Protection of red snapper (Lutjanus sanguineus) against Vibrio alginolyticus with a DNA vaccine containing flagellin flaA gene

Aims: The main aims of this study were to construct a DNA vaccine containing flagellin flaA gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of pcDNA‐flaA as a DNA vaccine candidate for red snapper (Lutjanus sanguineus). Methods and Results: Plasmid DNA encoding flagellin flaA gene (designated as pcDNA‐flaA) was used as a DNA vaccine to immunize red snapper. The distribution, expression and immunoprotection of the DNA vaccine were analysed in tissues of the red snapper by PCR, RT‐PCR and challenge test. PCR results indicated that pcDNA‐flaA distributed in liver, spleen, kidney, gill and injection site muscle at 7–28 days after vaccination. RT‐PCR results indicated that the flaA gene was expressed in all above tissues of vaccinated fish at 7–28 days after vaccination. In addition, fish receiving the DNA vaccine developed a protective response to live V. alginolyticus challenge 28 days post inoculation, the relative per cent survival (RPS) was 88%. Conclusions: This study showed that injection of pcDNA‐flaA induced an efficient, systemic and antigen‐specific immune response in red snapper, which makes it an effective vaccine candidate against V. alginolyticus infection. Significance and Impact of the Study: The finding that red snapper does adequately respond to pcDNA‐flaA intramuscular injection makes pcDNA‐flaA a promising candidate for DNA vaccine treatment. Furthermore, the availability of red snapper for foreign gene expression represents a useful model to develop effective prophylactic strategies and opens new perspectives for the treatment of bacterial pathogens of marine cultured fish.     

Assessment of DNA vaccine potential for gilthead sea bream (Sparus aurata) by intramuscular injection of a reporter gene

Naked circular plasmid DNA containing the cytomegalovirus (CMV)-promoter-driven lacZ reporter gene (pCMV-LacZ) was injected in the epaxial muscle of gilthead sea bream (Sparus aurata). A mosaic pattern of expression of beta-galactosidase (beta-gal) in the myofibres at the site of injection was visualised by in situ histochemical staining using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. As measured by o-nitrophenyl-beta-D-galactopyranoside assay, beta-gal enzymatic activity was found to steadily increase for at least 50 days post injection (p.i.) in pCMV-LacZ-injected muscle. In parallel, foreign DNA was detected by polymerase chain reaction in injected muscles (but not in other tissues) up to 60 days p.i., persisting most probably in an extrachromosomal, non-replicative, circular form. Neither beta-gal activity nor pCMV-LacZ-related amplification products were found 90 days p.i. Antibodies against beta-gal were demonstrated in pCMV-LacZ-injected fish sampled 45 days p.i. The results suggest that intramuscular delivery of foreign genes represents a realistic approach for DNA vaccine technology for the prevention of infectious diseases in gilthead sea bream.     

Effects of dietary methylmercury on the zebrafish brain: histological, mitochondrial, and gene transcription analyses

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for wildlife and human health through fish consumption. To study the neurotoxic impact of MeHg on piscivorous fish, we contaminated the model fish species Danio rerio for 25 and 50 days with food containing 13.5 μg/g dry weight (dw) of MeHg (0.6 μg MeHg/fish/day), an environmentally relevant dose leading to brain mercury concentrations of 30 ± 4 μg of Hg g⁻¹ (dw) after 25 days of exposure and 46 ± 7 μg of Hg g⁻¹ (dw) after 50 days. Brain mitochondrial respiration was not modified by exposure to MeHg, contrary to what happens in skeletal muscles. A 6-fold increase in the expression of the sdh gene encoding the succinate dehydrogenase Fe/S protein subunit was detected in the contaminated brain after 50 days of exposure. An up regulation of 3 genes, atp2b3a, atp2b3b, and slc8a2b, encoding for calcium transporters was noticed after 25 days of exposure but the atp2b3a and atp2b3b were repressed and the slc8a2b gene expression returned to its basal level after 50 days, suggesting a perturbation of calcium homeostasis. After 50 days, we detected the up regulation of glial fibrillary acidic protein and glutathione S-transferase genes (gfap and gst), along with a repression of the glutathione peroxidase gene gpx1. These results match well with a MeHg-induced onset of oxidative stress and inflammation. A transmission electron microscopic observation confirmed an impairment of the optical tectum integrity, with a decrease of the nucleal area in contaminated granular cells compared to control cells, and a lower density of cells in the contaminated tissue. A potential functional significance of such changes observed in optical tectum when considering wild fish contaminated in their natural habitat might be an impaired vision and therefore a lowered adaptability to their environment.     

