Association of Chromosome and Enzyme Polymorphisms in Natural and Cage Populations of DROSOPHILA MELANOGASTER

The frequencies of a polymorphic inversion, In(2L)t, and of Adh and αGpdh alleles were analyzed in three natural populations of Drosophila melanogaster from Japan. Significant positive correlations between the frequencies of In(2L)t and Adh^S or αGpdh^F were detected due to tight linkage. An analysis of correlation with latitude showed that the negative cline of Adh^S frequency could be explained entirely by its linkage with In(2L)t; the frequency of Adh^S on the standard chromosome did not show a latitudinal cline. To the contrary, the cline of αGpdh^F frequency itself was positive, and its linkage with In(2L)t makes the positive cline unclear. These results suggest that the two allozymes themselves respond to latitudinal natural selection in different ways. When these populations were transferred to laboratory cages and maintained for a long time, they lost the chromosomal polymorphism but retained stable enzyme polymorphisms, although allele frequencies in the cage were not the same as in nature. The frequencies of Adh and αGpdh alleles were close to those in earlier cage populations of the same geographical origin.     

Mimicry of isozyme adaptive advantage by gene association I. Relationship between Adh genotypes and body dimension in Drosophila cage populations: A multivariate analysis

Experiments are described that are to prove that the apparent selective responses at the Adh locus in Drosophila melanogaster are not independent from its genetic background and from the variation at the gene pool level brought about by the changes of selection pressure. The dynamics of allozyme frequencies were observed at the Adh locus, of five metric traits and of reproductive fitness in two synthetic populations of Drosophila melanogaster originated from the same cross between Canton and Oregon strains, homozygotes for different Adh alleles and reared at different temperature (25 °C and 28 °C), until all above mentioned characters showed no more variations. The results obtained by univariate and multivariate statistical analyses can be summarized as follows:

- Adh ^s allele frequency keeps high and reaches very similar values in spite of the different environmental temperatures, whose selective effects at the Adh locus are therefore unlikely;

- both populations evolve toward the stabilization of Adh frequencies and other characters with a process strictly dependent on the permanence of coadapted blocks of genes which were contributed to the F₂ generation by the parental Canton and which are identified phenotypically by the association of Adh ^s/s with short wing;

- at the stabilization point the flies classified on the basis of their Adh genotype exhibit different shapes, namely their metric phenotypes can be discriminated considering all the respective traits together by means of multivariate analyses.

Owing to the presence in the initial populations of heterosis and epistatic interactions between loci, the observed differences between Adh genotype groups should represent the outcome of selection upon coadapted blocks of genes rather than on individual loci. Therefore, these results are argued as further evidence that each Adh genotype can be associated to different gene arrangements and its adaptive value cannot be isolated from that of its genetic background.     

Frequency of Cry1F Non-Recessive Resistance Alleles in North Carolina Field Populations of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a target species of transgenic corn (Zea mays L.) that expresses single and pyramided Bacillus thuringiensis (Bt) toxin. In 2014, S. frugiperda were collected from a light trap in North Carolina, and a total of 212 F₁/F₂ isofemale lines of S. frugiperda were screened for resistance to Bt and non-Bt corn. All of the 212 isolines were susceptible to corn tissue expressing Cry1A.105 + Cry2Ab, Cry1F + Cry1A.105 + Cry2Ab, and Cry1F + Cry1Ab + Vip3Aa20. Growth rate bioassays were performed to isolate non-recessive Bt resistance alleles. Seven individuals out of the 212 isofemale lines carried major non-recessive alleles conferring resistance to Cry1F. A pooled colony was created from the seven individuals. This colony was 151.21 times more resistant to Cry1F than a known-susceptible population and was also resistant to Cry1A.105, but was not resistant to Cry2Ab and Vip3Aa20. The results demonstrate that field populations of S. frugiperda collected from North Carolina are generally susceptible to Cry1F, but that some individuals carry resistant alleles. The data generated in this study can be used as baseline data for resistance monitoring.     

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.     

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.     

