Marine biotoxins in the Catalan littoral: could biosensors be integrated into monitoring programmes?

Aquest article descriu els sensors enzimàtics i immunosensors electroquímics que s’han desenvolupat als nostres grups per a la detecció de la biotoxina marina àcid okadaic (OA), i discuteix la possibilitat d’integrar-los en programes de seguiment. Els sensors enzimàtics per a OA que es presenten es basen en la inhibició de la proteïna fosfatasa (PP2A) per aquesta toxina i la mesura electroquímica de l’activitat enzimàtica mitjançant l’ús de substrats enzimàtics apropiats, electroquímicament actius després de la seva desfosforació per l’enzim. Els immunosensors electroquímics descrits en aquest article es basen en un enzimoimmunoassaig sobre fase sòlida competitiu indirecte (ciELISA), amb fosfatasa alcalina (ALP) o peroxidasa (HRP) com a marcatges, i un sistema de reciclatge enzimàtic amb diaforasa (DI). Els biosensors presentats aquí s’han aplicat a l’anàlisi de dinoflagel·lats, musclos i ostres. Les validacions preliminars amb assaigs colorimètrics i LC-MS/MS han demostrat la possibilitat d’utilitzar les bioeines desenvolupades per al cribratge preliminar de biotoxines marines en mostres de camp o de cultiu, que ofereixen informació complementària a la cromatografia. En conclusió, tot i que encara cal optimitzar alguns paràmetres experimentals, la integració dels biosensors a programes de seguiment és viable i podria proporcionar avantatges respecte a altres tècniques analítiques pel que fa al temps d’anàlisi, la simplicitat, la selectivitat, la sensibilitat, el fet de poder ser d’un sol ús i l’efectivitat de cost. This article describes the electrochemical enzyme sensors and immunosensors that have been developed by our groups for the detection of marine biotoxin okadaic acid (OA), and discusses the possibility of integrating them into monitoring programmes. The enzyme sensors for OA reported herein are based on the inhibition of immobilised protein phosphatase 2A (PP2A) by this toxin and the electrochemical measurement of the enzyme activity through the use of appropriate enzyme substrates, which are electrochemically active after dephosphorylation by the enzyme. The electrochemical immunosensors described in this article are based on a competitive indirect Enzyme- Linked ImmunoSorbent Assay (ciELISA), using alkaline phosphatase (ALP) or horseradish peroxidase (HRP) as labels, and an enzymatic recycling system with diaphorase (DI). The biosensors presented herein have been applied to the analysis of dinoflagellates, mussels and oysters. Preliminary validations with colorimetric assays and LC-MS/MS have demonstrated the possibility of using the developed biotools for the preliminary screening of marine biotoxins in field or cultured samples, offering complementary information to chromatography. In conclusion, although optimisation of some experimental parameters is still required, the integration of biosensors into monitoring programmes is viable and may provide advantages over other analytical techniques in terms of analysis time, simplicity, selectivity, sensitivity, disposability of electrodes and cost effectiveness.

The effects of feeding Karlodinium veneficum (PLY # 103; Gymnodinium veneficum Ballantine) to the blue mussel Mytilus edulis

The effects of exposure to the type species for Karlodinium veneficum (PLY # 103) on immune function and histopathology in the blue mussel Mytilus edulis were investigated. Mussels from Whitsand Bay, Cornwall (UK) were exposed to K. veneficum (PLY # 103) for 3 and 6 days. Assays for immune function included total and differential cells counts, phagocytosis and release of extra cellular reactive oxygen species. Histology was carried out on digestive gland and mantle tissues. The toxin cell quota for K. veneficum (PLY #103) was measured by liquid chromatography-mass spectrometry detecting two separable toxins KvTx1 (11.6 ± 5.4 ng/ml) and KvTx2 (47.7 ± 4.2 ng/ml). There were significant effects of K. veneficum exposure with increasing phagocytosis and release of reactive oxygen species following 6 days exposure. There were no significant effects on total cell counts. However, differential cell counts did show significant effects after 3 days exposure to the toxic alga. All mussels produced faeces but not pseudofaeces indicating that algae were not rejected prior to ingestion. Digestive glands showed ingestion of the algae and hemocyte infiltration after 3 days of exposure, whereas mantle tissue did not show differences between treatments. As the effects of K. veneficum were not observed in the mantle tissue it can be hypothesized that the algal concentration was not high enough, or exposure long enough, to affect all the tissues. Despite being in culture for more than 50 years the original K. veneficum isolate obtained by Mary Parke still showed toxic effects on mussels.

