Effects of graded dietary levels of soy protein concentrate supplemented with methionine and phosphate on the immune and antioxidant responses of gilthead sea bream (Sparus aurata L.).

The effects of a dietary soy protein concentrate (SPC) as a fish meal (FM) substitute, on selected innate immune responses, the oxidative status, hepatic and intestinal morphology of gilthead sea bream, Sparus aurata, were evaluated after a three-month feeding trial. Isonitrogenous (45% crude protein) and isoenergetic (23 kJ/g gross energy) diets with 20% (SPC20), 40% (SPC40) and 60% (SPC60) of SPC inclusion, supplemented with methionine and phosphate, were evaluated against a diet containing FM as the sole protein source. Diets were allocated in triplicate groups of 26-g fish (8 kg m-3/tank) and administered for three months. Immune responses were evaluated by performing immunological assays in blood (respiratory burst activity) and serum (myeloperoxidase content, bacteriolytic and lysozyme activity), as well as by gene expression analysis of immune-associated genes (MHCIIα, ß2m, CSF-1R, NCCRP-1, TGF-ß1, HSP70) in the head kidney and distal intestine. In addition, oxidative stress was evaluated by measuring the activity of liver enzymes associated with the antioxidant system. The respiratory burst activity of blood was significantly decreased in the SPC40 group, while serum myeloperoxidase content and bacteriolytic and lysozyme activities were affected. Significantly higher expression levels of NCCRP-1 and HSP70 were found in SPC60 head kidneys, while increased intestinal MHCIIα and NCCRP-1 transcripts were observed in SPC40. Hepatic antioxidant enzyme activity of glutathione reductase and glutathione-S-transferase was significantly enhanced in the SPC40 and SPC60 groups, while superoxide dismutase activity was increased only in the SPC40 group. Moreover, increased lipid accumulation in the enterocytes of the distal intestine was observed in the SPC60 group. Overall, a three-month feeding period with diets over 40% of dietary SPC inclusion as a FM substitute, indicated increases on immune and antioxidant enzyme responses, suggesting the dietary SPC levels that gilthead sea bream can tolerate.

Comparative study of biochemical and immunological biomarkers in three marine bivalves exposed at a polluted site.

A battery of biochemical and immunological biomarkers used for pollution assessment were measured for first time in the clams Venus verrucosa and Callista chione and were compared with those of the mussel Mytilus galloprovincialis, a well-established indicator organism utilized in numerous environmental monitoring programs. Clams and mussel were transplanted at a polluted and a reference site or maintained at the laboratory. Among biochemical biomarkers, acetylcholinesterase did not differ at the polluted site in all species, but there was a significant difference between the mussel and the clams, glutathione S-transferase showed a clear inhibition at the polluted site in all species and a significant difference between the two clams was also indicated, while catalase activities were increased only in V. verrucosa at the polluted site and not in mussel or the other clam. Immunological biomarkers responses were also pronounced at the polluted site. Lysozyme activity was species-dependent whereas respiratory burst activity measured as luminol-dependent chemiluminescence (CL) was site and stimulus dependent, and it was evident in M. galloprovincialis and V. verrucosa and not in C. chione. Further investigation focused on biochemical and immunological biomarkers related with the oxidative mechanisms in clams will strengthen and expand their use as bioindicators for pollution assessment.

Short-term exposure of the European sea bass Dicentrarchus labrax to copper-based antifouling treated nets: copper bioavailability and biomarkers responses.

We studied if the levels of copper released from antifouling treated nets used in finfish mariculture could affect the immune defense mechanism and/or induce oxidative stress in Dicentrarchus labrax, after short term exposure in laboratory experiments. Dissolved copper concentration released from the treated nets, copper bioavailability and a set of biomarkers responses were measured. Biomarkers included hemoglobin concentration, activities of lysozyme, total complement, respiratory burst, glutathione S-transferase and acetycholinesterase and concentration of thiobarbituric acid reactive substances. Results indicated elevated copper concentration in seawater (184 µg L(-1)) but low concentration in muscle (1.5 µg g(-1)) and liver (117 µg g(-1)). Copper bioavailability was independent of copper complexes with dissolved organic carbon. However, formation of copper complexes with other matrices could neither be excluded nor justified. The released copper from the treated nets did not induce oxidative stress but affected the immediate immune defense mechanism of the exposed fish making them more easily vulnerable to diseases. Consequently, copper-based antifouling treated nets could be a risk factor for D. labrax health.

