Francesc Padrós

Ontogeny of the lymphoid organs in the turbot Scophthalmus maximus: a light and electron microscope study

Abstract A histological and ultrastructural study was made of the development of the kidney, thymus and spleen in the turbot Scophthalmus maximus , from hatching until the end of metamorphosis. Primordial haemopoietic stem cells are first observed in the pronephric kidney very early after hatch and rapidly differentiate into different cellular types. The spleen develops later, soon becoming rich in blood capillaries, red blood cells and thrombocytes. The thymus is the last lymphoid organ to appear but shows a quick development. This organ seems to originate from haemopoietic stem cells migrating from the head region of the kidney. Lymphoid organs become lymphoid in the sequence thymus, kidney and spleen. Although a small number of lymphocytes appear only in the later stages, cellular types involved in non-specific defense mechanisms, such as macrophagic and reticular cells, originate in early stages. These observations suggest that non-specific systems may play an important role in the immunocompetence mechanisms of the turbot during early larval development.

Ecological relevance of biomarkers in monitoring studies of macro-invertebrates and fish in Mediterranean rivers.

Mediterranean rivers are probably one of the most singular and endangered ecosystems worldwide due to the presence of many endemic species and a long history of anthropogenic impacts. Besides a conservation value per se, biodiversity is related to the services that ecosystems provide to society and the ability of these to cope with stressors, including climate change. Using macro-invertebrates and fish as sentinel organisms, this overview presents a synthesis of the state of the art in the application of biomarkers (stress and enzymatic responses, endocrine disruptors, trophic tracers, energy and bile metabolites, genotoxic indicators, histopathological and behavioural alterations, and genetic and cutting edge omic markers) to determine the causes and effects of anthropogenic stressors on the biodiversity of European Mediterranean rivers. We also discuss how a careful selection of sentinel species according to their ecological traits and the food-web structure of Mediterranean rivers could increase the ecological relevance of biomarker responses. Further, we provide suggestions to better harmonise ecological realism with experimental design in biomarker studies, including statistical analyses, which may also deliver a more comprehensible message to managers and policy makers. By keeping on the safe side the health status of populations of multiple-species in a community, we advocate to increase the resilience of fluvial ecosystems to face present and forecasted stressors. In conclusion, this review provides evidence that multi-biomarker approaches detect early signs of impairment in populations, and supports their incorporation in the standardised procedures of the Water Frame Work Directive to better appraise the status of European water bodies.

Effects of the gill parasite Zeuxapta seriolae (Monogenea: Heteraxinidae) on the amberjack Seriola dumerili Risso (Teleostei: Carangidae)

Zeuxapta seriolae (Monogenea: Heteraxinidae) infection was associated with important mortalities of amberjacks (Seriola dumerili) reared in tanks in the experimental facilities of the Spanish Institute of Oceanography (Western Mediterranean) during the period 1998–2000. Fish infested by this parasite presented characteristics similar to those described for Seriola quinqueradiata parasitized by Heteraxine heterocerca in Japan. All dead amberjacks had high parasite abundance (mean abundanceFS.D.=686.7F125.4) and egg strings entangled in gills. The parasitological analysis of 17 live amberjacks collected from the infested tanks showed that 41.2% of fish harboured Z. seriolae with intensities ranging between 5 and 731 parasites per fish. No apparent relationship was observed between the abundance of the parasite and the condition factor (rS=0.175, n=17, p>0.5). However, the haematocrit values were significantly lower in the fish infested with monogeneans. The relationship between parasite abundance and haematocrit values was negative and statistically significant (rS=0.625, n=17, p<0.01). The light and scanning electron microscope studies of affected gills revealed that the parasites attached to the host grasping one or two lamellae with each clamp of the haptor, which led to lamellar synechiae, lamellar clubbing and disruption of epithelial and vascular structures. A mild to moderate epitheliocystis infection was also

Effects of temperature decrease on feeding rates, immune indicators and histopathological changes of gilthead sea bream Sparus aurata fed with an experimental diet

Gilthead sea bream Sparus aurata were fed with either a commercial feed or a specifically prepared “winter” feed in order to assess whether fish showed signs similar to the so-called winter syndrome and whether the experimental diet showed protection against the syndrome. Fish were subjected to a ramp of temperature decrease and after 2 weeks, a further recovery of temperature. The temperature ramp produced changes on most immune indicators and feed intake was affected more severely than predicted by feeding tables. However, no generalised signs of the syndrome were detected. The experimental diet tested showed some degree of immune protection related to complement and phagocytosis activity and slight changes in the levels of leucocytic infiltration of the intestinal mucosa and submucosa.

