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1.
J Fish Dis ; 43(1): 23-38, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31663143

RESUMO

Using the approach of sequencing the V3-V4 region of the 16S rRNA gene, we have analysed the bacterial diversity associated with the distinct compartments of the gastrointestinal tract of perch (Perca fluviatilis) and cestodes (Proteocephalus sp.) parasitizing their digestive tract. The dominant microbiota associated with cestodes (Proteocephalus sp.) was represented by bacteria from the genera Serratia, Pseudomonas and Mycoplasma. By comparing the associated microbiota of perch and cestodes, a clear difference in bacterial composition and diversity was revealed between the community from the stomach content and other parts of the gastrointestinal tract of fish. Microbiota associated with cestodes was not significantly different in comparison with microbiota of different subcompartments of perch (mucosa and content of intestine and pyloric caeca) (ADONIS, p > .05) excluding microbiota of stomach content (ADONIS, p ≤ .05). PICRUSt-based functional assessments of the microbial communities of perch and cestodes indicated that they mainly linked in terms of metabolism and environmental information processing and could play an important role in the nutrition and health of host.


Assuntos
Infecções por Cestoides/veterinária , Doenças dos Peixes/epidemiologia , Gastroenteropatias/veterinária , Microbioma Gastrointestinal , Percas , Animais , Infecções por Cestoides/epidemiologia , Infecções por Cestoides/parasitologia , Feminino , Doenças dos Peixes/parasitologia , Gastroenteropatias/epidemiologia , Gastroenteropatias/parasitologia , Masculino , Percas/microbiologia , Sibéria/epidemiologia
2.
PeerJ ; 7: e8005, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824755

RESUMO

In the Coregonus lavaretus complex may be found lacustrine sympatric pairs, which serves as an intriguing model for studying different aspects of fish evolutionary biology. One such sympatric whitefish pair inhabits Teletskoye Lake (West Siberia, Russia) and includes a "large" form (Coregonus lavaretus pidschian (Gmelin, 1789)) and a "small" form (C. l. pravdinellus (Dulkeit, 1949)). C. l. pravdinellus has a narrow trophic specialization and feeds on zooplankton, whereas the diet of C. l. pidschian is based on benthic prey. In the present study we aimed to address the question of how the gut microbial community reflects the divergence in diet of a sympatric pair of whitefish. Studied samples included the mucosa and content were collected for cardiac and pyloric stomach, anterior, middle, and posterior intestine, but only mucosa was collected for the pyloric caeca. In addition, water, sediment, macrophyte (environmental microbiota) and invertebrate (microbiota of prey) samples were collected in the same location. The V3-V4 region of the 16S rRNA genes was chosen for microbiome analysis and the software PICRUSt used to estimate the difference functional roles of the microbiota. The number of OTUs and Chao1 index in mucosa and content of cardiac and pyloric stomach were significantly different between whitefish. Significant differences were observed between whitefish for content from different parts of the intestine in terms of OTU number and Chao1 indices, whereas for mucosa from the same parts of intestine these differences were absent. No significant differences were found for diversity estimates of mucosa and content of different parts of the gut (there were a few exceptions) between whitefish. The form of whitefish and the segment of the digestive system were factors with a significant determinative effect on the structure of the microbiota from gut mucosa and content. The most dominant phyla in mucosa and content of cardiac and pyloric stomach was Proteobacteria (57.0-84.0%) for both whitefish. Throughout the intestine of C. l. pidschian the dominant phyla in mucosa were Proteobacteria (38.8%) and Firmicutes (15.6%), whereas for C. l. pravdinellus-Tenericutes (49.6%) and Proteobacteria (28.1%). For both forms, the phylum Spirochaetes was found in a significant amount (20.0-25.0%) in the mucosa of the posterior intestine. While for the content obtained from anterior, middle and posterior intestines, the dominant bacterial phyla were the same as those described for mucosa from the same parts of the intestine for both whitefish. The bacterial community of the prey and environment was significantly different from bacterial communities found for all parts of the gut mucosa for both whitefish, with the exception of the mucosa of the cardiac stomach. According to PICRUSt the highest level of differences between whitefish at the L3 level were found for the intestinal mucosa (75.3%), whereas the lowest one was registered for stomach content (38.8%).

