Extension of life duration by dietary ethanol in Drosophila melanogaster: response to selection in two strains of different origins

Lines homozygous for the Adh ^S and Adh ^F alleles were extracted from a French and an African natural population of D. melanogaster. The lines from each geographic region were then crossed and the Mendelian F₂ constituted the first generation subjected to selection for an increase in adult survival in the presence of 2% ethanol.The responses to selection were similar in the two strains, in spite of their different ethanol tolerance. In less than 10 generations, life span with ethanol increased from about 7 to more than 12 days, with realized heritabilities of 0.14 and 0.18. This extension of longevity was also observed with other concentrations of ethanol but not with water alone. The increase in life span in presence of alcohol appears to result from unknown metabolic processes, which are not obligatorily related to the capacity of the flies to tolerate starvation, nor to their size, their lipid content of their ethanol tolerance. In the two lines, however, the Adh ^S allele was quickly eliminated, suggesting a selective advantage for the F allele.     

Alcohol dehydrogenase (ADH) isozymes in the AdhN/AdhN strain ofPeromyscus maniculatus (ADH− deermouse) and a possible role of class III ADH in alcohol metabolism

Although the Adh^N/Adh^N strain ofPeromyscus maniculatus (so-called ADH^? deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the Adh^F/Adh^F strain (so-called ADH⁺ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH^? strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (<100 mM) greatly increased (about sevenfold), to more than 50% that of ADH⁺ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH^? deermouse. It was also found that even the ADH⁺ strain has low liver ADH activity (<40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH^? and ADH⁺ deermouse were different not only from each other but also from that in the ddY mouse; the ADH^? strain was deficient in only Class I ADH, and the ADH⁺ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH^? strain.     

Partial Correction of Structural Defects in Alcohol Dehydrogenase through Interallelic Complementation in Drosophila melanogaster

Alcohol dehydrogenases (ADH) from the F₁ progeny of all pairwise crosses between 12 null-activity mutants and crosses between these mutants and four active variants, ADHⁿ⁵ ADH^F, ADH^D and ADH^S, were analyzed for the presence of active or inactive heterodimers. Gels were stained for ADH enzyme activity, and protein blots of duplicate gels were probed with ADH-specific antibody to detect cross-reacting material. Crosses between the three major electrophoretic variants. ADH^F, ADH^S and ADH^D, all produced active heterodimers. Four mutant proteins (ADHⁿ², ADHⁿ⁴, ADHⁿ¹⁰ and ADHⁿ¹³) did not form heterodimers with any other ADH subunit tested. Of the 28 crosses involving the remaining null activity mutants, 22 produce heterodimers. Twelve of these exhibit partial restoration of enzyme activity. In five cases of active heterodimers from null-activity crosses, Adhⁿ¹¹ supplied one of the subunits. In two crosses involving the active variant ADH^D, the null activity mutant subunits (ADHⁿ⁸ and ADHⁿ³) destabilized the heterodimer sufficiently to cause inactivation of the ADH^D subunit. In the cross between Adh^F and Adhⁿ³, the activity of the ADH^F subunit was also greatly reduced in association with the ADHⁿ³ subunit. Two crosses (Adhⁿ¹ x Adhⁿ¹¹ and Adhⁿ⁵ x Adhⁿ¹²) result in partial restoration of one of the homodimeric proteins (ADH ⁿ¹ and ADHⁿ¹², respectively), as well as forming active heterodimers.     

Latitudinal Adh allozymic variation and ethanol tolerance in Indian populations of Drosophila melanogaster

Ten Indian geographical populations of D. melanogaster were assayed electrophoretically for Adh genic variation. The Indian geographical populations of D. melanogaster revealed significant clinal variation (3 % for 1 d? latitude) at Adh locus and Adh^F allelic frequency correlated significantly with increase in latitude. It was suggested that the abundance of secondary alcohols in the southern Indian tropical and humid environment might exert selective pressure favouring higher frequency of Adh^S allele. Patterns of ethanol utilization as well as ethanol tolerance were analyzed in larval and adult individuals of six geographical populations of D. melanogaster. Latitudinal variation in ethanol tolerance was observed in D. melanogaster populations from India. The parallel occurrence of latitudinal variation it Adh locus as well as ethanol tolerance in Indian geographical populations of D. melanogaster could be maintained by balancing natural selection varying spatially along the north-south axis of the Indian sub-continent.     