Selection against homozygotes in the ADH polymorphic system of Drosophila melanogaster associated with the lethal factor l(2) Stm

In the natural populations ⁺Tüb, ⁺Prov, and ⁺Rov, similar Adh ^F allele frequencies occur (q _F=0.11, 0.18, and 0.08, respectively). However, there is a discrepancy in that the Adh ^F allele in ⁺Tüb is closely linked to the lethal factor 1(2)Stm, which reduces relative fitness of the F phenotype to zero. In spite of this, polymorphism is maintained also in ⁺Tüb, because the heterozygotes are superior to the homozygous S type (relative fitness=0.88). Under laboratory culture conditions, in ⁺Tüb the relative fitness of the S genotype further decreases to 0.6. After outcrossing the lethal factor, relative fitnesses for S, FS, and F become 0.6, 1, and 0.48, respectively, implying that fitness for S remains the same. Relative values for S, FS, and F in ⁺Prov, not affected by the lethal factor, are calculated by the maximum average fitness method to be 1, 1.2, and 0.2 under the assumption that heterozygous FS are similarly superior to S as in the natural ⁺Tüb population and all allele frequencies found are stable equilibrium values.     

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.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

Genetics of sunflower alcohol dehydrogenase: Adh 2 ,nonlinkage to Adh 1 and Adh 1 early alleles

Alcohol dehydrogenase (ADH) isozymes in annual sunflowers (Helianthus annuus) are dimers whose subunits are produced by two genes, Adh ₁ and Adh ₂ .The codominant F and S alleles of Adh ₁ produce the slower-migrating set of three isozymes. The faster-migrating set of three isozymes is controlled by Adh ₂ , which also has at least two alleles, F and S. Hybridization experiments indicated that the Adh ₂ alleles segregate in expected Mendelian fashion and that Adh ₁ and Adh ₂ are not linked. A third common 1-locus allele is designated early (E) because when homozygous it results in a blank at the 1FF isozyme position in mature seeds, but in developing seeds produces a normal-appearing band at the 1FF position. Hybridization studies showed that the early alleles segregated normally. Correlation between genotype and presence or absence of isozymes electrophoretically intermediate between those of Adh ₁ and Adh ₂ suggests that four intergenic isozymes may be formed as a result of dimerization of the four basic subunits. Studies of zymograms of developing seeds suggest that the remaining but inconstant zymogram bands are mature seed isozymes which have altered charges during early morphogenesis and thus are developmental artifacts.     

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.     

LATITUDINAL CLINE IN DROSOPHILA MELANOGASTER FOR KNOCKDOWN RESISTANCE TO ETHANOL FUMES AND FOR RATES OF RESPONSE TO SELECTION FOR FURTHER RESISTANCE

We have introduced a device for selecting Drosophila for increased resistance to very high concentrations of ethanol fumes. This device has enabled us to: 1) select quickly and easily over a thousand flies at a time, and 2) score the knockdown time of every fly in the distribution, while causing very little injury to the flies. A sample of nine west coast populations of Drosophila melanogaster showed a significant trend toward higher knockdown resistance in more northern populations. A population's level of knockdown resistance was virtually uncorrelated with its alcohol dehydrogenase (Adh) allele frequencies. Five of the above nine populations were then subjected to selection for further knockdown resistance. Each population was divided randomly into four groups of 256 flies: two lines to be selected, and two lines to remain unselected as control lines. In every generation each selected line was measured for knockdown resistance, and the last quartile of flies to be knocked down was saved to continue the selection cycle. Population sizes of the selected and unselected lines were all maintained at 256. Realized heritability, based on the responses to selection of the first four generations, was calculated for each selected line. The five populations were significantly heterogeneous for heritability estimates; the average heritability of the five populations pooled was 0.143 ± 0.019. Over the course of twelve generations, the ten selected lines increased their knockdown times by an average factor of 2.40. Before selection, the five populations were heterogeneous for knockdown resistance, and resistance was greatest among the most northern populations. The amount of change of knockdown resistance over the course of selection was also correlated with latitude: the most southern population increased its knockdown time by a factor of 2.23, and the most northern population increased it by a factor of 2.55. After ten generations of selection, the cline of knockdown resistance was about 4.5 times as steep as that before selection. Small phenotypic differences among populations before selection were thus exaggerated by the action of selection. The differences among populations in their rates of response to selection were attributed to genetic differences that existed before selection. The pattern of change of Adh frequencies over the course of selection was very inconsistent, both among and within populations. From this inconsistency of change of Adh alleles with selection, and the lack of correlation between Adh frequencies and knockdown resistance before selection, we concluded that Adh frequency changes could not have had much effect on the responses of the selected lines.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

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.     