3.
Fish Shellfish Immunol ; 94: 819-832, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31597086

RESUMO

The present study is the first report of some representative innate immune genes in meagre (Argyrosomus regius) larvae. This study has specifically focused on the growth period from hatching to the juvenile stage, a critical time in marine fish development when reliance on innate immune mechanisms are required for survival. We report molecular cloning of partial open reading frames and expression patterns for some innate immune genes (c3, cox2, met, lyzc, mxp, myd88, nod2, nod3). In addition, phylogenetic analyses of some of the sequences obtained was performed where confusion among closely allied isoforms may have existed. These results show the met isoform from meagre is met II, an isoform more similar to a homolog described in Larimichthys crocea; lysozyme (lyzc) corresponds to the c-type and NOD isoforms (nod2, nod3) separate into different clades confirming their distinctness within a common evolutionary history. Gene expression profiles of innate genes were investigated, for nine developmental stages, from 8 days post-hatching (dph) to 120 dph. Present results demonstrated that c3, cox2, met II, lyzc, mxp, myd88, nod2, and nod3 were expressed in all stages of larval development and displayed distinct expression profiles in separate tissues (kidney, spleen gut and gill). Moreover, expression patterns suggested theses innate immune genes may be influenced by feeding practices, i.e. switching from live prey (rotifer and Artemia) and weaning onto an inert commercial diet. In addition to evaluating changes in gene expression during early development, this study evaluated the modulation of gene expression by means of in vivo trials in juveniles that were stimulated with PAMPs (LPS, poly I:C, ß-glucan). These results revealed significant changes in mRNA levels of target genes in the kidney, spleen, gut and gills. However, expression profiles differed in magnitude depending on the stimulant and/or tissue. These results are discussed in terms of their relevance and potential application in aquaculture practices.


Assuntos
Proteínas de Peixes/genética , Imunidade Inata/genética , Perciformes/genética , Transcriptoma/imunologia , Animais , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Padrões Moleculares Associados a Patógenos/metabolismo , Perciformes/imunologia , Filogenia
4.
Front Physiol ; 10: 547, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31133878

RESUMO

In the context of intensive aquaculture, meagre (Argyrosomus regius) is one of the most important new aquaculture species in Southern Europe and several studies are focused on the optimization of its culture. Nevertheless, stressors such as handling during transport or culture maintenance may affect the immune system, thereby impairing some immune responses or provoking cellular damage. One strategy that has been used to avert this type of negative stress response is the supplementation of amino acids to improve resistance to stress. In this experiment, meagre (105.0 ± 2.6 g, mean ± standard deviation) juveniles were fed two diets for a period of 7 days, the first a commercial diet supplemented with 1% tryptophan (Trp) and second, the same commercial diet without tryptophan supplementation (control group). The effects of two types of handling stressors (air exposure and confinement/netting) on fish fed both diets was evaluated in terms of gene expression of the selected gut immunity markers, such as (1) innate immune response processes: c3 complement (c3), lysozyme (lys), and cyclooxygenase (cox2); (2) humoral immune response processes: interferon type 1 (ifn1), mx protein (mxp), interleukin 1b (il-1b), tumor necrosis factor 1a (tnf1a), and interleukin 10 (il-10); (3) antimicrobial peptides: defensin (def), hepcidin (hep), piscidin (pis), and a marker for mitochondrial respiration: glyceraldehyde 3-phosphate dehydrogenase (gapdh). Samples of the anterior intestine were collected at 1 and 6 h post-stress (hps). Results showed that in fish fed 1% Trp, the air exposure resulted in an upregulation of gene expression at 6 hps for c3, lys, cox2, ifn1, mxp, il-10 and gapdh, and il-1b and pis. The confinement/netting test for fish fed 1% Trp resulted in an upregulation of c3 and mxp and a downregulation of cox2, ifn1, il-1b, tnf1a, il-10, def, hep, and gapdh at both post-stress times (1 and 6 hps). According to the present study, dietary supplementation with 1% Trp may be considered as a proper nutritional strategy for improving tolerance and/or alleviating acute response to handling stressors.

