Interrelationships of heterozygosity, growth rate and heterozygote deficiencies in the coot clam, Mulinia lateralis.

Allozyme surveys of marine invertebrates commonly report heterozygote deficiencies, a correlation between multiple locus heterozygosity and size, or both. Hypotheses advanced to account for these phenomena include inbreeding, null alleles, selection, spatial or temporal Wahlund effects, aneuploidy and molecular imprinting. Previous studies have been unable to clearly distinguish among these alternative hypotheses. This report analyzes a large data set (1906 individuals, 15 allozyme loci) from a single field collection of the coot clam Mulinia lateralis and demonstrates (1) significant heterozygote deficiencies at 13 of 15 loci, (2) a correlation between the magnitude of heterozygote deficiency at a locus and the effect of heterozygosity at that locus on shell length, and (3) a distribution of multilocus heterozygosity which deviates from that predicted by observed single-locus heterozygosities. A critical examination of the abovementioned hypotheses as sources of these findings rules out inbreeding, null alleles, aneuploidy, population mixing and imprinting as sole causes. The pooling of larval subpopulations subjected to varying degrees of selection, aneuploidy or imprinting could account for the patterns observed in this study.

The effect of glucose-6-phosphate isomerase genotype on in vitro specific activity and in vivo flux in Mytilus edulis.

Four samples of the mussel Mytilus edulis were taken between 1984 and 1987 from Stony Brook, New York, and used to study the glucose-6-phosphate isomerase (GPI) polymorphism in this species. In vitro specific activity and in vivo flux measured in the same animals were found to be significantly correlated. A significant effect of GPI genotype on flux was observed in one of the samples; overall, significant evidence of effect of genotype on enzyme activity was also obtained. GPI activities of common genotypes tend to deviate less from the population mean than those of rare (frequency less than 5%) genotypes. This suggests the possibility that rare GPI genotypes are rare as a consequence of having biochemical properties that deviate from an optimum level and, therefore, having a lower fitness. In support of this hypothesis, we found in one of our samples that shell length is a concave function of GPI activity with an intermediate optimum activity level.

The differential contribution by individual enzymes of glycolysis and protein catabolism to the relationship between heterozygosity and growth rate in the coot clam, Mulinia lateralis.

The locus-specific effects of heterozygosity upon individual growth rate were determined for 15 polymorphic enzymes among 1906 individuals from a single cohort sample of the marine bivalve Mulinia lateralis. Two measures of individual growth rate (total wet weight and shell length) were made at collection and after a period of growth in the laboratory. The correlation between heterozygosity and growth rate was independently determined for each locus using multiple linear regression, thereby providing a rank of individual locus effects; these differed significantly. The four estimated rankings of relative locus effects (initial length, initial weight, length added in the laboratory, and added weight) were not statistically different. That is, a locus with a large effect of heterozygosity on growth rate in nature had a similarly large effect on laboratory growth rate. The effect of a locus was not related to heterozygosity per se; some highly heterozygous loci had no detectable correlation with growth rate. The data contained two pairs of relatively tightly linked loci; in both cases one locus of a pair had significant effects on growth rate, while the other had no effect. Loci with large and significant correlations with growth rate synthesize enzymes which function in protein catabolism or glycolysis; heterozygosity in enzymes of the pentose shunt, redox balance, or other miscellaneous metabolic roles was not correlated with growth rate. Since the metabolic basis for the correlation is known to derive from individual differences in net energy status, particularly energetic costs of whole-body protein turnover, these data indicate that phenotypic effects (e.g., variation in growth rate) are determined by heterozygosity at the studied genes, not other linked loci.

Dominance in physiological phenotypes and fitness at an enzyme locus.

Aminopeptidase-I allozymes, which are products of the Lap locus in the marine mussel, Mytilus edulis, differ in their catalytic efficiencies. These biochemical differences result in genotype-specific rates of change in the free amino acid pool, that is, cell volume regulation, when mussels are subjected to changes in salinity. A high degree of dominance was found among genotypes for these biochemical and physiological phenotypes. Selection models that incorporate dominance adequately predict observed genotypic properties at the Lap locus among natural populations that exhibit clinical allele frequency. This suggests that a high degree of dominance for fitness must also occur at this locus in natural populations. These results provide additional evidence that the maintenance of an allele frequency cline is operating by natural selection at the Lap locus.

Multiple-locus heterozygosity and the physiological energetics of growth in the coot clam, Mulinia lateralis, from a natural population.

