Inmaculada Varó

Effect of dichlorvos on cholinesterase activity of the European sea bass (Dicentrarchus labrax)

In this study, the acute toxicity of the organophosphorous pesticide (OP) dichlorvos and both in vitro and in vivo effects of dichlorvos on cholinesterase (ChE) activity of the European sea bass (Dicentrarchus labrax) were investigated. The characterisation of ChE and the “normal” range of activity in brain and muscle of non-exposed fish were determined in a first phase of the study. Acetylthiocholine was the substrate preferred of both brain and muscle ChE. Eserine sulphate and BW284C51 significantly inhibited the brain and muscle enzyme activity at low concentrations (μM range). Iso-OMPA had a significant effect in muscle, but not in brain tissue. These results suggest that acetylcholinesterase (AChE) is the predominant ChE form in brain tissue. In contrast, both acetylcholinesterase and butyrylcholinesterase seem to exist in muscle. Using acetylthiocholine as substrate, the “normal” range of fingerling head and muscle ChE were 58.05±2.11 and 118.03±8.67 U/mg protein, respectively. Corresponding values for juveniles were 43.32±4.42 and 19.44±2.44 U/mg protein for brain and muscle, respectively. Dichlorvos significantly inhibited the activity of ChE in the selected tissues, both in vitro and in vivo conditions. Differences in ChE sensitivity were found in relation to the age of the fish and the tissue analysed. The present study also showed that fingerlings of the European sea bass are relatively resistant to in vivo acute (96 h) dichlorvos exposure to concentrations between 0.125 and 1 mg/L, being able to tolerate high percentages of head ChE inhibition (37% and 76%) without lethal effects.

Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica

In this study, the acute toxicity of the organophosphorous pesticides dichlorvos and chlorpyrifos to two different species of Artemia (A. salina and A. parthenogenetica) was evaluated. In addition, the in vivo effect of these two pesticides on cholinesterase (ChE) activity of both A. salina and A. parthenogenetica was also determined. The characterisation of the ChE, using different substrates and specific inhibitors, and the normal range of activity in non-exposed individuals were previously investigated for both species. The results obtained indicate that the ChE of A. salina is different from that of A. parthenogenetica and that both enzymes cannot be classified neither as acetylcholinesterase nor as butyrylcholinesterase since they show intermediary characteristics between the two vertebrate forms. The range of normal ChE activity was 2.65+/-0.15 U/mg protein for A. salina, and 3.69+/-0.17 U/mg protein for A. parthenogenetica. Significant in vivo effects of both pesticides on Artemia ChE activity were found, at concentrations between 5.38 and 9.30 mg/l for dichlorvos and between 1.85 and 3.19 mg/l for chlorpyrifos. Both Artemia species are resistant to these pesticides and they are able to survive with more than 80% ChE inhibition. However, A. parthenogenetica is more resistant than A. salina, with about a 95% reduction in its ChE activity respect to the control for nauplii exposed to the median lethal concentrations (LC50), without lethal effects after 24 h of exposure.

Some aspects of Artemia biology affected by cestode parasitism

Artemia individuals from a wild population, bearing cestode cysticercoids (Hymenolepididae) were kept in the laboratory during four months to observe influences of the parasite on the host biology. No differences were found between parasitized and unparasitized shrimps regarding final adult size. The higher survival of parasitized animals suggests that parasitism is not pathogenic. The most dramatic effect was host castration. Parasitized individuals showed higher total lipid levels, probably linked to carotenoid pigments conveying a red color to parasitized shrimps.

Biomagnification Study on Organochlorine Compounds in Marine Aquaculture: The Sea Bass (Dicentrarchus labrax) as a Model