5.
Harmful Algae ; 84: 27-35, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31128810

RESUMO

Ostreopsis is a toxic benthic dinoflagellate largely distributed worldwide in tropical and temperate areas. In the Mediterranean Sea, periodic summer blooms have been reported and have become a serious concern due to their direct impact on human health and the environment. Current microalgae identification is performed via light microscopy, which is time-consuming and is not able to differentiate among Ostreopsis species. Therefore, there is mature need for rapid, specific and easy-to-use detection tools. In this work, a colorimetric assay exploiting a combination of recombinase polymerase amplification (RPA) and a sandwich hybridisation assay was developed for O. cf. ovata and O. cf. siamensis detection and quantification. The specificity of the system was demonstrated by cross-reactivity experiments and calibration curves were successfully constructed using genomic DNA, achieving limits of detection of 10 and 14 pg/µL for O. cf. ovata and O. cf. siamensis, respectively. The assay was applied to the analysis of planktonic and benthic environmental samples from different sites of the Catalan coast. Species-specific DNA quantifications were in agreement with qPCR analysis, demonstrating the reliability of the colorimetric approach. Significant correlations were also obtained between DNA quantifications and light microscopy counts. The approach may be a valuable tool to provide timely warnings, facilitate monitoring activities or study population dynamics, and paves the way towards the development of in situ tools for the monitoring of harmful algal blooms.


Assuntos
Colorimetria , Dinoflagelados , DNA , Humanos , Mar Mediterrâneo , Reprodutibilidade dos Testes
6.
Food Chem ; 290: 255-262, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31000045

RESUMO

Two small Lagocephalus sceleratus juveniles were captured in picarel targeting catches from North Aegean Sea (Greece) in the autumn of 2017. An electrochemical immunosensing tool using magnetic beads as immobilisation support was developed and applied to the rapid screening of tetrodotoxins (TTXs), potent neurotoxins that constitute a food safety hazard when present in seafood. This tool revealed the presence of TTXs in both individuals. Results were compared with those provided by mELISA and LC-HRMS, the latter confirming the presence of TTX. Some of the tissues contained TTX contents close to or above 2 mg/kg. L. sceleratus juveniles had been considered as non-toxic and, to our knowledge, this is the first report of high TTX levels in small L. sceleratus individuals. Such specimens can be mistaken with other edible species, posing a threat to consumers. The availability of low-cost and user-friendly tools for TTXs detection will contribute to guarantee seafood safety.


Assuntos
Técnicas Eletroquímicas/métodos , Tetraodontiformes/metabolismo , Tetrodotoxina/análise , Animais , Bactérias/isolamento & purificação , Grécia , Separação Imunomagnética/métodos , Oceanos e Mares , Alimentos Marinhos/análise , Alimentos Marinhos/microbiologia , Tetraodontiformes/crescimento & desenvolvimento , Tetrodotoxina/isolamento & purificação
7.
Front Microbiol ; 10: 368, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30873146

RESUMO

The genus Arcobacter (Vandamme et al., 1991), comprised of Campylobacter-related species, are considered zoonotic emergent pathogens. The presence of Arcobacter in food products like shellfish, has an elevated incidence worldwide. In this study, we developed a specific viable quantitative PCR (v-qPCR), using the dye propidium monoazide (PMA), for quantification of the viable Arcobacter spp. cells in raw oysters and mussels. The high selectivity of primers was demonstrated by using purified DNA from 38 different species, 20 of them from the genus Arcobacter. The optimization of PMA concentration showed that 20 µM was considered as an optimal concentration that inhibits the signal from dead cells at different concentrations (OD550 from 0.2 to 0.8) and at different ratios of live: dead cells (50:50 and 90:10). The v-qPCR results from shellfish samples were compared with those obtained in parallel using several culture isolation approaches (i.e., direct plating on marine and blood agar and by post-enrichment culturing in both media). The enrichment was performed in parallel in Arcobacter-CAT broth with and without adding NaCl. Additionally, the v-qPCR results were compared to those obtained with traditional quantitative (qPCR). The v-qPCR and the qPCR resulted in c.a. 94% of positive detection of Arcobacter vs. 41% obtained by culture approaches. When examining the reduction effect resulting from the use of v-qPCR, samples pre-enriched in Arcobacter-CAT broth supplemented with 2.5% NaCl showed a higher reduction (3.27 log copies) than that of samples obtained directly and those pre-enriched in Arcobacter-CAT broth isolation (1.05 and 1.04). When the v-qPCR was applied to detect arcobacter from real shellfish samples, 15/17 samples tested positive for viable Arcobacter with 3.41 to 8.70 log copies 1g-1. This study offers a new tool for Arcobacter surveillance in seafood.