The relationship between individual energy budgets and multiple-locus heterozygosity 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 efficiency (the ratio of energy available for growth to total energy absorbed) increased with individual heterozygosity. The positive relationship between observed growth and multiple-locus heterozygosity was associated with a negative relationship between routine metabolic costs and increasing heterozygosity. Reduction 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 physiological 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.

Biochemical studies of aminopeptidase polymorphism in Mytilus edulis. I. dependence of enzyme activity on season, tissue, and genotype.

Aminopeptidase-I is polymorphic in the marine bivalve Mytilus edulis and catalyzes the liberation of neutral an aromatic N-terminal amino acids from oligopeptides. The enzyme is abundant in the digestive gland, where it is lysosomal, but is present in several other tissues. Temporal variation in enzyme activity was monitored for 2.5 years in two natural populations. The temporal pattern of variation was similar in gill, mantle, and digestive gland tissues; variations occurred over both short and long time periods. Enzyme activity under ambient temperature conditions was seasonally related to temperature in gill and digestive gland, but varied with reproductive cycle in mantle tissue. In the last, maximum activity corresponded to the postreproductive period in each population. Enzyme activity varies in response to tissue-specific metabolic demands. Population difference in enzyme activity are due to both genotype-dependent enzyme activity, since allele frequencies differ between populations, and environmental salinity. High salinity induces high activity, which is a response to the need for higher intracellular concentrations of free amino acids for cell volume regulation. Salinity has comparable effects on enzyme activity in natural and experimental populations. Genotype-dependent specific activities are a consequence of both differing kinetic properties among genotypes [Koehn, R. K., and Siebenaller, J. S. (1981). Biochem. Genet. 19: 1143] and genotype-specific concentration of enzyme protein that change in response to environmental salinity.

Biochemical studies of aminopeptidase polymorphism in Mytilus edulis. II. Dependence of reaction rate on physical factors and enzyme concentration.

Enzymatic parameters of aminopeptidase-I that may be sensitive to temperature an solute variations were investigated to provide a functional explanation for specific activity differences among genotypes in natural populations. The effect of temperature on the apparent Km of L-leucyl-4-methoxy 2-naphthylamide and th dipeptide phenylalanyl-glycine was small, especially between 10 and 25 C. The apparent Km varied only between 36.7 and 49.8 microM at these temperatures and the six common genotypes did not differ in temperature-dependent substrate affinities. While pH had a significant effect on Km, no differences among genotypes were observed. Activation enthalpies were also identical among genotypes. Thermal inactivation was slowest at 15 C and the same for all genotypes. Of 18 tested amino acids, only phenylalanine inhibited aminopeptidase-I; K1 values ranged from 1.2 to 0.8 mM and were the same for all genotypes. Small differences among genotypes were detected in the inhibitory effect of zinc. The concentration of aminopeptidase-I enzyme was the same for all genotypes in a population exposed to oceanic salinity, but the concentration of Lap 94/94 was 15% lower than that of other genotypes in a population experiencing estuarine salinity. Genotypes with the Lap 94 allele exhibited higher apparent Kcat values in all population samples.The probable genotype-dependent effects of enzyme concentration and Kcat differences are discussed with regard to maintenance of the polymorphism and genetic differences among populations.

Maintenance of an aminopeptidase allele frequency cline by natural selection.

The product of the Lap locus in the marine bivalve Mytilus edulis is a neutral, membrane-associated aminopeptidase that is primarily localized on intestinal microvilli and in digestive cell lysosomes. Natural populations are genetically differentiated at the Lap locus between areas of differing salinity. A steep (0.55-0.15) allele frequency cline connects differentiated populations between the Atlantic Ocean and Long Island Sound. We demonstrate an annual gene flow/mortality cycle in cline populations whereby gene frequencies after mortality are correlated with salinity and enzyme activity. The cline is spatially and temporally unstable in immigrants, but stable in residents after mortality. Mortality is nonrandom with regard to the Lap locus; genotype-dependent properties of the aminopeptidase enzyme apparently led to a differential rate of the utilizaiton of nutrient reserves because selected genotypes exhibited an increased rate of tissue weight loss. Aminopeptidase genotypes are differentially adapted to different temperatures and salinities, which provides a mechanism for the relationship among biochemical, physiological, and population phenotypes.

Biochemical characterization of "LAP," a polymorphic aminopeptidase from the blue mussel, Mytilus edulis.