Low-temperature challenges to gilthead sea bream culture: review of cold-induced alterations and ‘Winter Syndrome’

Although gilthead sea bream have been cultured successfully for the last two decades they are particularly sensitive to low temperature. Especially in the northern Mediterranean area, cold affects fish health and decreases fish-farm production, and may even cause mortality through what is known as ‘Winter Disease’ or ‘Winter Syndrome’. This paper reviews the diagnosis and physiological effects of this disease, focusing on recent studies of cold-induced alterations in gilthead sea bream physiology. ‘Winter Syndrome’ is characterised by multi-organ dysfunction entailing hyposensitivity, erratic swimming, pale and friable livers, necrotic muscles, atrophy of the exocrine pancreas, and distended digestive tract. Its complex aetiology involves several factors such as thermal stress, metabolic depression, immune suppression, and occasional opportunistic pathogens. Low temperatures may be the initial cause of all these factors, except pathogen action. Indoor studies have demonstrated that a drop in temperature causes cold-induced fasting, thermal stress and metabolic depression. These immediate effects are related to an ionic imbalance caused by malfunctions of the gills and digestive system. They are also related to a fatty liver, which appeared steatotic and affected hepatic metabolism and blood composition. The result is a lower immune capacity and fish that are more susceptible to infection. There is no significant thermal compensation under cold conditions and in this situation any additional stress factors can cause fish to suffer metabolic collapse. This study reviews the physiological and zootechnical origins of the disease and, where possible, recommends ways of improving culture conditions during pre-cold, cold and recovery periods.

First evidence of polybrominated diphenyl ether (flame retardants) effects in feral barbel from the Ebro River basin (NE, Spain)

Polybrominated diphenyl ethers (PBDEs) are chemicals of environmental concern due to their lipophilic, persistent and bioaccumulable characteristics as well as for their potential endocrine disrupting role. Former studies carried out in a tributary of the Cinca river (Ebro basin, NE Spain) revealed high levels of PBDEs in fish due to the discharges of effluents rich in PBDEs coming from a nearby industrial park in Barbastro. In this study, several biomarkers of pollutants exposure were measured in barbel, Barbus graellsii, before (upstream) and after (downstream) the main industrial site (Barbastro) in the Vero river. The results evidenced an enhanced hepatic phase I and II metabolism (measured as reductases, glutathione S transferase and uridinediphospho-glucuronosyltransferase), and of the antioxidant enzyme glutathione peroxidase. Conversely, fishes collected from downstream reaches had their phase I CYP1A dependent ethoxyresorufin O-deethylase, antioxidant diaphorase and brain cholinesterase activities depleted. In addition, the histological study of the liver and kidney of these fish evidenced an increase of the number and size of macrophage aggregates in most individuals collected downstream. Bivariate correlated analyses showed that the above mentioned biomarkers were correlated to measured PBDE congeners, thus indicating that the observed biological effects were unlikely to be related to other environmental factors than PBDEs. Overall, the measured biochemical and histological markers provide new evidence that in field exposed fish, PBDEs levels were associated with high activities of phase I and II metabolic enzymes, oxidative stress in liver, neurotoxicity in brain and histopathological effects in both liver and kidney.

Zebrafish Models for Human Acute Organophosphorus Poisoning

Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

MARTX of Vibrio vulnificus biotype 2 is a virulence and survival factor.