Cadmium and copper contents in a freshwater fish species (brook trout, Salvelinus fontinalis) from the subantarctic Kerguelen Islands

The subantarctic Kerguelen Islands (49°S, 70°E) contain freshwaters among the most isolated in the world from direct human activities. Cadmium and copper concentrations were analyzed in muscle and liver tissues of 57 non-migratory brook trout (Salvelinus fontinalis) inhabiting the Sud River of Kerguelen Islands. The mean cadmium concentration in liver was 1.13 μg/g dry wt, within the range of levels measured in liver of marine fish from the Southern Ocean. Muscular Cd levels (0.12 μg/g dry wt) were roughly ten times higher than those measured in Kerguelen’s marine fish species. Copper levels were very high in the two organs (62.27 μg/g dry wt in liver and 3.02 μg/g dry wt in muscle) compared to those detected in fish from the Southern Ocean. Regarding the seasonal trend, the highest Cu and Cd muscular levels were measured in fish at the end of the austral winter, whereas the highest hepatic levels were observed at the end of the austral summer. Moreover, hepatic cadmium levels were higher in females than in males. These results could be related to brook trout spawning physiological preparations and foraging behavior during the summer period. We provide here the first results about Cu and Cd levels in liver and muscle of a freshwater fish species in an insular subantarctic context. They are in agreement with the high cadmium contamination found in fish of the Southern Ocean.     

A Streptococcus iniae DNA vaccine delivered by a live attenuated Edwardsiella tarda via natural infection induces cross‐genus protection

Edwardsiella tarda and Streptococcus iniae are important fish pathogens. We have reported previously a live E. tarda vaccine based on the attenuated strain TX5RM and a S. iniae DNA vaccine based on the antigen Sia10. In this study, we examined the possibility of constructing a cross‐genus vaccine by taking advantage of the residual infectivity of TX5RM and using it as a carrier host for the natural delivery of a S. iniae DNA vaccine. For this purpose, the recombinant TX5RM, TX5RMS10, was created, which harbours and retains stably the DNA vaccine plasmid pCS10 that expresses Sia10. When flounder were vaccinated with TX5RMS10 via oral and immersion routes, TX5RMS10 was detected in multiple tissues within 12–14 days postvaccination (p.v.). At 7 and 14 days p.v., expression of the DNA vaccine was detected in spleen, kidney and liver. Following E. tarda and S. iniae challenge at one and 2 months p.v., the vaccinated fish exhibited relative per cent survival rates of 69–83%. Immunological analysis indicated that TX5RMS10‐vaccinated fish produced specific serum antibodies and exhibited enhanced expression of a wide range of immune genes.     

DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size,dose, route of injection and duration of protection-early protection correlates with Mx expression