A genetically variable naphthylamidase enzyme, previously described as "leucine aminopeptidase," was purified approximately fiftyfold, and its biochemical properties were investigated. The enzyme was renamed "aminopeptidase I." Substrate affinities demonstrate that it is an alpha-aminoacyl peptide hydrolase (E.C. 3.4.11.-). Aminopeptidase I had a monomer molecular weight of 65--68,000, average of pI of pH 4.88, and broad pH optima between 6.5 and 8.0. The enzyme was inactivated rapidly between 40 and 50 C. Antibodies from purified enzyme did not cross-react with other naphthylamidases, but aminopeptidase I activity was inhibited by immune serum. The enzyme exhibited highest naphthylamidase activity for aromatic and hydrophobic aminoacyl naphthylamides. Aminopeptidase activity was highest for aromatic and hydrophobic N-terminal residues of tripeptides. Certain divalent metal cations, p-OH-mercuribenzoate, and N-ethylmaleimide were strongly inhibitory while chelating agents activated the enzyme.

Relationship between subunit size and number of rare electrophoretic alleles in human enzymes.

Data from published sources were used to compare the numbers of different electrophoretic alleles of 29 monomeric and dimeric human enzymes to their respective subunit molecular weights. Only those human enzymes were considered for which the total sample sizes were in excess of 2000 individuals. Correlations between these two variables were determined within sample size ranges of 2000 less than or equal to n less than or equal to 3000 and 4000 less than or equal to n less than or equal to 5000 individuals, and separately by quaternary class. There was no statistically significant correlation observed for the smaller sample size range in monomers; however, the correlations for the larger sample size range in monomers and both ranges in dimers were significant. Since there is no relationship between subunit size and heterozygosity, the relationships are due primarily to the incidence of rare alleles. These findings demonstrate the effect of locus-specific mutation rates, expected as a consequence of variation of cistron sizes, and imply that other forces are responsible for the relative frequencies of common alleles at some of the loci.

Morphological adaptation to thermal stress in a marine fish, Fundulus heteroclitus.

Populations of Fundulus heteroclitus (Cyprinodontidae), a coastal marine fish, were studied in control and artificially heated environments on the north shore of Long Island to determine patterns of variation in morphology and the extent to which this variation reflected adaptation to environmental characteristics. Principal components and discriminant function analyses were used to analyze variation in and among seventeen morphological characters. Fishes living in water artificially heated by a power plant exhibited marked divergence from control populations in head morphology, and convergence with a population sampled at more southern latitudes. Hence, these differences were interpreted as adaptations to warm environments. Greater morphological variation is detected at the heated locality than at control localities, and this may be partially due to a breakdown in developmental homeostasis, and partially due to selection favoring phenotypes that are rare in this environment.

Genetic organization and adaptive response of allozymes to ecological variables in Fundulus heteroclitus.

Populations of Fundulus heteroclitus, (Cyprinodontidae) a widespread coastal marine fish, were studied in control and artificially heated environments on the North Shore of Long Island, New York to determine (1) patterns of variation in biochemical phenotypes and (2) the extent to which this variation reflected adaptation to environmental characteristics. Variation at three of twelve polymorphic isoenzyme loci from the warm water population was beyond the range of variation among control populations, and resembled those determined for populations living at more southern latitudes. Hence, these differences were interpreted as adaptations to warm environments. Significant differences in allele frequencies and zygotic proportions at ten of twelve isoenzyme loci were found associated with differences in environments, sexes, and/or age classes. These data strongly support the view that protein polymorphisms are adaptive. Several observations suggested that selection acts upon multilocus phenotypes rather than upon those of single loci. Several di-locus phenotypic distributions were demonstrated to be nonrandom, and those that exhibited similar patterns of dependence over years were postulated to be maintained by selection. Highly heterozygous fish exhibited superior viability when cohorts were compared over successive years. The consequences of the polygynous mating system in this species for maintaining genetic variation and for allowing rapid evolutionary response to a variable environment are discussed.

Population genetics of marine pelecypods. 3. Epistasis between functionally related isoenzymes of Mytilus edulis.

The distribution of interlocus genotypic combinations was examined in Mytilus edulis for interdependence between two loci synthesizing functionally related isoenzymes. There is significant dependence between the Leucine Aminopeptidase and Aminopeptidase loci, which we attribute to epistasis, since the magnitude of dependency varies with age. Furthermore, dependency varies in magnitude with position in the intertidal zone from which samples were taken, suggesting that epistasis is a function of the combination of certain non-homologous alleles as well as of the environmental circumstance in which the combinations occur.