BOTTON, M.*; POGORZELSKA, M.; SMORAL, L.; SHEHATA, A.; HAMILTON, M.; Fordham Univ.; Fordham Univ.; Fordham Univ.; Fordham Univ.; Fordham Univ.: Thermal Biology of Horseshoe Crab Embryos and Larvae: A Role for Heat Shock Proteins
The sandy intertidal beaches where horseshoe crab (Limulus polyphemus) embryos and trilobite larvae live are thermally stressful habitats. Heat shock proteins (Hsp’s) are known to help protect a wide variety of organisms against temperature and other stresses by protecting proteins from denaturation, thereby preserving their physiological integrity. To study the potential importance of Hsp’s in the thermal biology of horseshoe crabs, stage 20 embryos and trilobite larvae were acclimated to 13 or 22°C and then heat shocked at 35°C for 3 hr, and the quantity of Hsp70 was assayed after 0, 2, 4, and 6 hr recovery times at 22°C. The temperature shocks were sublethal; 98% to 100% of the animals that were heat shocked under these conditions successfully molted. Embryos and trilobites were homogenized in tissue PE-LB buffer with DTT (reducing agent) and protease inhibitors. We then detected Hsp70 in tissue extracts using SDS-PAGE electrophoresis followed by Western blotting (using monoclonal mouse antibody against bovine Hsp70). We found high levels of constitutive Hsp70 in control crabs that were maintained at constant temperatures of 13 and 22°C. By using 2-D electrophoresis, which combines isoelectric focusing and SDS-PAGE, we found qualitative differences between the embryos and trilobites in their Hsp70 isoforms. However, levels of Hsp70 in control crabs were similar to those found in all heat shock treatments, regardless of the acclimation temperature or the duration of the recovery period. Temperature stress did not induce additional synthesis of Hsp70; rather, by maintaining a high constitutive level of Hsp’s, horseshoe crabs may be adapted to the cyclical tidal/diurnal temperature fluctuations that typify estuarine beaches.