Biomagnification of organochlorine compounds (pesticides and polychlorinated biphenyls) through the marine aquaculture food chain is investigated. From first-feeding, specimens of sea bass were exposed to commercial fish feed (that contained DDTs and PCBs residues) for ca. 24 months, and selected tissues (white and red muscle, liver, and visceral fat) were analyzed after 6 and 24 months of diet exposure. Data obtained showed that experimental fish tissues presented a similar contamination pattern to that of fish feed, and biomagnification processes of these compounds were proved. Additionally, commercial sea bass cultured in farms from the western Mediterranean were analyzed, their organochlorine concentrations being significantly lower than those of the 24 month old experimental fish. Thus, the exposition of human population to OCs through consumption of cultured fish would be lower than expected from experimental biomagnification studies, although red muscle presented similar OC levels in both cases, which were much higher than those of white muscle. Although levels of organochlorine compounds were found to be low, the persistence, ubiquity, and toxicity of these compounds, together with their presence in fish feed, make it necessary to monitor OC residues in the routine quality assurance programs of aquaculture activities, as this food chain is a source of these toxic compounds for human consumers. The development of sensitive analytical methodology based on GC-MS/MS has allowed for the reaching of low detection limits required to carry out the present study.

Biogeography of the genusArtemia (Crustacea, Branchiopoda, Anostraca) in Spain

This is an updated study on the biogeographic distribution of the populations of the genusArtemia (Branchiopoda, Anostraca) in Spain, with special focus on populations inhabiting salt lagoons and inland salterns. The populations recorded (40) belong to the bisexual speciesA. tunisiana and to the asexual groupA. parthenogenetica (diploid and tetraploid strains). They usually appear in three different hypersaline ecosystems: solar salterns and lagoons filled with sea water, solar salterns and lagoons containing brines of diluted mineral salts, and lagoons filled with athalassic (endorheic) brines. The brines in salinas are chemically characterized by high Cl− concentrations, especially in inland salinas, whose brine sources are geologically associated with evaporitic formations developed in the Triassic and Lower Liassic of eastern Spain. Brines in athalassic lagoons, geologically associated with Mesozoic and Cenozoic periods, show higher levels of sulfate (up to 40–50%) as well as Mg2+. From a geographical point of view, bisexual and diploid asexual populations are mainly found in coastal and inland salinas or lagoons below 40° N, while asexual tetraploid populations are found in inland salinas and athalassic lagoons above that latitude. These populations have been biologically characterized by the morphology of their adults through multivariate analysis.

Long-term effect of temperature on bioaccumulation of dietary metals and metallothionein induction in Sparus aurata

Previous studies have demonstrated that the commercial feed of aquacultured fish contains trace amounts of toxic and essential metals which can accumulate in tissues and finally be ingested by consumers. Recently rising temperatures, associated to the global warming phenomenon, have been reported as a factor to be taken into consideration in ecotoxicology, since temperature-dependent alterations in bioavailability, toxicokinetics and biotransformation rates can be expected. Sparus aurata were kept at 22°C, 27°C and 30°C for 3 months in order to determine the temperature effect on metallothionein induction and metal bioaccumulation from a non-experimentally contaminated commercial feed. A significant temperature-dependent accumulation of cadmium (Cd), copper (Cu), mercury (Hg), zinc (Zn), lead (Pb) and iron (Fe) was found in liver, together with that of manganese (Mn), Fe and Zn in muscle. Hg presented the highest bioaccumulation factor, and essential metal homeostasis was disturbed in both tissues at warm temperatures. An enhancement of hepatic metallothionein induction was found in fish exposed to the highest temperature.

Characterization of type ''B'' esterases and hepatic CYP450 isoenzimes in Senegalese sole for their further application in monitoring studies

In fish, the role that cholinesterases (ChEs) play in tissues other than those implicated in neural activity, as well as the involvement of carboxylesterases (CbEs) and cytochrome P450 isoenzymes (CYPs) in drug metabolism needs investigation. For that, Senegalese sole (Solea senegalensis) specimens were selected for characterization of several type B esterases and hepatic CYPs in order to further use this fish as sentinel. ChEs (acetylcholinesterase (AChE) and pseudocholinesterases (butyrylcholinesterase-BuChE and propionilcholinesterase-PrChE)) and CbEs were measured in brain, plasma, kidney, liver, gonad, muscle and gills. Moreover, seven fluorimetric substrates were selected to study CYP related activities in fish liver. The results showed that AChE was the dominant ChE form in brain whereas pseudocholinesterases were absent in most tissues, as demonstrated by low enzymatic activities using specific substrates and the lack of inhibition by iso-OMPA. Plasma exhibited trace activities of all the esterases assayed and no BuChE activity. CbEs were dominant in liver, but they were also present in kidney and brain. For CbE determination, α-naphtyl acetate (αNA) was seen as the most adequate substrate as it displayed higher enzymatic activities and showed more in vitro sensitivity to the carbamate eserine and the organophosphate pesticide dichlorvos. Alkoxyresorufin-O-dealkylase (EROD and BFCOD) activities, indicative in mammals of CYP1A and CYP3A subfamilies, respectively, were the highest microsomal CYP-related activities in liver. The results of this preliminary work allow us to select the most adequate esterase substrate, tissue and hepatic CYP substrate for further monitoring studies.