8.
Fish Shellfish Immunol ; 84: 509-520, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30227257

RESUMO

This study investigates the development of lymphoid organs and mucosal tissues in larval and juvenile meagre, Argyrosomus regius. For this purpose, meagre larvae were reared from hatch to the juvenile stage, under mesocosm conditions at 18-19 °C, using standard feeding sequences with live prey and artificial food. The kidney was evident upon hatch and included a visible pronephros, with undifferentiated stem cells and excretory tubules at 1 dph (3.15 ±â€¯0.1 mm SL). The thymus was first detected 8 dph (4.49 ±â€¯0.39 mm SL) and was clearly visible 12 dph (5.69 ±â€¯0.76 mm SL), 33 dph (15.69 ±â€¯1.81 mm SL) an outer thymocytic zone and inner epithelial zone were visible. The spleen was present 12 dph, located between exocrine pancreas and intestine and by 26 dph (11.84 ±â€¯1.3 mm SL) consisted of a mass of sinusoids filled with red blood cells. Melanomacrophage centers were found 83 dph (66.25 ±â€¯4.35 mm SL) in the spleen. Between 14-15 dph (6.9 ±â€¯1.1 mm SL), goblet and rodlet cells appear in the gill and intestinal epithelium. The lymphoid organs, which appear in the order of pronephric kidney (1 dph), thymus (8 dph) and spleen (12 dph) remarkably increase in size during the post-flexion stage. While functional studies are needed to confirm the activity of the immune response, the morphology of the lymphoid organs suggest that meagre is not immuno-competent until 83 dph.


Assuntos
Tecido Linfoide/crescimento & desenvolvimento , Membrana Mucosa/crescimento & desenvolvimento , Perciformes/crescimento & desenvolvimento , Animais , Tecido Linfoide/imunologia , Membrana Mucosa/imunologia , Perciformes/imunologia
9.
Anal Chim Acta ; 1039: 140-148, 2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30322545

RESUMO

Karlodinium is a dinoflagellate responsible for fish-killing events worldwide. In Alfacs Bay (NW Mediterranean Sea), the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. This work presents a method that combines recombinase polymerase amplification (RPA) with an enzyme-linked oligonucleotide assay (ELONA) to identify, discriminate and quantify these two species. The system was characterised using synthetic DNA and genomic DNA, and the specificity was confirmed by cross-reactivity experiments. Calibration curves were constructed using 10-fold dilutions of cultured cells, attaining a limit of detection of around 50,000 cells/L, far below the Karlodinium spp. alert threshold (200,000 cells/L). Finally, the assay was applied to spiked seawater samples, showing an excellent correlation with the spiking levels and light microscopy counts. This approach is more rapid, specific and user-friendly than traditional microscopy techniques, and shows great promise for the surveillance and management of harmful algal blooms.


Assuntos
Ensaio de Imunoadsorção Enzimática , Toxinas Marinhas/análise , Microalgas/química , Oligonucleotídeos/química , Reação em Cadeia da Polimerase em Tempo Real , Oligonucleotídeos/metabolismo
10.
PLoS One ; 13(10): e0205207, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30281676

RESUMO

Ostreid herpesvirus-1 (OsHV-1) has been involved in mass mortality episodes of Pacific oysters Crassostrea gigas throughout the world, causing important economic losses to the aquaculture industry. In the present study, magnetic beads (MBs) coated with an anionic polymer were used to capture viable OsHV-1 from two types of naturally infected matrix: oyster homogenate and seawater. Adsorption of the virus on the MBs and characterisation of the MB-virus conjugates was demonstrated by real-time quantitative PCR (qPCR). To study the infective capacity of the captured virus, MB-virus conjugates were injected in the adductor muscle of naïve spat oysters, using oyster homogenate and seawater without MBs as positive controls, and bare MBs and sterile water as negative controls. Mortalities were induced after injection with MB-virus conjugates and in positive controls, whereas no mortalities were recorded in negative controls. Subsequent OsHV-1 DNA and RNA analysis of the oysters by qPCR and reverse transcription qPCR (RT-qPCR), respectively, confirmed that the virus was the responsible for the mortality event and the ability of the MBs to capture viable viral particles. The capture of viable OsHV-1 using MBs is a rapid and easy isolation method and a promising tool, combined with qPCR, to be applied to OsHV-1 detection in aquaculture facilities.


