Evaluating the methodology for detecting stress proteins expression in corals exposed to drastic changes in the environment

Deborah Parrilla Hernández and Suhey Ortíz

INTRODUCTIONCoral reefs result from the interaction between symbiotic organisms composed of a dinoflagellate algae (zooxanthellae) and scleractinian corals. A central environmental issue in the context of global climate is the susceptibility of scleractinian corals to temperature changes, UV radiation and other natural and anthropogenic stress inducers. Such perturbations are likely the cause of the reported observations over the past two decades, especially in the 1990’s, when coral reefs experienced extensive degradation worldwide (Brown et. al., 1997). Most of these observations describe the ‘bleaching’ phenomenon as an immediate response of corals to drastic change in their normal growth conditions. Bleaching is a process whereby corals and other invertebrates in symbiotic relationship with the dinoflagellate algae, lose or experience degradation of the symbiont’s photosynthetic pigments. The main objective of this research is to use molecular biomarker system (i.e. stress proteins), that are already developed, to analyze specific parameters of coral cellular physiology and evaluate the methodology to detect stress proteins expression in corals.

METHODOLOGYField work consists of coral trasplantation from deep (7-10 m) to shallow and protected areas in the reef and the monitoring of UV light incidence and temperature. Experimental conditions are naturally exposed corals and corals with UV exclusion.At this time two different evaluation methodologies for stress proteins detection are analyzed in order to select that that is the most inexpensive, easy to implement and precise. One of the techniques includes the application of an immunological assay- ELISA (StressGen-Hsp 70 Kit) and the other employs traditional methodology: SDS-PAGE, immunoblotting and Immunodetection.

RESULTSELISA assay proves to be an easy to implement method to analyze the presence and quantify stress proteins; it is also less time-consuming. The ELISA assay uses the following standard concentrations for stress protein 70: 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0 (ng/ml). A calibration curve (Figure 1) was generated in order to determine the sample stress protein concentration. A limitation of this Hsp 70 kit is that antibodies used are not specific for cnidarians and they are specific for hsp 70. Recently developed antibodies for cnidarians allows us to evaluate stress proteins expression employing the traditional methodology, which involves three steps: protein separation in SDS-PAGE (Figure 2), immunoblotting and immunodetection using specific antibodies for different stress proteins: sp 90, sp 70, and sp 45 and small stress proteins 25 kDa (Figure 3). This method is tedious and time-consuming compared to the Hsp 70 kit.

CITED LITERATUREBlack, N., R. Voellmy, and A Szmant. 1995. Heat shock protein induction in Montastrea faveolata and Aiptasia pallida exposed to elevated temperatures. Biol. Bull. 188:234-240.Brown, B.E. 1997. Coral bleaching: causes and consequences. Coral Reefs 16 (suppl.):S129-S138.Downs, C.A., E. Mueller, S. Phillips, J.E. Fauth and C. Woodley. 2000. A molecular biomarker system for assessing the health of coral (Montastrea faveolata) during heat stress. Mar. Biotechnology 2. pp:1-12.Downs, C.A., J.E. Fauth, J.C. Halas, P. Dustan, J. Bemiss and C.M. Woodley. 2002. Oxidative stress and seasonal coral bleaching. Free Radical Biology and Medicine. 33(4):533-543.Fang, L., S. Huang and K. Lin. 1997. High temperature induces the synthesis of heat shock proteins and the elevation of intracellular calcium in coral Acropora grandis. Coral Reefs. 16:127-131.Hayes, R. and C. King. 1995. Induction of 70 kD heat shock protein in scleractinian corals by elevated temperature: significance for coral bleaching. Mol. Mar. and Biotech. 4(1):36-42.Tom, M., J. Douek, I. Yankelevich, T.C.G. Bosch and B. Rinkevich. 1999. Molecular characterization of the first heat shock protein 70 from a reef coral. Biochemical and Biophysical Research Communications. 262:103-108.

ACKNOWLEDGEMENTSThanks to the team of women of the ADVANCE IT program at UPR-H for their invaluable help. This material is based upon work supported by the National Science Foundation under Grant No. SBE-0123654.  Any opinions, findings, and conclusions or recommendations expressed in

this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Figura 1. Curva de calibración de Hsp 70

y = 0.9464x + 7.9108R2 = 0.9794

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Figura 1. Curva de calibración de Hsp 70

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Evaluating the methodology for detecting stress proteins expression in corals exposed to drastic changes in the environment

Deborah Parrilla Hernández and Suhey Ortiz Biology Department, University of Puerto Rico-Humacao, Postal Station CUH, Humacao,

Puerto Rico 00791INTRODUCTION

Coral reefs result from the interaction between symbiotic organisms composed of a dinoflagellate algae (zooxanthellae) and scleractinian corals. A central environmental issue in the context of global climate is the susceptibility of scleractinian corals to temperature changes, UV radiation, and other natural and anthropogenic stress inducers. Such perturbations are likely the cause of the reported observations over the past two decades, especially in the 1990’s, of coral reefs experiencing extensive degradation worldwide (Brown et. al., 1997). Most of these observations describe the ‘bleaching’ phenomenon as an immediate response of corals to drastic change in their normal growth conditions. Bleaching is a process whereby corals and other invertebrates in symbiotic relationship with the dinoflagellate algae, lose or experience degradation of the symbiont’s photosynthetic pigments. The main objective of this research is to use molecular biomarker system (i.e. stress proteins), that are already developed, to analyze specific parameters of coral cellular physiology and evaluate the methodology to detect stress proteins expression in corals.

METHODOLOGY•Field work consists of corals trasplantation from deep (7-10 m) to shallow and protected areas in the reef and the monitoring of UV light incidence and temperature. Experimental conditions are naturally exposed corals and corals with UV exclusion.•At this moment two different evaluation methodologies for stress proteins detection are analyzed in order to select the most inexpensive, easy to implement and precise. One of the techniques includes the application of an immunological assay- ELISA (StressGen-Hsp 70 Kit) and the other employs traditional methodology: SDS-PAGE, immunoblotting and Immunodetection.

RESULTSELISA assay proves to be an easy to implement method to analyze the presence and quantify stress proteins; it is also less time-consuming. The ELISA assay uses the following standard concentrations for stress protein 70: 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0 (ng/ml). A calibration curve (Figure 1) was generated in order to determine the sample stress protein concentration. A limitation of this Hsp 70 kit is that antibodies used are not specific for cnidarians and they are specific for hsp 70. Recently developed antibodies for cnidarians allows us to evaluate stress proteins expression employing the traditional methodology, which involves three steps: protein separation in SDS-PAGE (Figure 2), immunoblotting and immunodetection using specific antibodies for different stress proteins: sp 90, sp 70, and sp 45 and small stress proteins 25 kDa (Figure 3). This method is tedious and time- consuming compared to the Hsp 70 kit.

CITED LITERATUREAvailable upon request

ACKNOWLEDGEMENTSThanks to the team of women at ADVANCE IT Program UPR-H for their invaluable help. This material is based upon work supported by the National Science Foundation under Grant No. SBE-0123654.  Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Figure 2. SDS-PAGE analysis

Figure 3. Comparison of immunodetected stress proteins

70 kDa

25 kDaFigure 1