Rainbow trout of different sizes (10 and 100g) were injected intramuscularly (i.m.) or intraperitoneally (i.p.) with different doses (range 10 ng-10 microg) of a viral haemorrhagic septicaemia (VHS)-DNA vaccine (pcDNA3vhsG). As controls, fish were injected with the pcDNA3 plasmid alone, or with inactivated VHS virus. Fish were challenged at different times post-vaccination (p.v.) to assess protection. At certain times p.v., serum samples were analysed for neutralising antibody and liver tissue was analysed for Mx mRNA expression. A DNA dose of 0.5 microg injected by the i.m. route induced protection in fish of all sizes in challenges performed either 1 or 4 weeks p.v. This dose also conferred effective protection up to 9 months p.v. in fish >100 g. With lower doses of DNA (0.1 and 0.01 microg) and challenge at 4 weeks p.v., 10 g fish were partially protected but protection was not observed in 100 g fish. Vaccination by the i.p. route induced no or lower levels of protection compared with the i.m. route. Fish vaccinated with 0.5 microg DNA i.m. had no detectable serum neutralising antibody (NAb) at 4 weeks p.v. (with the exception of a single 10 g fish) but antibody was detected at 8 weeks and 6 months p.v. but not at 9 months p.v. However, cohorts of these fish showed effective protection at all timepoints. Lack of detectable levels of NAb (at 9 weeks p.v.) despite partial protection in challenge at 4 weeks p.v. was also observed with 0.01 microg doses of DNA i.m. NAb was detected in sera of fish at 8 weeks after vaccination with 0.1 microg i.m. but not in fish vaccinated with doses of 0.01-0.5 microg i.p. Early protection (1 week p.v.) correlated with elevated Mx gene expression.     

Immune Response in Goats to Different Payloads of FMDV Monovalent Vaccine: Protection Against Virulent Challenge and Development of Carrier Status

The relationship of Foot-and-Mouth Disease virus antigen payload and number of dose of vaccine conferring protection against virus challenge in goats was studied. Goats vaccinated with oil adjuvant Foot-and-Mouth Disease vaccines containing different antigen payloads with or without booster resisted virulent challenge at 21 days post-vaccination or 7 days after booster respectively. However, localized sub-clinical infection was observed in two vaccinated goats on 35 days post-challenge. RNA could be detected from 31.8% of vaccinated goats (10^2.69–10^4.99 viral RNA copies per cotton swab of nasal secretions) on day 35 post-challenge. Since no live virus could be isolated after 5 days post-challenge, the risk of these animals transmitting the disease was probably very low. The finding showed that oil adjuvant Foot-and-Mouth Disease vaccines containing antigen payload of 1.88 μg may prevent or reduce the local virus replication at the oropharynx and shedding of virus from nasal secretions and thereby reduce the amount of virus released into the environment subsequent to exposure to live virus. This study also showed that goats with poor sero conversion to vaccination can be infected without overt clinical signs and became carriers like sheep.     

Fish DNA vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunisation

The DNA vaccine, pIHNVw-G, contains the gene for the glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV), a major pathogen of salmon and trout. The relative efficacy of various routes of immunisation with pIHNVw-G was evaluated using 1.8 g rainbow trout fry vaccinated via intramuscular injection, scarification of the skin, intraperitoneal injection, intrabuccal administration, cutaneous particle bombardment using a gene gun, or immersion in water containing DNA vaccine-coated beads. Twenty-seven days after vaccination neutralising antibody titres were determined, and 2 days later groups of vaccinated and control unvaccinated fish were subjected to an IHNV immersion challenge. Results of the virus challenge showed that the intramuscular injection and the gene gun immunisation induced protective immunity in fry, while intraperitoneal injection provided partial protection. Neutralising antibodies were not detected in sera of vaccinated fish regardless of the route of immunisation used, suggesting that cell mediated immunity may be at least partially responsible for the observed protection.     

Selenium and Mercury in Native and Introduced Fish Species of Patagonian Lakes,Argentina

