PATRICK K.K. LEONG and DONAL T. MANAHAN, Department of Biological Sciences, University of Southern California, Los Angeles,California 90089-0371
The seawater of McMurdo Sound is characterized by low and constant temperatures and by high seasonal fluctuations in the availability of phytoplankton food sources. The sea urchin Sterechinus neumayeri , which is widely distributed and abundant in McMurdo Sound, has feeding larval forms that have to survive for long periods (weeks to months) in the water column in the near absence of algal foods. The lifespan of these larval forms without food is set by their maternally endowed energy reserves and their rate of utilization of these reserves (i.e., their metabolic rate) (Shilling and Manahan 1994). The aim of this study is to understand the major biochemical processes that establish the metabolic rates for these larval forms. In particular, we have focused on the role of the sodium pump (Na+,K+-ATPase). This enzyme is an important transmembrane protein responsible for maintaining ion gradients in animal cells. Previously, we have shown that the physiological activity of this single enzyme could account for over 40 percent of the metabolic rate of larvae of temperate species of sea urchin (Leong and Manahan 1997). In this study, we quantify the changes in activity of the sodium pump during development of the antarctic sea urchin S. neumayeri . In addition, we present the developmental changes in the activity of Na+,K+-ATPase for fed and starved larvae.
Adult sea urchins were collected from McMurdo Sound (off Cape Evans) by scuba divers in October 1996. Males and females were induced to spawn by standard methods (injection of 0.5 molar potassium chloride) and fertilized eggs were placed in 200-liter culture vessels at a concentration of 7 per milliliter. All subsequent culturing over a 2-month period was done using ambient seawater from McMurdo Sound (-1.5°C) that had been passed through a 0.2-micrometer pore-size filter prior to use. The culture water was changed every 3 to 4 days by gently sieving the animals onto mesh screens. Under these rearing conditions, the embryos developed to the first larval feeding stage (early pluteus) after 22 days. Once the feeding larval stage was reached, two experimental treatments were set up for which larvae were either
The total (potential) Na+,K+-ATPase activity and the physiologically active fraction of the total were measured in both the fed and starved larvae (figure 1 A and B ). The in vivo Na+,K+-ATPase activity of the enzyme (physiological activity at -1.0°C) in larvae was measured as the difference in transport rates of potassium ion (K+) from seawater [rubidium-86 ion (86Rb+) used as a radiotracer] in the presence and absence of ouabain, a specific inhibitor of Na+,K+-ATPase. The total Na+,K+-ATPase activity was measured in vitro using tissue homogenates of larvae (with and without ouabain). The method of Esmann (1988) was used for the in vitro measurements, where the rate of release by Na+,K+-ATPase of inorganic phosphate (Pi) from ATP was measured. All in vitro assays were conducted at 15°C. The activities so obtained were converted to their corresponding values at physiological temperature (-1.0°C) using a Q10 of 2.9. This value was previously determined for the effect of temperature on the activity of Na+,K+-ATPase in S. neumayeri (data not shown). The protein contents of prefeeding stages and of fed and starved larvae were determined using a modified Bradford method (Leong and Manahan 1997) to allow for calculations of developmental changes in the protein-specific activity of the enzyme.
Fed and starved larvae had increases during development in total (figure 1 A ) and in vivo (figure 1 B ) Na+,K+-ATPase activities; the fed larvae had higher activities for both sets of measurements. The protein content of the fed larvae increased with feeding at a rate of 8.1 nanograms per larva per day (figure 2 A ). The starved larvae continuously lost protein at -1.27 nanograms per larva per day (negative slope of regression for loss of protein in starved larvae is statistically significant: F(0.05,1,90) = 5.2, variance ratio = 71.34, P<0.001). Due to this loss of protein, the starved larvae had higher protein-specific total Na+,K+-ATPase activities compared to the fed larvae (figure 2 B ). This finding, which shows that the Na+,K+-ATPase protein was retained in starved larvae relative to the loss of total protein, resulting in higher specific activities of this enzyme in starved larvae, is further illustrated in figure 2 C , where the relationship is shown for enzyme activity as a function of the change in protein content per larva. This figure shows that the data set for the starved larvae was higher than would be predicted from the regression shown for the fed larvae. These data suggest that a critical amount of Na+,K+-ATPase activity has to be maintained in larvae of S. neumayeri . The consequence of this for starved larvae is a higher metabolic rate which will affect the lifespan of these antarctic sea urchin larvae.
We extend our thanks to Tracy Hamilton (University of Southern California) and Robert Robbins (Antarctic Support Associates) for diving operations necessary to collect adult sea urchins and to Adam Marsh and Tracy Hamilton for assistance with larval culturing. This research was supported by National Science Foundation grant OPP 94-20803 to D.T. Manahan.
Esmann, M. 1988. ATPase and phosphatase activity of Na+,K+-ATPase: Molar and specific activity, protein determination. Methods in Enzymology , 156, 105-115
Leong, P.K.K., and D.T. Manahan. 1997. Metabolic importance of Na+, K+-ATPase activity during sea urchin development. Journal of Experimental Biology , 200(22), 2881-2892.
Shilling, F.M., and D.T. Manahan. 1994. Energy metabolism and amino acid transport during early development of antarctic and temperate echinoderms. Biological Bulletin , 187(3), 398-407.