Copyright 0 1984 by the Genetics Society of America MULTIPLE-LOCUS HETEROZYGOSITY AND THE PHYSIOLOGICAL ENERGETICS OF GROWTH IN THE COOT CLAM, MULINIA LATERALIS, FROM A NATURAL POPULATION DAVID W. CARTON, RICHARD K. KOEHN AND TIMOTHY M. SCOTT Department of Ecology and Evolution, State University of Nno York at Stony Brook, Stony Brook, New York 11 794 Manuscript received February 29, 1984 Revised copy accepted June 4, 1984 ABSTRACT The relationship between individual energy budgets and multiple-locus het- erozygosity at six polymorphic enzyme loci was examined in Mulinia lateralis. Energy budgets were determined by measuring growth rates, rates of oxygen consumption, ammonia excretion and clearance rates. Enzyme genotypes were determined using starch gel electrophoresis. Growth rate and net growth effi- ciency (the ratio of energy available for growth to total energy absorbed) increased with individual heterozygosity. The positive relationship between ob- served growth and multiple-locus heterozygosity was associated with a negative relationship between routine metabolic costs and increasing heterozygosity. Re- duction in routine metabolic costs explained 60% of the observed increased growth of more heterozygous individuals. When routine metabolic costs were standardized for differences in feeding rates, these standard metabolic costs explained 97% of the differences in growth rate. Lower standard metabolic costs, associated with increasing heterozygosity, have been proposed as a phys- iological mechanism for the relationship between multiple-locus heterozygosity and growth rate that has been reported for a variety of organisms, ranging in diversity from aspens to humans. This study demonstrates that reduction of standard metabolic costs, at least in clams, accounts for virtually all of the differences in growth rate among individuals of differing heterozygosity. VIDENCE of increasing growth rate and metabolic efficiency with greater E multiple-locus heterozygosity at electrophoretically detectable enzyme loci has been accumulating for a wide variety of plant and animal species (KOEHN and GAFFNEY1984). A positive relationship between multiple-locus heterozy- gosity and growth, or the energy available for growth, has recently been dem- onstrated in a variety of marine invertebrates, including oysters (SINGHand ZOUROS 1978; ZOUROS, SINCHand MILES 1980; KOEHN and SHUMWAY1982), mussels (KOEHN and GAFFNEY1984) and oyster drills (GARTON1984). The results of these studies on natural, outbred populations have been similar: growth rate increases with levels of heterozygosity. Except in one case (KOEHN and SHUMWAY1982), the underlying causes for these differences in growth have not been identified. Genetics 108 445-455 October, 1984 446 D. W. GARTON, R. K. KOEHN AND T. M. SCOTT Scope for growth equals the energy available for growth and reproduction and is expressed as the difference between absorbed energy and energy lost through metabolic processes (WINBERG1956; BAYNEand NEWELL1983). En- ergy available for growth can be increased, in principle, by increasing metabolic efficiency (reducing weight-specific metabolic rates) and/or increasing absorp- tion (by increasing feeding rate or absorption efficiency). KOEHN and SHUMWAY (1 982) demonstrated that weight-specific basal oxygen uptake declined signif- icantly with increasing heterozygosity in starved oysters, Crassostrea wirginica. They hypothesized that the greater metabolic efficiency associated with het- erozygosity would also be reflected in greater scope for growth. RODHOUSE and GAFFNEY(1 984) further demonstrated in starved oysters that the increased metabolic efficiency with increasing heterozygosity is reflected in reduced rates of dry weight loss (lower negative scope for growth). These studies demonstrate qualitatively and indirectly that relatively high scopes for growth can be due to increased metabolic efficiency. Growth, however, is also influenced by feeding rate and there are metabolic costs associated with feeding activities. In the oyster drills, Thais haemastoma and T. lamellosa, increased feeding rates were the most important factor as- sociated with size and heterozygosity affecting scope for growth (GARTON 1984; CARTONand STICKLE1984). Although routine metabolic costs were inversely related to multiple-locus heterozygosity, increased metabolic eff- ciency could account for only 20% of the increased scope for growth associated with increased individual heterozygosity. The energetic cost of feeding was not determined in the oyster drill study (CARTON1984). To date there has been no investigation of the relative quantitative contri- butions of standard metabolic efficiency, feeding rate and the metabolic cost of feeding to the observed relationship between