Vibrio vulnificus biotype 2 is a polyphyletic group whose virulence for fish relies on a plasmid. This plasmid contains an rtxA gene duplicated in the small chromosome that encodes a MARTX (Multifunctional, Autoprocessing Repeats-in-Toxin) unique within the species in domain structure (MARTX type III). To discover the role of this toxin in the fitness of this biotype in the fish-farming environment, single- and double-knockout mutants were isolated from a zoonotic strain and analysed in a series of in vivo and in vitro experiments with eel, fish cell lines and amoebae isolated from gills. Mice, murine and human cell lines were also assayed for comparative purposes. The results suggest that MARTX type III is involved in the lysis of a wide range of eukaryotic cells, including the amoebae, erythrocytes, epithelial cells and phagocytes after bacterium-cell contact. In fish, MARTX type III may act as a toxin involved in the onset of septic shock, while in mice it may promote bacterial colonization by preventing phagocytosis of bacterial cells. Moreover, this toxin could protect bacteria from predation by amoebae, which would increase bacterial survival outside the host and would explain the fitness of this biotype in the fish-farming environment.

Parasitisation by Bathycreadium elongatum (Digenea, Opecoelidae) in pyloric caeca of Trachyrincus scabrus (Teleostei, Macrouridae)

A novel process of transmural passive displacement of a digenean parasite was studied in the digestive tract of the roughsnout grenadier Trachyrincus scabrus, which is found in the northwestern Mediterranean Sea. This mechanism seems to facilitate the elimination of a significant portion of intestinal parasites. The digenean parasite Bathycreadium elongatum was found in the intestine, mainly within pyloric caeca, in 74.4% of T. scabrus, with a mean abundance of 44 individuals per fish. Nodule-like lesions were also found in the mesentery of pyloric caeca of infected T. scabrus. Histological sections of the nodules revealed granulomatous inflammatory responses surrounding degraded digeneans. Partial nucleotide sequences of the 28S rRNA gene obtained from intracaecal B. elongatum and from the core of the nodules of the mesentery of pyloric caeca showed 100% mutual identity with an overlap of 971 bp. The greatest abundance of both intracaecal B. elongatum and nodules occurred in spring. During summer, and especially autumn, the abundance of intracaecal B. elongatum decreased. Prevalence and abundance of nodules increased in autumn. In winter intracaecal parasite abundance and prevalence began to increase, but decreased again in nodules. During spring and summer, parasites pass into the visceral cavity, hypothetically owing to the fragility of the wall of pyloric caeca in their apical zone, and become degraded through a granulomatous inflammatory response. This process seems to have a detrimental effect on the B. elongatum cycle since some of parasites are trapped and degrade in the connective tissue in which they are unable to complete their life cycle.

Microbial and histopathological study of the vibriosis caused by Vibrio vulnificus serovar E in eels: the metalloprotease Vvp is not an essential lesional factor.

Vibrio vulnificus biotype 2 serovar E (Bt2-serE) is a zoonotic pathogen that causes a haemorrhagic septicaemia in eels, called warm water vibriosis. The main objective of the present work was to study the onset of the eel vibriosis from the microbiological and histopathological viewpoint, as well as to ascertain the role of the protease Vvp as a lesional factor by comparing the histopathological lesions caused by the wild strain and its vvp deficient derivative. The wild-type strain was observed to attach to the gills, where it multiplied following saturation dynamics, subsequently invading the blood stream and reaching the internal organs. Here it reached population sizes that are notably lower than those associated with other fish septicaemia. Parallel to bacterial growth, there was a notable decrease in haematocrit values and haemoglobin concentration in blood as well as extensive haemorrhages in all the analysed organs. The main histopathological lesions were detected in the head kidney in the form of extensive necrosis affecting the haematopoietic tissue. Very few bacteria were visualized in the different organs, most of which were close to blood cells and capillary vessels, which is compatible with the results obtained in the microbiological study. The same lesions were produced when extracellular products (ECPs) were injected instead of bacteria or when the vvp-defective mutant or its ECPs were injected. The overall results suggest that the pathology caused by V. vulnificus in the eel is not caused by massive bacterial growth in the blood and internal organs but, rather, by the effect of potent toxic factors other than the metalloprotease, which have yet to be determined.