Assuntos
Aquicultura/métodos , Crassostrea/virologia , Vírus de DNA/isolamento & purificação , Alimentos Marinhos/virologia , Água do Mar/virologia , Animais , Vírus de DNA/genética , Vírus de DNA/patogenicidade , DNA Viral/isolamento & purificação , Separação Imunomagnética/métodos , Oceano Pacífico , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo
11.
Sci Total Environ ; 624: 1171-1179, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29929229

RESUMO

The genus Arcobacter comprises Campylobacter-related species, considered zoonotic emergent pathogens, the presence of which in water has been associated with fecal pollution. Discharges of fecal polluted water into the sea have been considered as one of the main reasons for the presence of Arcobacter in shellfish, and this may represent a risk for public health. In this study, the European Union shellfish food safety criteria based on levels of Escherichia coli were studied in relation to their capacity to predict the presence of Arcobacter species. In addition, the accumulation factor (AF) that measures the concentration ratio between the microbes present in the shellfish and in the water, was also studied for both bacteria. The results show that the presence of E. coli correlated with the presence of the potentially pathogenic species A. butzleri and A. cryaerophilus. However, in 26.1% of the shellfish samples (corresponding to those taken during summer months) E. coli failed to predict the presence of, for instance A. butzleri and A. skirrowii, among other species. In the rest of the samples a significant correlation between the concentration of E. coli and Arcobacter spp. (mussels and oyster; R2=0.744) was found. This study indicates that the presence of E. coli can predict the presence of pathogenic Arcobacter species in shellfish samples harvested from water with temperatures lower than 26.2°C. Consumption of shellfish collected at higher temperatures which may not be permissive to the growth of E. coli but does allow growth of Arcobacter spp., may represent a risk for consumers.


Assuntos
Arcobacter/crescimento & desenvolvimento , Contaminação de Alimentos/estatística & dados numéricos , Inocuidade dos Alimentos/métodos , Frutos do Mar/normas , Escherichia coli , União Europeia , Contaminação de Alimentos/legislação & jurisprudência , Frutos do Mar/microbiologia , Frutos do Mar/estatística & dados numéricos
12.
BMC Genomics ; 19(1): 396, 2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29793421

RESUMO

BACKGROUND: Aquaculture production is expected to double by 2030, and demands for aquafeeds and raw materials are expected to increase accordingly. Sustainable growth of aquaculture will require the development of highly nutritive and functional raw materials to efficiently replace fish meal. Enzymatic hydrolysis of marine and aquaculture raw materials could bring new functionalities to finished products. The aim of this study was to determine the zootechnical and transcriptomic performances of protein hydrolysates of different origins (tilapia, shrimp, and a combination of the two) in European seabass (Dicentrarchux labrax) fed a low fish meal diet (5%), for 65 days. RESULTS: Results were compared to a positive control fed with 20% of fish meal. Growth performances, anterior intestine histological organization and transcriptomic responses were monitored and analyzed. Dietary inclusion of protein hydrolysates in the low fish meal diet restored similar growth performances to those of the positive control. Inclusion of dietary shrimp hydrolysate resulted in larger villi and more goblet cells, even better than the positive control. Transcriptomic analysis of the anterior intestine showed that dietary hydrolysate inclusion restored a pattern of intestinal gene expression very close to the pattern of the positive control. However, as compared to the low fish meal diet and depending on their origin, the different hydrolysates did not modulate metabolic pathways in the same way. Dietary shrimp hydrolysate inclusion modulated more metabolic pathways related to immunity, while nutritional metabolism was more impacted by dietary tilapia hydrolysate. Interestingly, the combination of the two hydrolysates enhanced the benefits of hydrolysate inclusion in diets: more genes and metabolic pathways were regulated by the combined hydrolysates than by each hydrolysate tested independently. CONCLUSIONS: Protein hydrolysates manufactured from aquaculture by-products are promising candidates to help replace fish meal in aquaculture feeds without disrupting animal metabolism and performances.


