1. EFFECT OF MERCURY ON THE BRINE SHRIMP ARTEMIA FROM THE GREAT SALT LAKE Nicole Berthelemy-Okazaki and Dustin Ingraham Department of Zoology, Weber State University, Ogden, Utah, USA nokazaki@weber.edunokazaki@weber.edu Abstract Mercury (Hg), a toxic metal accumulating in the Great Salt Lake (GSL), has been found in high levels in shrimp and bird tissues. We investigated the effect of Hg on various brine shrimp life cycle and biochemical parameters. Larvae and adult survival were tested after 24-hour acute exposure in Hg concs. ranging from 1 g to 1 μg/l. Survival, maturation time, number of eggs per brood and type of brood were observed in shrimp exposed to Hg concs. ranging from 1 mg to 1 μg/l. 1)SDS-PAGE on shrimp tissue showed protein profiles under Hg stress. 2)Mortality levels increased sharply from 0 to 50% in adult & larvae e exposed to Hg concs. increasing from 1 to 10 mg/l. 3 ) Survival rates were minimally affected in shrimp raised at 1 mg/l Hg. 4) However, maturation time increased while the #eggs/broods decreased in shrimp raised at 0.1 mg/l Hg. 5) No significant effect was demonstrated in shrimp raised in 0.1 μg/l Hg. 6) Protein profiles suggest that some proteins are down-regulated while others are up-regulated. Introduction - Toxic compounds accumulating in the lake, among them Hg 1 - Hg: source from local industries & airborne particles 3. - Hg: deleterious effects of the health of the brine shrimp & on bird populations 1,2. - Objectives: 1) quantify mortality rate in shrimp exposed a wide range of Hg concs. for 24 hr 2) monitoring several life parameters (survival, maturation & # eggs/brood) in brine shrimp exposed to chronic Hg - Correlations between acute stress & biochemical changes in Artemia were studied Materials & Methods Life parameters: - Acute exposure: Five sets of 20 adults and 32-day old larvae were exposed for 24 hrs to Hg concs. ranging from 1 g to 1 μg/l of 3% seawater at 24 o C (by 10 fold increment). Mortality rates were calculated. - Chronic exposure: Five sets of 50 larvae were raised to adulthood in Hg concs. ranging from 1 μg/l to 1 mg/l. Survival, maturation time & fertility were monitored. Biochemical studies: - In each study, for each Hg conc., brine shrimp samples were run on a SDS-PAGE for protein profile. Heat shock proteins 70 & 90 (hsp 70 & 90) were measured by western blot. Statistical analysis: - One way ANOVA was applied to test effect of treatments. Results --Acute exposure - A sharp increase in mortality is seen when Hg concs. increase from 10 to 100 mg/l. - This effect is similar in adults and larvae (Fig. 1). --Chronic exposure Maturation time Maturation times not significantly different when shrimp raised in Hg Concs. ranging from 1 mg to 1 µg/l (Figs. 2 & 3). Note: Survival rates to maturity in control, 1, 10, 100 and 1000 μg/l were respectively 43, 94, 92, 38 and 27%. # eggs/brood (fertility index) S\strongly affected by Hg levels at & above 0.1 mg/l (Figs. 4 & 5). - Biochemical responses – protein profiles - A SDS-PAGE gel with a stain for general proteins shows the protein profile in Artemia placed in varying Hg concs. (Fig. 6) - For clarity, Fig. 7 shows a scanning of Lanes 1 & 5 (0 & 10 mg/l Hg). Some proteins in the vicinity of hsp 90 & 70 appear to be up-regulated. Fewer proteins are down-regulated. - A western blot (Fig. 8) with anti hsp70 shows a correlation between increasing Hg concs. & darker staining. Discussion & Conclusion # eggs/brood - Increasing Hg conc. in the Great Salt Lake are likely to impact the shrimp & therefore the bird populations. - Hg conc. of 10 ug/l, while not killing the shrimp sharply reduces their fertility. - Some proteins might indicate stress in the shrimp. - A potential stress indicator might be hsp 70; further research to confirm & quantify this effect. Literature Cited Acknowledgements This research was funded by the WSU Undergraduate Research Office & Research, Scholarship & Growth Committee. 1Naftz, D., B. Waddell, D. Krabbenhoft. 2005. Mercury in water and biota from Great Salt Lake, Utah: Reconnaissance-phase results. USGS 2 Sarabia, R., A. Torreblanca, J.J. Del Ramo and J. Diaz-Mayans. 1998. Effects of low mercury concentration exposure on hatching, growth and survival in the Artemia strain La Mata parthenogenetic diploid. Comp. Biochem. Physiol. (A) 120: 93-97. 3 Schuster, P., D.P. Krabbenhoft, D.L. Naftz, L. DeWaynececil, M.L. Olson, J. Dewild, D. Susong, J. Green and M. Abbot. 2002. Atmospheric Mercury Deposition during the Last 270 Years: A Glacial Ice Core Record of Natural and Anthropogenic Sources. Environ. Sci. Technol. 2002, 36, 2303-2310. 10 -3 = 1000 mg/l 10 -4 = 100 mg/l 10 -5 = 10 mg/l 10 -6 = 1 mg/l 10 -7 = 0.1 mg/l 10 -8 = 0.01 mg/l 10 -9 = 0.001 mg/l Fig. 6. Protein profile (SDS-PAGE) of shrimp exposed to varying levels of Hg for 24 hrs. Fig. 7. Scanning profile of lanes 0 & 5 (0 &10 mg/l Hg) Fig. 8. Hsp 70 western blot of shrimp exposed to varying levels of Hg for 24 hrs. - Life parameters