Organization and promoter analysis of the grouper (Epinephelus coioides) epinecidin-1 gene.

Epinecidin-1, an antimicrobial peptide documented in some fish, is an essential component of the innate immune response in fish, but little is known about its gene regulation. To better understand the molecular mechanism controlling transcription of the epinecidin-1 gene, we cloned and sequenced the genes and promoter regions of three epinecidin-1 peptides from the grouper (Epinephelus coioides). These genes have the potential to encode three putative epinecidin-1 peptides with either a short or a long 5'-untranslated region (UTR). These epinecidin-1 genes, numbered 124-1 (gene structure: 5 exons), 124-2 (gene structure: 5 exons), and 961 (gene structure: 4 exons), have 3' UTR sequences that dramatically differ by being located on different exons in clones 124 and 961. To address the roles of lipopolysaccharide (LPS) and poly(I):poly(C) in regulating epinecidin-1 expression, serial deletions were prepared in the promoter region of two clones that contained three genes. Different fragments of the epinecidin-1 5'-flanking region were transfected into U937 (human histiocytic lymphoma) and ZFL (zebrafish liver) cells and then treated with 0, 1, 10, and 100 mug/mL LPS or poly(I):poly(C). The results showed that after treatment with 10 mug/mL LPS, high promoter activity was observed in the 0.6-kb promoter fragment (of clone 961). Promoter deletions showed that hepatocyte nuclear factor (HNF)-1 was required for a maximal response of epinecidin-1 961 promoter activity after LPS treatment in ZFL cells. Morphological studies of transgenic zebrafish indicated that the 2-kb epinecidin-1 124-1 promoter-driven GFP transcripts appeared in the eye and skin as confirmed by immunohistochemical staining. These results indicate that the 2-kb epinecidin-1 124-1 promoter is active in a tissue-specific manner.

Gene expression and localization of the epinecidin-1 antimicrobial peptide in the grouper (Epinephelus coioides), and its role in protecting fish against pathogenic infection.

Epinecidin-1 is an antimicrobial peptide and plays a vital role in protecting fish against pathogenic infection. As a mimic of a grouper epinecidin-1 peptide, it has tertiary structures that closely resemble those of pleurocidin found in the winter flounder (Pleuronectes americanus). The tissue-specific, lipopolysaccharide (LPS)-stimulation-specific, and poly(I):poly(C)-stimulation-specific expressions of the grouper (Epinephelus coioides) epinecidin-1 antimicrobial peptide were determined using a comparative reverse-transcription polymerase chain reaction. Results of the tissue distribution analysis revealed high levels of epinecidin-1 messenger RNA (mRNA) in the head kidneys, intestines, and skin. Expression of epinecidin-1 mRNA was dose-dependently stimulated by both LPS and poly(I):poly(C). Immunohistochemical analysis with the polyclonal antiserum of a grouper epinecidin-1 peptide (rabbit polyclonal antibody) showed that the peptide was localized with the epinecidin-1 antibody in the gills and intestines. Two synthetic peptides of the grouper epinecidin-1 peptide (g-ple 22-51 and g-ple 22-42) and one winter flounder pleurocidin as a control exhibited high antimicrobial activities against gram-negative or gram-positive bacteria. In addition, peptide treatment was effective in promoting a significant increase in fish survival after the injection of Vibrio vulnificus in tilapia (Oreochromis mossambicus) and grouper. These results are relevant to the design of prophylactic and therapeutic strategies to counter bacterial infections, especially for preventing or ameliorating immune defects in fish during bacterial infections.

Influences of salinity on the biokinetics of Cd, Se, and Zn in the intertidal mudskipper Periophthalmus cantonensis.

