Development of a fluorescent transgenic zebrafish biosensor for sensing aquatic heavy metal pollution.

We report a transgenic zebrafish (Danio rerio) designed to respond to heavy metals using a metal-responsive promoter linked to a fluorescent reporter gene (DsRed2). The metallothionein MT-Ia1 promoter containing metal-responsive elements was derived from the Asian green mussel, Perna viridis. The promoter is known to be induced by a broad spectrum of heavy metals. The promoter-reporter cassette cloned into the Tol2 transposon vector was microinjected into zebrafish embryos that were then reared to maturity. A transgene integration rate of 28 % was observed. The confirmed transgenics were mated with wild-type counterparts, and pools of F1 embryos were exposed to sub-lethal doses of Cd(2+), Cu(2+), Hg(2+), Pb(2+) and Zn(2+). The red fluorescence response of zebrafish embryos was observed 8 h post- exposure to these sub-lethal doses of heavy metals using a fluorescence microscope. Reporter expression estimated by real-time PCR revealed eightfold, sixfold and twofold increase on exposure to highest concentrations of Hg(2+), Cd(2+) and Cu(2+), while Pb(2+) and Zn(2+) had no effect. This biosensor could be a first-level screening method for confirming aquatic heavy metal bio-toxicity to eukaryotes.

Expression studies on NA+/K(+)-ATPase in gills of Penaeus monodon (Fabricius) acclimated to different salinities.

The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K(+)-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35 per thousand salinities and maintained for 20 days to observe long-term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5 per thousand compared to 25 per thousand (isosmotic salinity) and 0.48 folds lower at 35 per thousand. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5 per thousand the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25 per thousand and 35 per thousand NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25 per thousand. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5 per thousand measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.