Assuntos
Ração Animal/análise , Aquicultura , Bass/genética , Proteínas na Dieta/química , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Transcriptoma/efeitos dos fármacos , Animais , Hidrólise , Redes e Vias Metabólicas/efeitos dos fármacos
13.
Mar Pollut Bull ; 127: 437-444, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29475682

RESUMO

Chilean waters are often affected by Alexandrium catenella, one of the leading organisms behind Harmful Algae Blooms (HABs). Genetic variability for this species are commonly carried out from cultured samples, approach that may not accurately quantify genetic variability of this organism in the water column. In this study, genetic variability of A. catenella was determined by sequencing the rDNA region, in water samples from the Canal Puyuhuapi (South Austral Ecosystem of Chile). A. catenella was detected in 8,8% of samples analysed. All sequences obtained were A. catenella (Tamara complex group I), with three highly frequent haplotypes (34%), and twenty new haplotypes. These haplotypes increase the genetic variability from 2.8% to 3.14% in this area. Through this new method, genetic determination of A. catenella can accurately be monitored and ecological studies of this species can be implemented.


Assuntos
Dinoflagelados/genética , Ecossistema , Água/parasitologia , Chile , Variação Genética , Haplótipos
14.
Harmful Algae ; 71: 57-77, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29306397

RESUMO

Strains of a dinoflagellate from the Salton Sea, previously identified as Protoceratium reticulatum and yessotoxin producing, have been reexamined morphologically and genetically and Pentaplacodinium saltonense n. gen. et sp. is erected to accommodate this species. Pentaplacodinium saltonense differs from Protoceratium reticulatum (Claparède et Lachmann 1859) Bütschli 1885 in the number of precingular plates (five vs. six), cingular displacement (two widths vs. one), and distinct cyst morphology. Incubation experiments (excystment and encystment) show that the resting cyst of Pentaplacodinium saltonense is morphologically most similar to the cyst-defined species Operculodinium israelianum (Rossignol, 1962) Wall (1967) and O. psilatum Wall (1967). Collections of comparative material from around the globe (including Protoceratium reticulatum and the genus Ceratocorys) and single cell PCR were used to clarify molecular phylogenies. Variable regions in the LSU (three new sequences), SSU (12 new sequences) and intergenic ITS 1-2 (14 new sequences) were obtained. These show that Pentaplacodinium saltonense and Protoceratium reticulatum form two distinct clades. Pentaplacodinium saltonense forms a monophyletic clade with several unidentified strains from Malaysia. LSU and SSU rDNA sequences of three species of Ceratocorys (C. armata, C. gourreti, C. horrida) from the Mediterranean and several other unidentified strains from Malaysia form a well-supported sister clade. The unique phylogenetic position of an unidentified strain from Hawaii is also documented and requires further examination. In addition, based on the V9 SSU topology (bootstrap values >80%), specimens from Elands Bay (South Africa), originally described as Gonyaulax grindleyi by Reinecke (1967), cluster with Protoceratium reticulatum. The known range of Pentaplacodinium saltonense is tropical to subtropical, and its cyst is recorded as a fossil in upper Cenozoic sediments. Protoceratium reticulatum and Pentaplacodinium saltonense seem to inhabit different niches: motile stages of these dinoflagellates have not been found in the same plankton sample.


Assuntos
Dinoflagelados/classificação , California , DNA de Algas/análise , DNA de Protozoário/análise , DNA Ribossômico , Dinoflagelados/genética , Dinoflagelados/ultraestrutura , Genes de Protozoários , Microscopia Eletrônica de Varredura , Filogenia , Análise de Sequência de DNA
15.
Mar Environ Res ; 133: 6-14, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29174400

RESUMO

Rapid and cost-effective methods to monitor the presence of diarrhetic shellfish poisoning (DSP) toxins in seawater samples in an easy and reliable manner are required to protect human health and avoid economic losses to shellfish industry. Immunoassays for the detection of okadaic acid (OA) and dinophysistoxin-1 and dinophysistoxin-2 are developed by immobilising OA on self-assembled monothiols or dithiols in an ordered and oriented way, providing an effective limit of detection of ∼1 ng OA equiv./mL seawater. The immunoassays are applied to the analysis of the particulate fraction of seawater samples from two Catalan harbours (NW Mediterranean) and samples collected periodically from the Galician Rias (E Atlantic), as well as a reference mussel sample. Results are in agreement with LC-MS/MS and the certified values. OA concentration in seawater correlates with Dinophysis cell abundance, with a 1-2 weeks lag. The immunoassays provide powerful high-throughput analytical methods potentially applicable as alternative monitoring tools.