A survey of mercury (Hg) and selenium (Se) contents was performed in fish collected from lakes located in two National Parks of the northern patagonian Andean range. Two native species, catfish (Diplomystes viedmensis) and creole perch (Percichthys trucha), and three introduced species, brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), and brook trout (Salvelinus fontinalis), were caught from lakes Nahuel Huapi, Moreno, Traful, Espejo Chico, and Guillelmo belonging to Nahuel Huapi National Park and from lakes Futalaufquen and Rivadavia, Los Alerces National Park. In lake Moreno, fish diet items were analyzed and rainbow trout grown in a farm. Hg and Se were measured in muscle and liver tissues by instrumental neutron activation analysis. The average concentrations in muscle of Hg for all species, ages, and lakes are between 0.4 to 1.0 μg g⁻¹ dry weight (DW) with a few fish, mainly native, exceeding the United States Environmental Protection Agency health advisory for freshwater fish limited consumption, and from 0.8 to 1.5 μg g⁻¹ DW for Se. Average concentrations in liver of Hg in all species range from 0.4 to 0.9 μg g⁻¹ DW. Brown trout, the top predator in these lakes, showed the lowest average Hg burden in both tissues. Se concentrations in the liver of brown and rainbow trout, up to 279 μg g⁻¹ DW, are higher than those expected for nearly pristine lakes, exceeding 20 μg g⁻¹ DW, the threshold concentration associated with Se toxicity. These species show lower Hg contents in muscle, suggesting a possible detoxification of Hg by a Se-rich diet. Creole perch and velvet catfish livers have lower Se concentrations, with a narrower span of values (2.3 to 8.5 μg g⁻¹ and 3.3 to 5.5 μg g⁻¹ DW respectively).     

Schistosoma japonicum: The design and experimental evaluation of a multivalent DNA vaccine

The aim of this study was to construct and evaluate the immunity efficacy of the DNA multivalent vaccine pVIVO₂SjFABP-23. The vaccine was constructed and produced as follows. Forty BALB/c mice were divided into four groups designated pVIVO₂, pVIVO₂Sj23, pVIVO₂SjFABP and pVIVO₂SjFABP-23. Each mouse was immunized with 100 μg of the corresponding plasmid DNA by intramuscular injection. 28 days post-vaccination, the mice were challenged with S. japonicum cercariae, and the worm and egg burdens were determined 42 days post-challenge. Serum samples were collected from all the mice before and after vaccination and at the end of the experiment, and used for antibody detection. The IFN-γ and IL-4 levels were quantified in the supernatants of specifically stimulated spleen cells. The number of worms was reduced by 52%, 40% and 42% in mice respectively immunized with pVIVO₂SjFABP-23, pVIVO₂Sj23 or pVIVO₂SjFABP. A respective 61%, 38% and 39% egg reduction was determined relative to those mice that only received the empty pVIVO2 plasmid. pVIVO₂SjFABP-23 immunization increased IgG levels against SWAP and SEA. Increased IFN-γ levels were detected in the supernatant of specific stimulated spleen cells from mice immunized with the 3 different constructs. The multivalent DNA vaccine developed induced higher levels of protection than the two monovalent tested vaccines.     

Gene-Gun-Mediated Transfer of Reporter Genes to Somatic Zebrafish (Danio rerio) Tissues

We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-μm microcarriers and 1 μg of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 μg of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when using the biolistic DNA delivery parameters and target tissues described. Received August 20, 1999; accepted January 6, 2000.     

Characterization of terminal deoxynucleotidyl transferase and polymerase μ in zebrafish

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase μ (polμ), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and polμ and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish polμ. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Polμ expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Polμ is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and polμ in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and polμ provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and polμ themselves.     

Electroporation-mediated transient gene expression in intact cells of sweetpotato

Summary Transient expression of the β-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweetpotatoIpomoea batatas L. (Lam.) by electroporation. The influence of several factors including electric field strength, buffer composition, time course of transientGUS gene expression, DNA concentration, enzyme, and polyethylene glycol (PEG) treatment was examined onGUS gene expression (number of blue spots). MaximumGUS gene expression (an average of 90 blue spots/fifty mg fresh weight callus tissue) was observed after 48 h when callus pieces were preincubated with electroporation (EPR) buffer for 1 h, followed by electroporation with a single electric pulse of 500 V/cm discharged from a 960-μF capacitor in the presence of 20 μg DNA/ml and 8.3 μl NaCl (3M). Changing the electroporation buffer conductivity (by varying the buffer composition with low-high salt concentrations), had only slight effect on the number of blue spots. Similarly, the time course study ofGUS gene expression revealed that GUS activity could be detected 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 μg/ml in the EPR buffer had a slight effect on the expression frequency (from 20–110 blue spots, and 112 blue spots with 20 μg/ml). The number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 μl PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation can be used for producing transgenic sweetpotato tissue.     