Chemical selection of mutants that affect ADH activity in Drosophila. III. Effects of ethanol

A chemical selection scheme is presented for the isolation of rare Adh-positive Drosophila. It makes use of the fact that flies lacking detectable ADH activity die as adults or larvae on relatively low concentrations of ethanol in the medium. We have demonstrated that this procedure is a practical one by crossing two Adh-negative alleles, screening 1.5×10⁶ embryos, and isolating 14 Adh-positive survivors.Supported by Postdoctoral Fellowship GM-57 from the National Institutes of Health to C. V. and by Grant GM-18254 from the NIH.Contribution No. 841 from the Department of Biology, The Johns Hopkins University.     

The alcohol dehydrogenase alleloenzymes AdhS and AdhF from the fruitfly Drosophila melanogaster: An enzymatic rate assay to determine the active-site concentration

A rapid and reproducible enzymatic rate assay for the quantitative determination of the concentration of active sites is presented for the alleloenzymes Adh^S and Adh^F from Drosophila melanogaster. Using this procedure the turnover numbers as catalytic-center activities were found to be 12.2 sec^–1 for Adh^F and 3.4 sec^–1 for Adh^S with secondary alcohols. This showed a slower dissociation of the coenzyme from the binary enzyme-NADH complex with Adh^S and hence a stronger binding of NADH to this alleloenzyme. With ethanol, the catalytic-center activity was 1.4 sec^–1 for Adh^S and 2.8 sec^–1 for Adh^F, and hence the single amino acid mutation distinguishing the two alleloenzymes also affected hydride transfer.     

Association and the Adh locus with a lethal factor (l(2) Stm) in Drosophila melanogaster

Keeping Drosophila cultures at 28 C results in elimination of all minor multiple ADH bands, thought to be due to conformational change. Thus in diploid and triploid adults heterozygous for the Adh ^F and Adh ^Salleles, relative staining intensities are found for the three bands which were in conformity with the assumption that both alleles are equally expressed. Among all polymorphic strains derived from natural Central European and Mediterranean populations, the strain ⁺Tüb is unique in that its Adh ^Fallele is closely linked to a new recessive lethal factor, named 1(2)Stm. All Adh ^F 1/Adh^F 1 pupae are unable to emerge, and die. The lethal effect is obvious 50 hr earlier by retarded eye, bristle, and body wall pigmentation. Although all pupae of the phenotype F die, Adh ^F allele frequency scarcely seems to be lowered in this natural population.     

Mimicry of isozyme adaptive advantage by gene association II. Adh genotype frequency variations in Drosophila cage populations reared at different temperatures

The adaptive response of the Adh locus to different temperatures was determined in an experimental population with a stabilized gene pool in order to confirm the conclusions reached in a previous paper (Pieragostini et al., Genetica 56: 27–37, 1981) using as experimental material a laboratory population in which heterosis and strong interaction between loci were present.By sampling a stable lab population, three cage populations were constructed, heterozygous at the Adh locus and genetically identical but reared at different temperatures (P 18 °C, P 25 °C and P 28 °C). On these populations we studied the dynamics of Adh allele frequencies and of five metric traits up to the fifth generation of caging. The most interesting result is represented by the differences between P 28 °C and the other two populations (P 25 °C and P 18 °C). In particular P 25 °C and P 18 °C show a strong similarity with the original population both in Adh allele frequency and relationship between Adh genotype and the respective metric phenotype; this is not the case for P28 °C which differed from the original population and the other two newly founded ones not only in Adh allele frequencies but even in gene arrangements.Comparing these results with those obtained in the previous work it can be concluded that Adh frequency variations can be a side effect of the evolutionary pattern of the populations which in turn depends on the initial genetic composition.     

