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

1
Evaluating the methodology for detecting stress
proteins expression in corals exposed to drastic
changes in the environmentDeborah Parrilla
Hernández and Suhey Ortíz

• INTRODUCTION
• 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 1990s, 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 symbionts 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 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.
• RESULTS
• ELISA 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 LITERATURE Black, N., R. Voellmy, and A
Szmant. 1995. Heat shock protein induction in
Montastrea faveolata and Aiptasia pallida exposed
to elevated temperatures. Biol. Bull.
188234-240. 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.
pp1-12. 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. 16127-131. 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. 262103-108. ACKNOWLEDGEMENTS Tha
nks 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.
Figure 3. Comparison of immunodetected stress
proteins
2
Evaluating the methodology for detecting
stress proteins expression in corals exposed to
drastic changes in the environmentDeborah
Parrilla Hernández and Suhey Ortiz Biology
Department, University of Puerto Rico-Humacao,
Postal Station CUH, Humacao, Puerto Rico 00791

• INTRODUCTION
• 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 1990s, 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
  symbionts 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.

Figure 2. SDS-PAGE analysis
RESULTS ELISA 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.
70 kDa
25 kDa
Figure 1
Figure 3. Comparison of immunodetected stress
proteins
CITED LITERATURE Available upon
request ACKNOWLEDGEMENTS Thanks 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.