mitotane have shown that in some patients a complete remission is observed both of the tumor and metastasis. Side effects due to these drugs are common and adrenal insufficiency may occur. Glucocorticoid and mineralocorticoid reposition should be done with 2 to 3 times the physiological doses.

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain).

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.

Accumulation of paralytic shellfish toxins (PST) from the cyanobacterium Aphanizomenon issatschenkoi by the cladoceran Daphnia magna.

In order to access the effects of Paralytic Shellfish Toxins (PST) in freshwater environment, the accumulation of PST produced by the cyanobacteria Aphanizomenon issatschenkoi in juvenile Daphnia magna was investigated. D. magna was exposed to A. issatschenkoi cells (1.2 x 10(6) cells ml(-1)) for 6, 8, 12, 24 and 30 h and also to lyophilised material (1 mg ml(-1)) for 24h. Survival and somatic growth of the juvenile D. magna was investigated, as was the activity of the biotransformation enzyme system glutathione-S-transferases (GSTs). Between 643+/-65.35 and 1170+/-51.72 pmol PST ml(-1) were detected by HPLC-FLD in D. magna culture medium containing cells and 2745+/-64.61 pmol PST toxin ml(-1), in the medium containing lyophilised material. PST were detected in D. magna tissues in cells exposure (between 6.51 x 10(-2)+/-1.37 x 10(-2) and 3.78 x 10(-1)+/-1.15 x 10(-2)pmol PST animal(-1)). In D. magna exposed to lyophilised material the mean (+/-SD) PST concentration was found to be 6.96 x 10(-3) (+/-3.84 x 10(-3)) pmol PST animal(-1). Following exposure to 1.2 x 10(6) cells ml(-1)A. issatschenkoi fresh cells growth and survival of D. magna were reduced. D. magna exposed to the two A. issatschenkoi treatments (fresh cells and lyophilised material), showed a reduction in activity of the cytosolic glutathione-S-transferases (cGSTs). The results of this study indicate that D. magna can accumulate PST toxins and that the cyanobacterium A. issatschenkoi affects both the fitness and growth potential of juvenile D. magna.

Tumores do córtex adrenal na infância / Childhood adrenocortical tumors

O tumor do córtex adrenal (TCA) na infância é raro. Entretanto, no Paraná sua incidência é 15 vezes maior do que a observada em outros países. Descrevemos as características clínicas, laboratoriais, tratamento e evolução de 125 pacientes atendidos em uma única instituição no Paraná. A média de idade ao diagnóstico foi de 4,3 anos, com uma relação feminino:masculino de 2,6:1. As formas clínicas mais comuns foram virilização isolada (51,2 por cento) e virilização e síndrome de Cushing (42 por cento). Em 4,8 por cento dos casos, os tumores foram não-funcionantes. Dois pacientes (1,6 por cento) apresentaram síndrome de Cushing isolado e 1 (0,8 por cento) síndrome de Conn. Hipertensão arterial ocorreu em 56 por cento dos pacientes. O único tratamento curativo é a cirurgia. Os dados da nossa casuística mostram que estádio clínico 1 do TCA, ausência de trombo intra-venoso e ausência de spillage durante a cirurgia estão associados a uma maior sobrevida.

Accumulation and depuration of cyanobacterial paralytic shellfish toxins by the freshwater mussel Anodonta cygnea.