A long-term study on interactions between the Adh and αGpdh allozyme polymorphisms and the chromosomal inversion In(2L)t in a seminatural population of D. melanogaster

The Adh and αGpdh allozyme loci (both located on the second chromosome) showed considerable fluctuations in allele frequencies in a seminatural population of Drosophila melanogaster during 1972–97. Both long-term and short-term fluctuations were observed. The short-term fluctuations occurred within almost all years and comparison of allele frequencies between winters and summers showed significantly higher Adh^S (P < 0.001) and αGpdh^F (P < 0.01) allele frequencies in summers. Frequencies of these alleles were significantly positively correlated with environmental temperature, suggesting the adaptive significance of these allozyme polymorphisms. Frequency changes of the Odh locus (located on the third chromosome) showed no seasonal pattern and were not correlated with environmental temperature. Almost all short-term and long-term increases in Adh^S frequency were accompanied by a corresponding decrease in αGpdh^S frequency (r = –0.82, P < 0.001) and vice versa. Further analysis showed that gametic disequilibria between the Adh and αGpdh loci, which frequently occurred, were due to the presence of inversion In(2L)t located on the same chromosome arm and In(2L)t frequencies were positively correlated with environmental temperature. Gametic disequilibria between Adh and Odh and between Odh and αGpdh were hardly observed. Because In(2L)t is exclusively associated with the Adh^S/αGpdh^F allele combination, the observed correlated response in Adh/αGpdh allele frequencies is (at least partly) explained by hitchhiking effects with In(2L)t. This means that the adaptive value of the allozyme polymorphisms has been overestimated by ignoring In(2L)t polymorphism. Fluctuations in Adh allele frequencies are fully explained by selection on In(2L)t polymorphism, whereas we have shown that αGpdh frequency fluctuations are only partly explained by chromosomal hitchhiking, indicating the presence of selective differences among αGpdh genotypes in relation with temperature and independent of In(2L)t. Frequency fluctuations of αGpdh and In(2L)t are consistent with their latitudinal distributions, assuming that temperature is the main environmental factor varying with latitude that causes directly or indirectly these frequency distributions. However, the results of the tropical greenhouse population show no correlation of Adh (independent of In(2L)t) and Odh allele frequencies with environmental temperature, which may indicate that the latitudinal distribution in allele frequencies for these loci is not the result of selection on the F/S polymorphism in a direct way.     

Perturbation of gene frequencies in a natural population of Drosophila melanogaster: evidence for selection at the Adh locus

The cellar population of Drosophila melanogaster at the Chateau Tahbilk Winery (Victoria, Australia) was perturbed for alcohol dehydrogenase (Adh) gene frequencies. Phenol oxidase (Phox) frequencies were also perturbed and monitored as a control. Subsequent gene frequency changes, together with information on population structure, indicated that selection acted on the chromosome regions of both loci. Adh gene frequencies returned to preperturbation levels in a predictable manner. A model in which the relative fitness of Adh phenotypes was determined by temperature-dependent specific activities of enzymes of Adh genotypes adequately accounts for the rate of gene frequency change at this locus. Thus temperature behaves as a selective agent in modulating Adh gene frequencies in this cellar environment.     

Respiration of individual honeybee larvae in relation to age and ambient temperature

The CO₂ production of individual larvae of Apis mellifera carnica, which were incubated within their cells at a natural air humidity of 60–80%, was determined by an open-flow gas analyzer in relation to larval age and ambient temperature. In larvae incubated at 34 °C the amount of CO₂ produced appeared to fall only moderately from 3.89±1.57 µl mg^–1 h^–1 in 0.5-day-old larvae to 2.98±0.57 µl mg^–1 h^–1 in 3.5-day-old larvae. The decline was steeper up to an age of 5.5 days (0.95±1.15 µl mg^–1 h^–1). Our measurements show that the respiration and energy turnover of larvae younger than about 80 h is considerably lower (up to 35%) than expected from extrapolations of data determined in older larvae. The temperature dependency of CO₂ production was determined in 3.5-day-old larvae, which were incubated at temperatures varying from 18 to 38 °C in steps of 4 °C. The larvae generated 0.48±0.03 µl mg^–1 h^–1 CO₂ at 18 °C, and 3.97±0.50 µl mg^–1 h^–1 CO₂ at 38 °C. The temperature-dependent respiration rate was fitted to a logistic curve. We found that the inflection point of this curve (32.5 °C) is below the normal brood nest temperature (33–36 °C). The average Q₁₀ was 3.13, which is higher than in freshly emerged resting honeybees but similar to adult bees. This strong temperature dependency enables the bees to speed up brood development by achieving high temperatures. On the other hand, the results suggest that the strong temperature dependency forces the bees to maintain thermal homeostasis of the brood nest to avoid delayed brood development during periods of low temperature.Abbreviations m body mass - R rate of development or respiration - T_I inflexion point of a logistic (sigmoid) curve - T_L lethal temperature - T_O temperature of optimum (maximum) developmentCommunicated by G. Heldmaier     