Pathology and immune response of the blue mussel (Mytilus edulis L.) after an exposure to the harmful dinoflagellate Prorocentrum minimum

The harmful dinoflagellate Prorocentrum minimum has different effects upon various species of grazing bivalves, and these effects also vary with life-history stage. Possible effects of this dinoflagellate upon mussels have not been reported; therefore, experiments exposing adult blue mussels, Mytilus edulis, to P. minimum were conducted. Mussels were exposed to cultures of toxic P. minimum or benign Rhodomonas sp. in glass aquaria. After a short period of acclimation, samples were collected on day 0 (before the exposure) and after 3, 6, and 9 days of continuous-exposure experiment. Hemolymph was extracted for flow-cytometric analyses of hemocyte, immune-response functions, and soft tissues were excised for histopathology. Mussels responded to P. minimum exposure with diapedesis of hemocytes into the intestine, presumably to isolate P. minimum cells within the gut, thereby minimizing damage to other tissues. This immune response appeared to have been sustained throughout the 9-day exposure period, as circulating hemocytes retained hematological and functional properties. Bacteria proliferated in the intestines of the P. minimum-exposed mussels. Hemocytes within the intestine appeared to be either overwhelmed by the large number of bacteria or fully occupied in the encapsulating response to P. minimum cells; when hemocytes reached the intestine lumina, they underwent apoptosis and bacterial degradation. This experiment demonstrated that M. edulis is affected by ingestion of toxic P. minimum; however, the specific responses observed in the blue mussel differed from those reported for other bivalve species. This finding highlights the need to study effects of HABs on different bivalve species, rather than inferring that results from one species reflect the exposure responses of all bivalves.

Experimental exposure of the blue mussel (Mytilus edulis, L.) to the toxic dinoflagellate Alexandrium fundyense: Histopathology, immune responses, and recovery

Mussels (Mytilus edulis) were exposed to cultures of the toxic dinoflagellate Alexandrium fundyense or the non-toxic alga Rhodomonas sp. to evaluate the effects of the harmful alga on the mussels and to study recovery after discontinuation of the A. fundyense exposure. Mussels were exposed for 9 days to the different algae and then all were fed Rhodomonas sp. for 6 more days. Samples of hemolymph for hemocyte analyses and tissues for histology were collected before the exposure and periodically during exposure and recovery periods. Mussels filtered and ingested both microalgal cultures, producing fecal pellets containing degraded, partially degraded, and intact cells of both algae. Mussels exposed to A. fundyense had an inflammatory response consisting of degranulation and diapedesis of hemocytes into the alimentary canal and, as the exposure continued, hemocyte migration into the connective tissue between the gonadal follicles. Evidence of lipid peroxidation, similar to the detoxification pathway described for various xenobiotics, was found; insoluble lipofuchsin granules formed (ceroidosis), and hemocytes carried the granules to the alimentary canal, thus eliminating putative dinoflagellate toxins in feces. As the number of circulating hemocytes in A. fundyense-exposed mussels became depleted, mussels were immunocompromised, and pathological changes followed, i.e., increased prevalences of ceroidosis and trematodes after 9 days of exposure. Moreover, the total number of pathological changes increased from the beginning of the exposure until the last day (day 9). After 6 days of the exposure, mussels in one of the three tanks exposed to A. fundyense mass spawned; these mussels showed more severe effects of the toxic algae than non-spawning mussels exposed to A. fundyense. No significant differences were found between the two treatments during the recovery period, indicating rapid homeostatic processes in tissues and circulating hemocytes.

An integrated approach for the assessment of HAB dynamics in two NW Mediterranean bays from a GEOHAB perspective

Alfacs and Fangar Bay in the Ebro Delta, NW Mediterranean are the major sites in Catalonia for shellfish cultivation. These bays are subject to occasional closures in shellfish harvesting due to the presence of phycotoxins. Fish kills have also been associated with harmful algal blooms. The comparison of phytoplankton dynamics in both bays offers the opportunity to reveal differences in bloom patterns of species known to be harmful for the ecosystem and aquaculture activities. Field research is underway under the GEOHAB framework within the Core Research Project on HABs in Fjords and Coastal Embayments. The overall objective of this study is to improve our understanding of HAB biogeographical patterns, and key elements driving bloom dynamics in time and space within these semi-constrained embayments. Via the comparative approach we aim to improve the prediction for monitoring purposes, with a focus on Karlodinium spp. associated with massive kills of aquaculture species. This objective is addressed by incorporating long-term time series of phytoplankton identification and enumeration with the first results of recent field work in both bays. The latter includes the application of optical sensors, to yield a complementary view with enhanced spatial and temporal resolution of bloom phenomena.

Is Microtox R toxicity related to potentially harmful algae proliferation in Mediterranean salt marshes?

