High-resolution electrophoresis has revealed 10 allozymes of esterase-6 (EC 3.1.1.1) in Drosophila melanogaster. The sequences of 13 isolates of the Est6 gene covering all 10 allozymes were obtained and 52 nucleotide differences were found. Sixteen of these cause amino acid replacements, of which three result in charge differences whose size and direction are consistent with the electrophoretic mobilities of the allozymes in which they occur. The smeared electrophoretic phenotype of one allozyme can be explained by the loss of a cysteine residue involved in a disulfide bridge. Several minor mobility variants within the major F and S electrophoretic phenotypes differ by amino acid substitutions that are generally conservative for charge but not for some other properties (size, polarity, or hydrophobicity). Four amino acid differences are found among different isolates of the same allozymes and, overall, 12 amino acid haplotypes occur among the 13 isolates sequenced. Nevertheless, the most common variants within F and S are distinguished by only two amino acids (Asn/Asp at 237 and Thr/Ala at 247), and these are the most likely targets for the selection underlying complementary latitudinal clines in F and S frequencies.Two major difficulties have beset attempts to elucidate the adaptive significance of specific enzyme polymorphisms. The first concerns the lack of information on gametic disequilibrium between the locus of interest and variation at nearby but unknown loci; the second concerns the true amount and nature of molecular polymorphism underlying electrophoretic phenotypes (1). The first difficulty has largely been overcome for the esterase-6 (EST6; carboxylic-ester hydrolase, EC 3.1.1.1) polymorphism ofDrosophila because parallel latitudinal dines in gene frequency occur for the same allozymes in both D. melanogaster and its sibling species D. simulans (2). These species are thought to have diverged 8-10 million years ago (3), suggesting that the Est6 gene itself is probably the unit under selection, as intergenic gametic disequilibrium should have broken down since the divergence of the two species. This paper addresses the second question, concerning the number and nature of polymorphisms for Est6, by comparing the nucleotide sequences of several alleles from D. melanogaster.High-resolution cellulose acetate electrophoresis has shown five major classes of mobility variants for EST6 in D. melanogaster, with seven minor mobility variants within the most common major classes, F (fast) and S (slow) (4). Another seven variants within F and S have been resolved by in vitro thermostability criteria (5), and preliminary analyses of a small number of lines by both electrophoretic and thermostability procedures suggest that the variants detected by the two techniques are not the same (4). While the adaptive significance of the many minor mobility and thermostability variants is unknown, there is biogeographical (2), biochemical (6), and behavioral (7) evidence that the major F and S classes differ under nat...

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High resolution electrophoretic analyses of the polymorphic esterase 6 enzyme have been carried out on 133 isoallelic lines from three Australian populations of Drosophila melanogaster spanning 25° of latitude. These and previous data for 157 lines from another Australian population at an intermediate latitude reveal a total of 14 polymorphic esterase 6 allozymes, falling into five major mobility classes. Two classes, EST6-F and EST6-S, contain eleven of the allozymes but one allozyme, EST6-8 within the EST6-S class, is several times more common than any other. Variation in the frequency of this single allozyme can explain most of the latitudinal dines previously reported for the major EST6-F and EST6-S classes. Thermostability analyses of 52 of the Australian lines and 13 American lines reveal at least seven more EST6 variants within five of the allozymes, bringing the total number of variants to at least 21. Of the six allozymes for which more than one line was subjected to thermostability analyses, only EST6-8 could not be partitioned into additional variants. This corroborates a previous finding that two different isolates of the Est6-8 allele have identical DNA sequences and suggests that this allele, although now the most common, has nevertheless arisen relatively recently.

Several lines of evidence indicate that natural selection operates between the major EST6-F and EST6-S allozymes of Drosophila melanogaster. In particular, consistent latitudinal clines and seasonal variation in their relative frequencies strongly suggest that they are not selectively equivalent in field populations. Several laboratory studies have found frequency-dependent fitness differences among the Est6-F and Est6-S genotypes. Moreover, the purified EST6-F and EST6-S allozymes differ in biochemical properties and the physiology of the enzyme, as a major component of the seminal fluid, suggests that these differences could affect reproductive aspects of fitness. However, molecular analyses reveal high levels of variation in the EST6 protein both within and between the EST6-F and EST6-S allozymes. Limited thermostability and more sensitive electrophoretic analyses reveal at least 17 variants of the two allozymes and sequence comparisons among 13 isolates of the Est6 gene reveal 16 nucleotide polymorphisms that would lead to amino acid differences. Two closely linked amino acid differences are strongly associated with the major difference between EST6-F and EST6-S; either or both of these are likely to cause the observed biochemical differences between EST6-F and EST6-S and may be the primary targets for the selection between these allozymes. The functional and adaptive significance of the other amino acid polymorphisms is unclear, although the data suggest that the EST6-8 haplotype within EST6-S has both arisen and proliferated relatively recently.

Genotype frequencies at the alcohol dehydrogenase-1 (Adh-1) locus of D. buzzatii were analysed for deviations from Hardy-Weinberg equilibria in the progeny of laboratory populations established at five initial Adh-1" allele frequencies and kept at either 18°C, 25°C or 30°C. At 25°C, no observed genotype frequencies were significantly different from Hardy-Weinberg expectations. Observed frequencies of heterozygotes were generally less than expected for populations at 18°C and 30°C. Fitness differences among genotypes were greater in males than in females, with Adh-1' homozygotes having highest fitness at 18°C and AdIs-1' homozygotes having highest fitness at 30°C. The results are discussed in relation to previous field and laboratory studies on D. buzzatii and to the Adh polymorphism of D. melanogaster.