Assuntos
Monitoramento Ambiental/métodos , Imunoensaio , Toxinas Marinhas/análise , Ácido Okadáico/análise , Animais , Bivalves , Humanos , Água do Mar/química , Frutos do Mar , Intoxicação por Frutos do Mar
16.
Mar Drugs ; 15(12)2017 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-29258236

RESUMO

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.


Assuntos
Organismos Aquáticos/química , Dinoflagelados/química , Toxinas Marinhas/química , Cromatografia Líquida/métodos , Dinoflagelados/isolamento & purificação , Mar Mediterrâneo , Polienos/química , Piranos/química , Espectrometria de Massas em Tandem/métodos
17.
Fish Shellfish Immunol ; 69: 200-210, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28842373

RESUMO

Antimicrobial peptides (AMPs), components of innate immunity, play an important role in protecting fish. In this study we report the molecular cloning of full open reading frames and characterization of expression of three AMP genes (ß-defensin (defb), hepcidin (hep2), piscidin (pisc) in meagre (Argyrosomus regius). A phylogenetic analysis of the expressed sequences obtained shows the defensin isoform forms a clade with the other members of the beta class of this family, hepcidin corresponds to hepcidin 2, and piscidin corresponds to class I of its respective family. Gene expression profiles of AMPs was investigated, by means of quantification of mRNA in nine development stages, from 8 days post-hatching (dph) to accomplishment of juvenile form (120 dph). During development it was demonstrated defb, hep2, pisc were expressed in all stages of larval development and in juvenile tissues (kidney, spleen gut and gill). Moreover, expression patterns suggest the expression levels of theses AMPs are influenced by live prey (rotifer, Artemia) and first intake of commercial diet. Induction experiments in vivo (24 h) and in vitro (4, 12, 24 h) with PAMPs (LPS, poly (I:C), ß-glucan) revealed significant changes in gene expression of the three AMP genes, in kidney, spleen, gut and gill. However, expression profiles differed in magnitude and time course response. defb expression shows a similar trend in vivo and in vitro in kidney at 24 h after LPS and ß-glucan stimulation. The hep2 expression levels were up-regulated upon ß-glucan challenge in vivo, more in gut and gills than kidney, while in vitro hep2 expression was up-regulated in kidney cells by LPS, poly (I:C), ß-glucan (4 h). pisc expression was up-regulated in kidney cells, splenocytes by ß-glucan, but in gill cells by poly (I:C) and ß-glucan in vivo. However, pisc expression was upregulated in kidney cells by ß-glucan and gill cells by LPS at 4 post-stimulation in vitro. These data suggest that AMPs play an important role in defense against pathogens, with each AMP having differing efficacies against specific types of microorganisms, although follow-up studies focusing on the biological activities in fish are needed.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Padrões Moleculares Associados a Patógenos/farmacologia , Perciformes/genética , Perciformes/imunologia , Animais , Perfilação da Expressão Gênica/veterinária , Hepcidinas/genética , Hepcidinas/imunologia , Filogenia , beta-Defensinas/genética , beta-Defensinas/imunologia
18.
J Exp Biol ; 220(Pt 19): 3442-3454, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28733328