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities

Two unlinked genes, Adh ₁ and Adh ₂, control the production of alcohol dehydrogenase (ADH) in seeds of the annual sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels following D-R of an intergenic isozyme showed that the Adh ₂ isozymes were more than twice as active as those of Adh ₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh ₁ ^F /Adh ₁ ^F , Adh ₂ ^S /Adh ₂ ^S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh ₁, Adh₂, and intergenic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.Supported by Graduate School and BioMed grants and by NSF Grant GB35853.     

Allozyme variation in Greek wild populations of Drosophila melanogaster and D. simulans along a North-South gradient

Allozyme frequency data from five Greek wild sympatric populations of Drosophila melanogaster and D. simulans along a North-South gradient were analyzed for genotype-environment relationships. The regression coefficient of genetic distance on geographic distance indicates that there is a significant relationship between these parameters for D. melanogaster only. Highly significant differences in specific alleles at certain loci were found between the various local populations studied. The changes in Gpdh ^F of D. melanogaster and Est-6 ^F of D. simulans exhibited clinal patterns in allele frequencies. In addition, analysis of D. melanogaster Gpdh ^Fand Adh ^F allele frequencies shows that the Greek data do not have regression coefficients (regressing allele frequency on degrees North of latitude) of the same sign as East-and West-Coast United-States populations. These contradictory data are discussed in relation to what is known about the maintenance of the Adh and Gpdh polymorphisms.     

Selection at the alcohol dehydrogenase locus of Drosophila melanogaster: Effects of ethanol and temperature

Drosophila melanogaster larvae were subjected to 10 generations of selection on 6% ethanol at 17, 25, and 30°C. For each temperature there was a significant (P<0.01) increase in the frequency of the Adh isoallele. Controls with no ethanol showed no change in the frequency of the Adh ^F isoallele. Larvae subjected to stronger selection on 8% ethanol confirmed the results. When adults of various ages were subjected to 16 and 32°C, the ADH^F isoenzyme retained its twofold advantage in activity over ADH^S regardless of the temperature. The same result was obtained with larvae at 16 and 35°C. Although some effect of temperature was demonstrated, it was concluded that the effect was not strong enough for temperature to be a selective factor under the conditions studied. However, ethanol is a strong selective factor for laboratory populations.     

CHROMOSOMAL POLYMORPHISM IN ISOFEMALE LINES AND CAGE POPULATIONS OF DROSOPHILA MELANOGASTER

Drosophila melanogaster populations in nature usually carry inversion polymorphisms. When they were transferred to and maintained in the laboratory as large cage populations, frequencies of polymorphic inversions were drastically decreased and finally eliminated. This “cage effect” was observed irrespective of the geographical origin of the population or the initial frequency of each inversion. The decrease and elimination of inversions in the cage was not overcome by changing conditions such as medium, temperature, or the number of isofemale lines (40-600) introduced. On the other hand, in the sets of isofemale lines derived from the same geographical origins as the cage populations, each of which was maintained as a small vial population, the inversion frequencies, though decreased from the initial frequencies, were kept at significantly high levels. The cage populations initiated with one or two isofemale lines also maintained the inversion polymorphisms that were as high as vial populations.     

Effects on ADH activity and distribution,following selection for tolerance to ethanol in Drosophila melanogaster

Strains of Drosophila melanogaster homozygous for either the Adh ^F or the Adh ^S allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.     

Enzymatic responses of Drosophila melanogaster to long- and short-term exposures to ethanol

The effects of environmental ethanol on larva-to-pupa survival and on the activities of four enzymes were investigated in three Drosophila melanogaster strains. The strains had different allelic combinations at the Odh and Aldox loci on their third chromosomes, but they all carried the Adh ^S -Gpdh ^F allelic combination on the second chromosome. Replicates of each of the strains were exposed to three different ethanol treatments: (i) no ethanol in the medium (control); (ii) 5% ethanol for a single generation (short-term exposure); (iii) 5% ethanol for 20 generations (long-term exposure). In all experiments, the activities of four enzymes (ADH, ODH, GPDH and AOX) were measured in larvae, pupae and adults. The results showed that (i) the larval and adult metabolic responses to environmental ethanol were different; (ii) enzyme activity changes under short-term exposure differed from those measured under long-term exposure; (iii) the activities of the allozymes common to all strains (ADH-S and GPDH-F), differed depending on the genetic background. Changes in larva-to-pupa survival were seen when the larvae of control and exposed lines of the three strains were confronted with various concentrations of ethanol. In all three strains, the exposed lines had significantly higher initial survival rate and ethanol tolerance than the control lines. Strain-specific differences were observed in the ethanol tolerance of both types of line.