Stable Expression of a Foreign Gene, Delivered by Gene Gun, in the Muscle of Rainbow Trout Oncorhynchus mykiss

We report the efficient delivery of a foreign gene into muscle of rainbow trout Oncorhynchus mykiss with a gene gun. The foreign gene was a reporter gene, chloramphenicol acetyltransferase (CAT). Two CAT-containing plasmids were used: pCMV-CAT, which contains cytomegalovirus immediate early promoter, and pSV2-CAT, which contains the simian virus 40 early promoter. All plasmids were introduced by particle bombardment using a gene gun. During the 90-day sampling period following bombardment, CAT was strongly and stably expressed in the muscle of all the fish bombarded with pCMV-CAT and pSV2-CAT. No CAT expression was detected in the blood samples until 90 days after introduction, when it was found in only one fish from the pCMV-CAT group and one from the pSV2-CAT group. The stable and long-term expression of plasmid DNA in muscle makes muscle an attractive target tissue for the introduction of viral DNA for the purpose of DNA vaccination. Received June 5, 1999; accepted November 2, 1999.     

鼠巨细胞病毒 IE-1核酸疫苗和灭活疫苗联合免疫抗病毒感染的研究

巨细胞病毒（Cytomegalovirus,CMV）在人群中感染普遍,对婴幼儿及免疫低下人群中造成严重疾病,目前还没有针对该病毒的商品化疫苗。本研究以BALB/c小鼠为动物模型,探讨鼠巨细胞病毒（Murine cytomega-lovirus,MCMV）IE-1 DNA疫苗和MCMV灭活疫苗联合免疫抗MCMV感染的免疫保护效果。将编码IE-1基因的DNA疫苗（pIE-1）通过肌肉注射辅以电穿孔的方式对小鼠进行初免,再用全病毒灭活疫苗单独或者辅以MF59佐剂进行加强免疫,分别通过ELISA和ELISPOT方法检测到联合免疫策略在免疫组小鼠体内诱导了MC-MV特异性的抗体应答和CTL应答;免疫两周后用3＆#215;LD50致死剂量MCMV感染小鼠,疫苗对小鼠的免疫保护通过检测小鼠存活率、重要器官中的病毒滴度及体重丢失率来评价。结果显示,与单独免疫DNA疫苗或灭活疫苗相比,IE-1 DNA疫苗联合灭活疫苗组能同时在小鼠体内诱导体液免疫和细胞免疫应答,并提供小鼠完全保护;而且MF59辅以灭活疫苗免疫小鼠能增强疫苗的免疫效果。     

Atrazine Tolerance of Grass Species with Potential for Use in Vegetated Filters in Australia

The response of introduced (Pennisetum clandestinum Hochst.) and native (Stipa aristiglumis F. Muell., Themeda australis (R. Br.) Stapf, Danthonia spp. (L.) grasses to the herbicide atrazine was studied in plants with the potential for use in vegetated filters (biofilters) designed to reduce chemical loads in agricultural runoff. The response was detected by photosynthetic inhibition using leaf chlorophyll fluorescence. With continuous short-term (14 days) dosing with atrazine in sand culture, P. clandestinum showed the greatest tolerance, regardless of the dose (20–500 μg/L). In a clay vertosol soil in the glasshouse, the four species were tolerant to longer-term (84 days) application of three successive doses of simulated run-on, each dose containing 100 μg/L atrazine, a concentration which is comparable to the highest reported in runoff from agricultural land in Australia. Even with a subsequent single atrazine dose at ∼ ∼5000 μg/L, established plants of the four species showed signs of quick recovery (7–21 days) to normal photosynthetic activity. In a field experiment with simulated run-on, applied to P. clandestinum pasture in sandy loam soil, repeated doses at concentrations up to 1000 μg/L gave no significant response; only a subsequent single dose of 5000 μg/L had significant effects, from which plants soon recovered. Although damage from atrazine can be demonstrated with continuous dosing in non-adsorbing media, in soil culture the tolerance of the four selected species to repeat doses of atrazine shows they may be used confidently for biofiltering purposes. P. clandestinum was especially tolerant.     