RESUMO

Fish are ectotherms and temperature plays a determinant role in their physiology, biology and ecology, and is a driver of seasonal responses. The present study assessed how thermal imprinting during embryonic and larval stages modified the response of adult fish to low water temperature. We targeted the gilthead sea bream, which develops a condition known as winter syndrome when it is exposed to low water temperatures. Eggs and larvae of sea bream were exposed to four different thermal regimes and then the response of the resulting adults to a low temperature challenge was assessed. Sea bream exposed to a high-low thermal regime as eggs and larvae (HLT; 22°C until hatch and then 18°C until larvae-juvenile transition) had increased plasma cortisol and lower sodium and potassium in response to a cold challenge compared with the other thermal history groups. Plasma glucose and osmolality were increased in cold-challenged HLT fish relative to the unchallenged HLT fish. Cold challenge modified bone homeostasis/responsiveness in the low-high thermal regime group (LHT) relative to other groups, and ocn, ogn1/2, igf1, gr and trα/ß transcripts were all downregulated. In the low temperature group (LT) and HLT group challenged with a low temperature, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities were decreased relative to unchallenged groups, and bone calcium content also decreased in the LT group. Overall, the results indicate that thermal imprinting during early development of sea bream causes a change in the physiological response of adults to a cold challenge.


Assuntos
Osso e Ossos/fisiologia , Temperatura Baixa , Proteínas de Peixes/genética , Expressão Gênica , Homeostase , Dourada/fisiologia , Animais , Proteínas de Peixes/metabolismo , Dourada/genética , Dourada/crescimento & desenvolvimento
19.
Front Physiol ; 8: 458, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28713287

RESUMO

Vitamin A (VA) and retinoid derivatives are known morphogens controlling vertebrate development. Despite the research effort conducted during the last decade, the precise mechanism of how VA induces post-natal bone changes, and particularly those operating through crosstalk with the thyroid hormones (THs) remain to be fully understood. Since effects and mechanisms seem to be dose and time-dependent, flatfish are an interesting study model as they undergo a characteristic process of metamorphosis driven by THs that can be followed by external appearance. Here, we studied the effects of VA imbalance that might determine Senegalese sole (Solea senegalensis) skeletogenetic phenotype through development of thyroid follicles, THs homeostasis and signaling when a dietary VA excess was specifically provided during pre-, pro- or post-metamorphic stages using enriched rotifers and Artemia as carriers. The increased VA content in enriched live prey was associated to a higher VA content in fish at all developmental stages. Dietary VA content clearly affected thyroid follicle development, T3 and T4 immunoreactive staining, skeletogenesis and mineralization in a dose and time-dependent fashion. Gene expression analysis showed that VA levels modified the mRNA abundance of VA- and TH-specific nuclear receptors at specific developmental stages. Present results provide new and key knowledge to better understand how VA and TH pathways interact at tissue, cellular and nuclear level at different developmental periods in Senegalese sole, unveiling how dietary modulation might determine juvenile phenotype and physiology.

20.
J Endocrinol ; 233(3): 381-394, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28420709

RESUMO

The impact of thermal imprinting on the plasticity of the hypothalamic-pituitary-interrenal (HPI) axis and stress response in an adult ectotherm, the gilthead sea bream (Sparusaurata, L.), during its development was assessed. Fish were reared under 4 thermal regimes, and the resulting adults exposed to acute confinement stress and plasma cortisol levels and genes of the HPI axis were monitored. Changes in immune function, a common result of stress, were also evaluated using histomorphometric measurements of melanomacrophages centers (MMCs) in the head kidney and by monitoring macrophage-related transcripts. Thermal history significantly modified the HPI responsiveness in adult sea bream when eggs and larvae were reared at a higher than optimal temperature (HT, 22°C), and they had a reduced amplitude in their cortisol response and significantly upregulated pituitary pomc and head kidney star transcripts. Additionally, after an acute stress challenge, immune function was modified and the head kidney of adult fish reared during development at high temperatures (HT and LHT, 18-22°C) had a decreased number of MMCs and a significant downregulation of dopachrome tautomerase. Thermal imprinting during development influenced adult sea bream physiology and increased plasma levels of glucose and sodium even in the absence of an acute stress in fish reared under a high-low thermal regime (HLT, 22-18°C). Overall, the results demonstrate that temperature during early development influences the adult HPI axis and immune function in a teleost fish.


Assuntos
Temperatura Alta , Imunidade Inata/fisiologia , Dourada/imunologia , Estresse Fisiológico/imunologia , Animais , Hidrocortisona/sangue , Sistema Hipotálamo-Hipofisário/imunologia , Glândula Inter-Renal/imunologia , Dourada/sangue
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