The increasing frequency by which the production of paralytic shellfish toxins (PST) by freshwater bloom-forming cyanobacteria is being noticed world-wide raises the possibility of PST bioaccumulation by freshwater mussels. This study evaluates PST accumulation and depuration by the freshwater mussel Anodonta cygnea exposed over a 14-day period to high densities (mean = 1.4 x 10(9) cells1(-1), S.D. = 0.29 x 10(9) cellsl(-1)) of the toxic cyanobacterium Aphanizomenon issatschenkoi (corresponding to a mean toxin concentration of 25.5 nmol PSTl(-1), S.D. = 9.9 nmol PSTl(-1)). Mussels were subsequently detoxified either by starvation or by feeding on the non-toxic green-algae Ankistodesmus falcatus. Filter feeding activity and toxin uptake by the mussels were followed by cell counting and toxin analysis in water samples taken before and after each daily water renewal. The accumulation and depuration of PST as well as the anatomical distribution of toxins were monitored throughout the experiment by HPLC analysis of mussel extracts. Mussels fed the toxic cyanobacterium removed on average 65.3% of cells and 40.36% of total PST daily provided. Daily rates of cell clearance (% of initial) were negatively correlated with the amounts of PST daily provided (but not with the amount of cells). This suggests a negative effect of toxins on the feeding behaviour of mussels. Small amounts of toxins could be detected in the mussels after the second day of exposure, reaching a maximum of 26 microg PST100 g(-1) by day 7. The viscera contained the greatest proportion of toxins (78%) at the start of the toxification. However, increasing amounts of PST were found in the remaining tissues (gills, mantle and foot) over time. Toxins detected in the mussel extracts were the same provided in the dietary A. issatschenkoi. Nevertheless, mussels showed a higher proportion of saxitoxin and decarbomoylsaxitoxin and a lower proportion of gonyautoxin-5 than the fed cyanobacterium. Similar depuration efficiencies were observed among starved individuals (6.9% day(-1)) and those fed with A. falcatus (8.2% day(-1)) indicating that both treatments had comparable effects on toxin metabolism. Mussels showed a typical S shaped depuration kinetics curve consisting of a first short period of slow toxin decay followed by a rapid loss and a subsequent slower release of toxins. Trace to undetectable levels of PST were found in mussels after the 14-day depurating period. Although freshwater mussels are not widely consumed by humans, their capacity to accumulate PST points to the risk of PST propagation through the food chain of freshwater ecosystems via filter-feeding mussels.

The loss of PSP toxin production in a formerly toxic Alexandrium lusitanicum clone.

Toxin production has always been considered a constitutive characteristic of dinoflagellates in the genus Alexandrium. Here we demonstrate that saxitoxin production can be lost by an Alexandrium species during routine culture maintenance. This is the first report of any marine saxitoxin-producing alga ever to have completely lost the ability to produce toxins. A clonal toxic isolate of Alexandrium lusitanicum from Portugal has been maintained in culture since 1962. In 1992, a subculture was established and sent to a different laboratory. Recent comparisons of the parental strain and the subculture revealed that the former had lost its toxicity, whereas the latter still produces saxitoxins. This loss of toxicity was confirmed by three independent toxin detection methods: mouse bioassay, mouse neuroblastoma assay and HPLC. Sequence analyses of different rRNA domains demonstrated that the toxic and non-toxic cultures are genetically identical for those markers. Morphological analysis showed that both cultures have the same plate tabulation and are A. lusitanicum. These results strongly argue that the loss of toxicity is not a result of a culturing artifact or mistake, such as mislabeling or contamination. The clonal cultures also show a significant difference in growth. Possible explanations for the change include genetic mutations or the effects of prolonged treatment of the non-toxic culture with antibiotics.

[Childhood adrenocortical tumors]. / Tumores do córtex adrenal na infância.

Adrenocortical tumors (ACT) in children are uncommon. However, the incidence of these tumors in Paraná, Brazil, is 15 times higher than that worldwide. We describe the clinical, laboratory and treatment characteristics and outcome of 125 patients treated in a single institution in the State of Paraná. The median age at diagnosis was 4.3 years, with a female:male ratio of 2.6:1. The most common forms of presentation were isolated virilization (51.2%) and virilization and Cushing's syndrome (42%). Nonfunctioning tumors comprised 4.8% of the cases. Two patients (1.6%) had isolated Cushing's syndrome and 1 (0.8%) had Conn's syndrome. Fifty-six percent presented hypertension. Surgery is the only curative treatment. Our data show that disease stage 1, absence of spillage during surgery and absence of intravenous thrombus were associated with better survival rates.

Toxic cyanobacteria strains isolated from blooms in the Guadiana River (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 _ 21.86 mg mcyst-LR equivalentúL-1 in Valdelacalzada and 0.10-11.3 µg mcyst-LR equivalentúL-1 in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.

Reevaluation of production of paralytic shellfish toxin by bacteria associated with dinoflagellates of the Portuguese coast.

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.