The biokinetics (aqueous uptake, dietary assimilation, and elimination) of Cd, Se, and Zn in the intertidal mudskipper, Periophthalmus cantonensis, were examined at different acclimated salinities using the radiotracer technique. The dietary assimilation efficiency from ingested radiolabeled polychaetes was the highest for Se (32-40%), followed by Zn (5-7%) and Cd (2-3%), and was not influenced by salinity within a range of 10-30 psu. Uptake from the dissolved phase typically exhibited a linear pattern within the first 12 h of exposure, followed by a second slower uptake. The highest concentration factor (CF) was found for Zn, followed by Cd and Se. Differences in salinity did not significantly affect the CF of the three metals within the first 12 h, but the CFs were significantly higher at lower salinities (10-20 psu) than at the highest salinity (30 psu) by the end of 48 h exposure. Because the degrees to which the uptake of Se (a metalloid) and Cd/Zn were affected by salinity were comparable, we concluded that metal speciation as a result of salinity change was not important in leading to a change in metal CF. Physiological changes may be responsible for the increasing uptake at lowered salinity. The elimination rates of the three metals (0.01-0.06 d(-1)) were not significantly affected by salinity, but Se was eliminated at a faster rate following aqueous uptake than following dietary ingestion. There was no consistent influence of exposure routes on Cd and Zn elimination. The accumulated Cd was mainly associated with the gut, whereas the muscle was the dominant target site for Se and Zn accumulation.

The uptake of Cd, Cr, and Zn by the macroalga Enteromorpha crinita and subsequent transfer to the marine herbivorous rabbitfish, Siganus canaliculatus.

Marine macroalgae can appreciably accumulate metals out of seawater and are the principle food source for marine herbivorous fish, thus playing an important role in introducing metals into marine food webs. There have been few experimental studies on metal transfer from macroalgae up the trophic chain. In this study, we examined the assimilation efficiencies (AEs) of Cd, Cr, and Zn in the rabbitfish, Siganus canaliculatus, which feeds on the macroalga Enteromorpha crinita. The influences of metal concentration and nutrient conditions in the macroalga, and starvation on the assimilation of metals in fish were investigated. The macroalgae were radiolabeled by direct exposure to radiotracers in the dissolved phase. The uptake of metals in the macroalgae proceeded linearly and was directly proportional to the metal concentration in the ambient seawater. Ammonium and nitrate enrichment significantly increased the accumulation of Cd by the macroalgae, whereas Cr accumulation was not influenced by nutrient enrichment. The metal concentrations in the macroalgae did not have any effect on metal assimilation in the rabbitfish. The measured AEs of the metals were generally higher in starved rabbitfish than in fed fish, probably as a result of the prolonged retention of metals in the gut of fish. Rabbitfish fed macroalgae that had been previously exposed to ammonium or nitrate addition decreased their assimilation of Cd and Zn, but not Cr. Phosphate enrichment did not have any significant effect on metal AE in the rabbitfish. Our study demonstrated that marine herbivorous fish can appreciably assimilate trace metals from marine macroalgae and that trophic transfer should be considered as a source for metal accumulation in fish. Nutrient condition and starvation do have considerable effects on the AEs of Cd and Zn in rabbitfish that are fed macroalgae.

Trophic transfer of heavy metals from freshwater zooplankton Daphnia magna to zebrafish Danio reiro.

The trophic transfer of metals along the food chain has been recognized as an important issue in the study of water quality in recent years. Feeding experiments were conducted to examine the assimilation of three metals (Cd, Cr and Zn) by the zebrafish Danio reiro feeding on the freshwater zooplankton Daphnia magna. The zooplankton were exposed to radiotracers from both the aqueous and dietary phases for different duration, and then pulse-fed to the zebrafish for measurements of metal assimilation efficiency (AE). The calculated AEs were 3-8% for Cd, 2-39% for Cr, and 17-36% for Zn in the zebrafish. For Cd and Zn, there was no statistically significant difference between the two different radiolabeling routes (aqueous and dietary exposure). For Cr, the AEs were higher when it was accumulated by D. magna from the dietary source than when it was accumulated from the aqueous phase. The gut passage time (GPT) was 6-10 h for all metals, with less variation for Zn among the different treatments. There was no obvious relationship between metal GPT and metal AE, presumably due to the narrow range of variation of metal gut passage. About 5-36%, 20-31%, and 8-30% of the total Cd, Cr and Zn was found in the soft tissue of D. magna after the radiolabeling. A much higher fraction of Cd and Zn was found in the soft tissue of D. magna when the metals were accumulated from the dietary phase. No significant relationship between the metal AE and the metal distribution in the soft tissue of D. magna was however documented in this study. Our results demonstrated that there was major difference in metal AE in freshwater fish among different metals. Metal localization in prey organisms and GPT appear to have little influence on metal assimilation by the zebrafish.