Adaptive significance of the action of the Drosophila melanogaster alcohol dehydrogenase locus through the heat shock protein system

Four Drosophila melanogaster strains, each homozygous for one of the two major ADH aliozymes, Fast and Slow (Adh ^F1, Adh ^S , Adh ^F2 and Adh ^S2) were used to study the interaction of the Adh locus with ethanol and temperature. The separate and especially the combined effects of these two parameters allow the conclusion that the Adh locus of D. melanogaster intervenes in the adaptation process through the heat shock protein system.     

Genetic regulation of liver alcohol dehydrogenase in Peromyscus

Data from genetic crosses of Peromyscus maniculatus and P. polionotus suggest that electrophoretic variants of liver alcohol dehydrogenase are coded by alleles at a single locus. These alleles, designated Adh ^F , Adh ^S , and Adh ^N , determine, respectively, the fast, slow, and not detectable (null) ADH electrophoretic phenotypes. Heterozygotes (Adh ^F /Adh ^S ) exhibit three bands on zymograms, suggesting a dimeric subunit structure for the enzyme. However, Adh ^F /Adh ^N and Adh ^S /Adh ^N animals exhibit a single band, suggesting that the Adh ^N allele does not produce a polypeptide subunit capable of dimerizing into an active molecule. Fast and slow electrophoretic phenotypes exhibit multiple bands which can be converted into single major fast and slow bands, respectively, upon treatment with oxidized or reduced NAD. Addition of NAD also stabilizes both the fast and slow enzyme to heat inactivation at 60 C for at least 30 min.This work was supported by Predoctoral Fellowship AA-05067 from the National Institute of Alcohol Abuse and Alcoholism to K. G. B. and South Carolina Commission on Alcohol and Drug Abuse Grant 7607. Also, partial support was provided by NIH Grant CA-16184.     

Effect of rearing temperature and larval density on larval survival, age at pupation and adult size of Anopheles gambiae

The effects of temperature and larval density on survival of larvae, growth rate, age at pupation, and adult size (measured as wing length and dry weight) of laboratory-reared Anopheles gambiae (Diptera: Culicidae) were studied. Larvae were reared at three temperatures (24, 27 and 30°C) and three densities (0.5, 1 and 2 larvae/cm²). The effects of density and temperature strongly interacted to determine the mosquitoes' life-history parameters. Survival was highest at the intermediate temperature of 27°C. The differences between the temperatures increased with increasing density. At 30°C survival decreased as density increased, but at 27°C increasing density led to higher survival. Age at pupation increased as temperature decreased from 30°C to 24°C and as density decreased from 2 to 0.5 larvae/cm². Adult size also increased as temperature decreased, but showed a negative correlation with density only at 27°C. In contrast, at 24°C and 30°C a decrease in density led to a decrease in adult size. Growth rate showed a similar pattern. At 27°C growth rate decreased as density increased, but at other temperatures the opposite trend was observed.     