Effects of salmon calcitonin and anti-salmon calcitonin antibody on plasma calcium concentrations in the goldfish,Carassius auratus

The role of calcitonin (CT) in plasma calcium regulation was studied by the administration of exogenous CT and anti-salmon(s) CT antibody using goldfish,Carasius auratus, loaded or otherwise with calcium. CT elicited a decrease in plasma calcium concentrations at a dose of 10 ng/g body weight 1 h after administration. However, no effects were observed following doses of 30 ng and 50 ng/g 1 h, nor for the three doses 3 h after administration. In calcium-loaded fish, the effect of CT was different depending on the dosage of CT. Ten ng and 50 ng/g induced a decrease and an increase in plasma calcium concentrations, respectively, 3 h after administration. Anti-sCT antibody (0.02 μg or 0.1 μg/g) did not affect plasma calcium concentrations. In calcium-loaded fish, neither dose of anti-sCT antibody changed plasma calcium concentrations 1 h after administration. However, following a dose of 0.1 μg/g, plasma calcium concentrations decreased after 3 h. A positive correlation between plasma calcium concentrations and the gonad somatic index (GSI) in females was no longer apparent after administration of anti-sCT antibody. There was no relationship between plasma calcium concentrations and GSI in control and anti-sCT antibody-treated males. These results suggested that CT regulates plasma calcium concentrations in different ways depending on the dosage with CT having a role in calcium physiology during vitellogenesis.     

Cadmium,cobalt, chromium,and experimental myocardial infarction

In the case of experimental heart muscle infarction, the infarcted tissue of 18 pigs had a cadmium content of 0.38 μg/g dry weight and a cobalt content of 0.45 μg/g dry weight. In 25 non-infarcted pig hearts, the cadmium concentration amounted to 0.27 μg/g dry weight and the cobalt concentration to 0.37 μg/g dry weight. Thus, as far as the infarcted heart muscle tissue is concerned, there is a highly significant increase in the cadmium content (p<0.01) and a significant increase in cobalt content (p<0.05) compared to a non-infarcted heart. No differences were established with regard to chromium concentrations.     

Comparative Effects of Direct Cadmium Contamination on Gene Expression in Gills, Liver, Skeletal Muscles and Brain of the Zebrafish (Danio rerio)

The effects of cadmium (Cd) on gene expression were examined in four organs (gills, liver, skeletal muscles and brain) of the zebrafish. Adult male fish were subjected to three different water contamination pressures over periods of 7 and 21 days: control medium (C₀: no Cd added) and two contaminated media (C₁: 1.9 ± 0.6 μg Cd l⁻¹, and C₂: 9.6 ± 2.9 μg Cd l⁻¹). Fourteen genes involved in antioxidant defences, metal chelation, active efflux of organic compounds, mitochondrial metabolism, DNA repair and apoptosis were selected and their expression levels investigated by quantitative real-time PCR. Cadmium concentrations were determined in the four organs and metallothionein (MT) protein levels investigated in brain, liver and gills. Although skeletal muscle was a poor Cd-accumulating tissue, many genes were up-regulated at day 7: mt1, cyt, bax, gadd and rad51 genes. Three additional genes, c-jun, pyc and tap, were up-regulated in muscles at day 21 whereas bax, gadd and rad51 had returned to basal levels. Surprisingly, mt1 and c-jun were the only genes displaying a differential induction after 21 days in liver, although this organ accumulated the highest cadmium concentration. In brain, only mt1, mt2 and c-jun genes were up-regulated after 21 days. In gills, the highest response was observed after 7 days, featuring the differential expression of oxidative stress-response hsp70 and mitochondrial sod genes, along with genes involved in mitochondrial metabolism and metal detoxification. Then, after 21 days, the expression of almost every genes returned to basal levels while both mt1 and mt2 genes were up-regulated.