Bioassays and biochemical biomarkers for assessing olive mill and citrus processing wastewater toxicity.

The toxicity and the biochemical effects of olive mill wastewater and citrus processing wastewater were evaluated using acute toxicity bioassays (Gammarus pulex and Hydropsyche peristerica) and biochemical biomarkers (acetylcholinesterase [AChE] and glutathione S-transferase [GST]). The bioassays indicated toxic properties of olive mill and citrus processing wastewaters. The 24 h LC(50) values of olive mill wastewater ranged from 2.64% to 3.36% for G. pulex and 3.62% to 3.88% for H. peristerica, while the LC(50) of citrus processing wastewater was 25.26% for G. pulex and 17.16% for H. peristerica. Based on a five-class hazard classification system applied for wastewaters discharged into the aquatic environment, olive mill wastewater and citrus processing wastewater were classified as highly toxic and toxic, respectively. Results of the biochemical biomarkers showed that both agroindustrial effluents at increasing sublethal wastewater concentrations could cause inhibition of the AChE and induction of the GST activities. These first results showed that both species as well as their AChE and GST activities have the potential to be used as indicators and biomarkers for assessing olive mill and citrus processing wastewaters quality.

Assessment of contaminant impacts in a semi-enclosed estuary (Amvrakikos Gulf, NW Greece): bioenergetics and biochemical biomarkers in mussels.

A combination of bioenergetics and biochemical biomarkers in mussels was applied to assess possible pollution impacts in a protected semi-enclosed estuary (Amvrakikos Gulf, NW Greece) that receives pesticide discharges through riverine transport. Scope for growth, a physiological condition index representing the energy budget of the organism, was applied to detect general stress effects on the health status of mussels. The low energy budgets of mussels revealed stress conditions and provided early warning signals of possible consequences at higher levels of biological organization. Biochemical markers of exposure confirmed a risk of pesticide contamination. Decreased acetylcholinesterase activities indicated exposure to organophosphate and carbamate pesticides. Responses of the antioxidant enzyme glutathione peroxidase suggested the presence of contaminants capable of reactive oxygen species production that could be related to organochlorine pesticide contamination in the area. On the other hand, metallothionein levels implied low metal contamination.

Assessment of the impact of heavy metal pollution from a ferro-nickel smelting plant using biomarkers.

A set of biomarkers was used to assess the impact of heavy metal pollution by a ferro-nickel smelting plant in Larymna bay (North Evoikos Gulf, Greece). These included a biomarker reflecting health status of an organism (scope for growth, SFG), a cellular biomarker of heavy metal exposure (composition of metal-containing granules), and two biochemical biomarkers reflecting oxidative stress (glutathione peroxidase, GPX) and neurotoxicity (acetylcholinesterase, AChE) measured in mussels (Mytilus galloprovincialis) both native and transplanted for 1 and 6 months at the coastal area of Larymna. All biomarkers in mussels at Larymna revealed differences from mussels at a reference site, signaling effects of the increased heavy metal levels on the biota. While effects on SFG and GPX in Larymna mussels were obvious on short-term exposure and persistent during chronic exposure, only chronic exposure induced a possibly cumulative effect on AChE. To validate the causal relationship between heavy metal exposure and effects observed in Larymna, SFG, GPX, and ACHE were examined in mussels exposed to a mixture of heavy metals (Ni, Cr, and Fe) under controlled laboratory conditions. The laboratory experiment verified the causal relationship between SFG and GPX responses and heavy metals but this was not demonstrated for AChE. Results from field-collected and laboratory-exposed mussels indicated a potential of GPX as predictive biomarker of population-level effects of heavy metal exposure.

Assessing the quality of marine coastal environments: comparison of scope for growth and Microtox bioassay results of pollution gradient areas in eastern Mediterranean (Greece).

Methods for assessing the quality of marine coastal environments are numerous and rapidly evolving. The integration of physiological parameters termed as Scope for Growth (SfG) and the luminescent bioassay Microtox were applied in several sites of Saronikos and Amvrakikos Gulfs (Greece) to assess the environmental quality and compare the results of the two methods. Each site in the two areas undergoes different types and levels of pollution. Both methods could identify a pollution gradient reflecting the quality of each site. A very good linear correlation was noticed between the two methods (r = 0.98). The stress order, which was similar in both methods, exhibited the expected pollution gradient according to the pressure that each site undergoes. Results suggest that for screening and fast isolation of polluted sites before further analysis, the rapid and simple Microtox assay could operate as an alternative to the more complex SfG method.