THE ASSOCIATION BETWEEN THE POLYMORPHISMS AT THE Adh AND αGpdh LOCI AND THE In(2L)t INVERSION IN DROSOPHILA MELANOGASTER IN RELATION TO TEMPERATURE

Substantial allele-frequency changes were observed at the Adh and αGpdh loci in a seminatural population of Drosophila melanogaster kept in a tropical greenhouse during 1972–1985. Further analysis of the changes at the Adh and αGpdh loci showed that linkage disequilibrium between these loci occurred for a prolonged period due to the presence of In(2L)t, a long inversion on the left arm of the second chromosome. We observed increases in the frequencies of In(2L)t and of short inversions on the left arm of the second chromosome in subpopulations kept at 29.5°C or 33°C. These inversion-frequency increases were accompanied by an increase in Adh^s and a decrease in αGpdh^s frequency. In populations kept at 20°C and 25°C, inversion frequencies decreased, while αGpdh^s allele frequencies decreased at 25°C and increased at 20°C. At 33°C, egg-to-adult survival of individuals possessing In(2L)t, either in the homokaryotypic or the heterokaryotypic state, was higher than that of the other karyotypes of identical allozyme constitution (i.e., Adh^s αGpdh^F). Thus it seems that In(2L)t has a selective advantage at high temperature. We argue that the observed changes in allele frequencies at the Adh and αGpdh loci are, in part, due to genic selection and are not merely the result of selection acting on the chromosome rearrangements and hitchhiking of the allozymes. The results are discussed with respect to the latitudinal clines found for In(2L)t, Adh, and αGpdh.     

A laboratory study on the life cycle of Sericostoma personatum (Kirby & Spence), and light dark-dependent food consumption

The life cycle of Sericostoma personatum (Spence) was studied at 6 °C, 10 °C and 14 °C and at each temperature at 8 and 14 hrs daylength. Embryogenesis was not temperature dependent in the 12°–18°C range. Only 7 of 38 (app. 18%) had a direct development, the rest remained in diapause with partly developed larvae. Hatching success of single egg masses was over 95%. At 6 °C at both LDs, about 452 days are required for larval development. At 10 °C 370 days (LD 8/16), or 320 days (LD 14/10) and at 14° C 319 days (LD 8/16) and 295 days (LD 14/10) were required. Duration of instars III and IV was longer at 6 °C (both LDs), compared with all other groups. Vth instar larvae of the 14 °C (LD 14/10) group grew fastest. Instar VI larvae of the 10 °C short day group developed faster than all others. Instar VII larvae of both 14 °C groups and of the 10 °C long day group develop faster than the rest. Duration of pupal instar is only temperature dependent, regardless of light regime. The field life cycle of S. personatum may require 2–5 years. Larvae are night active. They feed on Coarse Particular Organic Material (CPOM) on the sediment surface at night. They release faeces (Fine Particular Organic Material, FPOM) into the sediment where they rest by day at a few cm depth. Their burrowing behavior thus contributes to the retention of FPOM in the stream channel. Daily food consumption at constant 10 °C is significantly dependent on night length (r ² = 0.979, p < 0.05). Two factors thus may limit food consumption: in winter, low temperatures, and in summer short nights. The species thus avoids competition by day-active shredders and predation by day-active predators.     

Isolation and characterization of temperature-sensitivelethal(2)giant larva alleles

Summary In this paper we present an analysis of the behavior ofl(2)gl ^tsimaginal wing discs during culture in adult hosts. Thel(2)gl ^tslarvae reared at 29° C contain two types of wing discs, those that are morphologically normal and those that are abnormal. When discs of both types are cultured in adult hosts at 29° C, the restrictive temperature, they give rise to transplantable neoplastic tissue. However, when the 29° C reared discs are cultured at 15° C, the permissive temperature, the morphologically normal discs maintain their morphology, but the morphologically abnormal discs give rise to neoplasms. Thel(2)gl ^tslarvae reared at 15° C contain only morphologically normal discs. When these discs are cultured in adult hosts at 29° C they give rise to neoplasms, however if the discs are cultured at 15° C they maintain their normal morphology. These results demonstrate: (1) that all wing imaginal discs obtained from 29° C rearedl(2)gl ^tslarvae are competent to undergo neoplastic development, (2) the morphologically abnormal discs obtained from the 29° C rearedl(2)gl ^tslarvae are committed to neoplastic development, (3) the neoplastic development of the morphologically normal discs is temperature dependent, (4) once the neoplastic development of thel(2)gl ^tsdiscs has been initiated the process is not readily reversible. In addition, the ability ofl(2)gl ^tswing discs to perform epimorphic regulation was tested by amputating morphologically normal permissively rearedl(2)gl ^tswing discs and culturing both fragiments at the permissive temperature. Fragments of control wild-type discs maintained their morphology during culture at the permissive temperature. However, both fragments of txel(2)gl ^tsdiscs became neoplastic. This result is discussed with respect to a possible role for thel(2)gl ⁺function in epimorphic regulation and with respect to the phenomena of tumor promotion in vertebrates.     