Sublethal zinc exposure has a detrimental effect on reproductive performance but not on the cyst hatching success of Artemia parthenogenetica

The sublethal zinc toxicity to Artemia parthenogenetica as regards the possibility of colonization of zinc polluted salterns by means of cysts has been assessed by a cyst hatching assay and a life table approach. Emergence and hatching at different times as well as the whole hatching profile were taken as end-points for evaluating success of development. Demographic and reproductive parameters calculated according to the Lotka equation were used as an indicator of the chronic toxicity of the population. No adverse effects of waterborne zinc were found on hatching and emergence of cysts of A. parthenogenetica at any of the concentrations tested (0.01 mg/l, 0.1 mg/l, 0.5 mg/l, 1 mg/l, 5 mg/l). Chronic zinc exposure at 0.08 mg/l had detrimental effects on A. parthenogenetica fecundity, as detected by a decrease in the percentage of fertile females, which in turn produces a decrease in r. Chronic toxicity of zinc may be a limiting step for A. parthenogenetica colonization and the establishing of permanent populations in zinc-polluted brine ponds.

Dietary Effect on the Proteome of the Common Octopus (Octopus vulgaris) Paralarvae

Nowadays, the common octopus (Octopus vulgaris) culture is hampered by massive mortalities occurring during early life-cycle stages (paralarvae). Despite the causes of the high paralarvae mortality are not yet well-defined and understood, the nutritional stress caused by inadequate diets is pointed out as one of the main factors. In this study, the effects of diet on paralarvae is analyzed through a proteomic approach, to search for novel biomarkers of nutritional stress. A total of 43 proteins showing differential expression in the different conditions studied have been identified. The analysis highlights proteins related with the carbohydrate metabolism: glyceraldehyde-3-phosphate-dedydrogenase (GAPDH), triosephosphate isomerase; other ways of energetic metabolism: NADP+-specific isocitrate dehydrogenase, arginine kinase; detoxification: glutathione-S-transferase (GST); stress: heat shock proteins (HSP70); structural constituent of eye lens: S-crystallin 3; and cytoskeleton: actin, actin-beta/gamma1, beta actin. These results allow defining characteristic proteomes of paralarvae depending on the diet; as well as the use of several of these proteins as novel biomarkers to evaluate their welfare linked to nutritional stress. Notably, the changes of proteins like S-crystallin 3, arginine kinase and NAD+ specific isocitrate dehydrogenase, may be related to fed vs. starving paralarvae, particularly in the first 4 days of development.

Effect of ivermectin on the liver of gilthead sea bream Sparus aurata: A proteomic approach

Gilthead sea bream Sparus aurata is the most commercialized Mediterranean aquacultured fish species. Ivermectin has recently (experimentally) started to be used to control ectoparasitic infestations in Mediterranean cultured marine fish. The potential hepatotoxicity of ivermectin was investigated in gilthead sea bream juveniles (35g) following oral administration at the recommended dose of 0.2 mgkg(-1) fish for 10d. Difference Gel Electrophoresis Technology (DIGE) was used to study the effect of this treatment in gilthead sea bream liver protein profile under routine culture conditions. The 2D-DIGE protein maps obtained were analyzed using the DeCyder 6.5 software. The results obtained showed significant changes in the expression of 36 proteins respect to the control group. Among these proteins, six increased in abundance, and 30 decreased. Spot showing differential expression respect to the control were analyzed by mass spectrometry and database search, which resulted in three positive identifications corresponding to hepatic proteins involved in lipid metabolism (apoA-I), oxidative stress responses and energy generation (beta-globin, ATP synthase subunit beta). These proteins have not been previously associated to invermectin effect.