Mediterranean salt marshes are ecosystems that are highly influenced by sea changes and freshwater inputs from runoff. In these ecosystems, toxic and non-toxic algae blooms often produce large and unpredictable biomasses of phytoplankton. The Microtox R test has been described as a successful, quick method for detecting toxicity in various phytoplankton taxa. Ourstudy sought to test the efficiency of Microtox R in detecting toxic HAB in Mediterranean salt marshes. The results showed that the Microtox R test was able to detect toxic substances in the particulate matter of several lagoons in the Empordà salt marshes. This Microtox R toxicity coincided with periods when potentially harmful cyanobacteria, dinoflagellates andhaptophytes had a high biomass. The results suggest that potentially harmful phytoplankton cannot be ruled out as a source of Microtox R

Is Microtox R toxicity related to potentially harmful algae proliferation in Mediterranean salt marshes?

Mediterranean salt marshes are ecosystems that are highly influenced by sea changes and freshwater inputs from runoff. In these ecosystems, toxic and non-toxic algae blooms often produce large and unpredictable biomasses of phytoplankton. The Microtox R test has been described as a successful, quick method for detecting toxicity in various phytoplankton taxa. Ourstudy sought to test the efficiency of Microtox R in detecting toxic HAB in Mediterranean salt marshes. The results showed that the Microtox R test was able to detect toxic substances in the particulate matter of several lagoons in the Empordà salt marshes. This Microtox R toxicity coincided with periods when potentially harmful cyanobacteria, dinoflagellates and haptophytes had a high biomass. The results suggest that potentially harmful phytoplankton cannot be ruled out as a source of Microtox R

Evolució de les poblacions d'algues i dels paràmetres fisicoquímics de dos bassals de la Riera de la Valleta

Treball de recerca realitzat per un alumne d’ensenyament secundari i guardonat amb un Premi CIRIT per fomentar l'esperit científic del Jovent l’any 2008. S’ha observat l’evolució de les poblacions d’algues (i d’altres microorganismes) de dos bassals de la riera de la Valleta –Alt Empordà – que, durant l’estiu, quan aquesta riera s’asseca, conserven l’aigua. S’han mesurat diversos paràmetres fisicoquímics de diverses mostres (temperatura, pH, percentatge d’oxigen i lluminositat) intentant trobar relacions entre alguns d’aquests paràmetres, i entre aquests i les poblacions d’algues observades.

El Consum de drogues tòxiques, estupefaents i substàncies pisoctròpiques, per menors d'edat, en llocs públics del districte d'Horta-Guinardó de la ciutat de Barcelona

Com ja he indicat en més d’una ocasió al llarg d’aquest Màster, l’elecció del tema sobre el que versa aquest Projecte final respon, en bona part, a l’interès que em va suscitar la qüestió durant el temps que vaig estar ocupant el lloc de cap del Servei d’Assessorament Jurídic de la Direcció General de la Policia. Entre les funcions que aquesta unitat tenia assignades hi havia la tramitació dels procediments sancionadors incoats per les denúncies efectuades pels cossos de policia local i la policia de la Generalitat-mossos d’esquadra, per infraccions a la Llei orgànica 1/1992, de 21 de febrer, de protecció de la seguretat ciutadana. Al Servei d’Assessorament Jurídic li va correspondre la responsabilitat d’iniciar la tramitació de les denúncies esmentades, tan bon punt es va engegar el procés de desplegament de la policia de la Generalitat-mossos d’esquadra com a policia ordinària i integral, en substitució de les forces i els cossos de seguretat de l’Estat, a l’octubre de 1994. A mesura que s’anava produint el desplegament, la funció de tramitar les actes esteses per la policia de la Generalitat-mossos d’esquadra i per les policies locals, es va anar assumint pels serveis territorials d’Administració en què es va estructurar la Direcció General de Seguretat Ciutadana (en l’actualitat, Direcció General de la Policia).

Cianofícies i algues aerofítiques de substrats carbonatats.

Atmophytic Cyanophyta and Algae from limestone substrata. The walls of caves and tombs support an abundant colonization of light-dependent organisms. A total of 4 liverworts, 3 mosses, 7 lichens and 28 cyanophyta and algae are reported from 3 collecting sites. The development and specific composition of these organisms is related to microclimatic parameters. Opportunistic species present in soils are found on the mouth of hypogeal niches. Light attenuation allows the development of calcifying cyanophyta following a succesional pattern, each level having a different dominant species. Communities dominated by Scytonema julianum are replaced by Herpyzonema pulverulentum and, towards the less iluminated area, by Geitleria calcarea and Loriella sp. Key words: Caves, Light attenuation, Non vascular plants, Colonization, Calcifying cyanophyta, Scytonema julianum, Herpyzonema pulverulentum, Geitleria calcarea, Chlorophyta, Diatoms, Microclimatic gradient.

Aportació al coneixement de les algues marines de les costes del País Valencià

En aquest treball donem a coneixer els primers resultats sobre la flora algologica del Pais Valencia. El material que hem pogut recollir i determinar fins aquest moment procedeix de setze localitats de la costa que estem estudiant, compresa entre Peniscola i el Cap de Santa Pola.