Photoinhibition at chilling temperature

The effects of moderate light at chilling temperature on the photosynthesis of unhardened (acclimated to +18° C) and hardened (cold-acclimated) spinach (Spinacea oleracea L.) leaves were studied by means of fluorescence-induction measurements at 20° C and 77K and by determination of quantum yield of O₂ evolution. Exposure to 550 mol photons·m^-2·s^-1 at +4° C induced a strong photoinhibition in the unhardened leaves within a few hours. Photoinhibition manifested by a decline in quantum yield was characterized by an increase in initial fluorescence (F _o) and a decrease in variable fluorescence (F _v) and in the ratio of variable to maximum fluorescence (F _V/F _M), both at 77K and 20° C. The decline in quantum yield was more closely related to the decrease in the F _V/F _M ratio measured at 20° C, as compared with F _V/F _M at 77K. Quenching of the variable fluorescence of photosystem II was accompanied by a decline in photosystem-I fluorescence at 77K, indicating increased thermal de-excitation of pigments as the main consequence of the light treatment. All these changes detected in fluorescence parameters as well as in the quantum yield of O₂ evolution were fully reversible within 1–3 h at a higher temperature in low light. The fast recovery led us to the view that this photoinhibition represents a regulatory mechanism protecting the photosynthetic apparatus from the adverse effects of excess light by increasing thermal energy dissipation. Long-term cold acclimation probably enforces other protective mechanisms, as the hardened leaves were insensitive to the same light treatment that induced strong inhibition of photosynthesis in unhardened leaves.Abbreviations F ₀ initial fluorescence - F _M maximum fluorescence - F _V variable fluorescence (F _M-F ₀ - PFD photon flux density - PS photosystem     

Alcohol dehydrogenase polymorphism in the seaweed fly,Coelopa frigida

Seaweed flies (Coelopa frigida) inhabit piles of decaying seaweed on the seashore. All populations so far studied have been found to be polymorphic at the alcohol dehydrogenase locus (Adh). This article reports an attempt to identify some of the forces of natural selection that may be maintaining this polymorphism. First, the genetic determination of the rather complex isozyme system is described. Several inbred lines homozygous at the Adh locus were derived and the biochemical properties of their allozymes compared. Significant differences in both specific activities and thermal stabilities were found between ADH allozymes. A simple experiment is reported in which individuals with different Adh genotypes were cultured in competition with each other in the presence of elevated levels of ethanol. Although the presence of ethanol resulted in greater mortality, there is no evidence that it was selective with respect to the Adh genotypes. The possible relevance of these results to the maintenance of the Adh polymorphism is discussed.This work was supported by a grant to T. H. D. from the Science Research Council.     

Studies of esterase 6 in Drosophila melanogaster XII. Evidence for temperature selection of Est 6 and Adh alleles

Changes in allele frequencies at the esterase 6 (Est 6) and alcohol dehydrogenase (Adh) enzyme loci of Drosophila melanogaster and simulans are examined in natural populations and artificial populations maintained at two temperatures. Results from cage populations at 18 °C and 25 °C provide evidence for temperature selection at both loci. Seasonal population samples show no significant change in gene frequencies for either locus, a reasonable outcome given the small selection coefficients found in cage populations. The temperature effect for the Adh locus appears to be direct: natural selection of the fast allele in cool environs and of the slow allele in warm environs. The temperature effect for Est 6 is weaker and complicated by sex differences and deviations from Hardy-Weinberg expectation. This evidence and different Est 6 frequencies found for melanogaster and simulans, in conjunction with evidence of the male reproductive function of this enzyme, suggest that Est 6 polymorphisms are maintained in natural populations by a complex form of sexual selection.