growth and multiple-locus heterozygosity. This study reports on the relative contributions of these com- ponents of the energy budget to the relationship between growth rate and multiple-locus heterozygosity in the clam, Mulinia lateralis. MATERIALS AND METHODS Clams: Individuals of M. lateralis were collected by dredging from Great South Bay, Long Island, New York, in July 1983. Clams were brought to the laboratory and placed in 5.5-liter plastic trays filled with filtered, aerated seawater at 23” and 26”/00 salinity. The seawater was changed every 2-4 days. Clams were fed a diet of mixed culture algae, Isochrysis galbana and Monochrysis sp., reared by the methods of GUILLARD(1975). Clams were fed an average of twice per day during the 30-day experimental period for determining growth rates. Individual clams (N = 150) were tagged with numbered discs, or “bee dots” (Chr. Graze AG, West Germany) and polyacrylate glue. Shell lengths and heights were measured to the nearest 0.01 mm using vernier calipers. Soft tissue dry weight was estimated using the allometric relationship between dry weight and shell height: flesh dry weight (milligrams) = 0.006 (shell height in millimeters)3-495,r2 = 0.777, n = 494 (S. E. SHUMWAYand T. M. SCOTT,unpublished data). Energy budgets: Equations describing the flow of energy through organisms have been developed (WINBERG1956; CRISP 1971) and applied to marine bivalves (reviewed by BAYNEand NEWELL 1983). “Scope for growth” represents the energy remaining after the metabolic demands of the organism have been met and can be positive or negative. Scope for growth (SG) was determined using the following form of the energy budget: SG = Ab - (R + U), HETEROZYGOSITY AND GROWTH IN CLAMS 44 7 where Ab (absorption) is that part of consumed energy that is not eliminated as feces, R (respiration) is that part of the assimilated energy that is converted directly to heat or to mechanical work performed by the organism and U (excretion) is that part of the assimilated energy that is passed out of the organism as excretory material, usually as urine. Direct measurement of growth rates were made over 4 wk of the experiment. Following the measurement of growth rate, the oxygen uptake, ammonia excretion and feeding rate of individual clams were determined. Respiration was measured using a Gilson differential respirometer and standard manometric techniques. Oxygen uptake was converted to energy equivalents by applying the oxycaloric coefficient of 4.73 cal/ml of oxygen consumed at standard temperature and pressure (STP) (CRISP 197 1). Ammonia excre- tion was determined by GRASHOFFand JOHANNSEN’S (1 972) modification of the phenol-hypo- chlorite method. ELLIOTand DAVISON(1975) give the energy equivalent of ammonia excretion at 5.94 cal fmg of ammonia nitrogen excreted. Feeding rates were determined for each clam (inde- pendent of oxygen uptake) by measuring the rate of disappearance of an algal suspension in a static system using a model ZB Coulter Counter. Therefore, routine metabolic costs (R + U) were measured directly and scope for growth (SG) was estimated from the change in dry weight over time, assuming a caloric equivalent of 4 calfmg of dry weight. Total energy absorption (Ab) was calculated from the difference. “Routine metabolism” refers to oxygen consumption measured with uncontrolled but minimum motor activity (PROSSER1973). As activity varies among individuals, oxygen uptake was extrapo- lated to zero activity. Since the primary activity of filter-feeding bivalves is feeding (clearance of algae from water) (BAYNEand NEWELL1983), oxygen uptake was standardized using the linear relationship between oxygen uptake and clearance rate: Oxygen uptake rate (VOs) = a + b (clearance rate) and adjusted intercepts were calculated for each individual as a = observed VOn - b (observed clearance rate). The intercept, a, represents oxygen uptake at zero activity and is termed “standard metabolism.” Similarly, growth rate is also related positively to feeding rate. Clearance rate-specific growth rates were calculated from the linear relationship: Growth rate = a + b (clearance rate), and the adjusted intercept was calculated for each individual as a = observed growth rate - b (observed clearance rate). This provides an estimate of growth rate on a “standard ration.” Therefore, both energetic costs (oxygen consumption) and energetic gains (growth rate) were standardized on a common variable, feeding rate. Weight-specific rate functions were calculated from the whole animal rate functions using the general form: V, = aWb where V, is the whole animal rate variable and W is the flesh dry weight; a and b are fitted constants calculated from the regression of the log of the rate variable on the log of dry weight,