Alcohol dehydrogenase polymorphism in the flour moth, Ephestia kühniella

Comp. Biochem. Physiol. Vol. 82B, No. 3, pp. 455-459, 1985

0305-0491/85 $3.00 + 0.00 :({~ 1985 Pergamon Press Ltd

Printed in Great Britain

ALCOHOL D E H Y D R O G E N A S E POLYMORPHISM IN THE FLOUR MOTH, EPHESTIA KfJHNIELL.4 F. LEIBENGUTH and U. KUTZ Fachrichtung Genetik der Universit~t des Saarlandes, D-6600 Saarbriicken, FRG (Tel: 0681-3021)

(Received 7 March 1985) Abstract--l. In horizontal starch-gel electropherograms strains of Ephestia kiihniella present different ADH patterns, which are governed by two codominant Adh alleles. 2. Homozygotes show one, either slow or fast migrating allozyme, each with one or sometimes two sub-bands, and in heterozygotes three prominent bands with two or sometimes four additional sub-bands are revealed. Sub-bands in both homo- and heterozygotes presumably originate as NAD-carbonylicaddition complexes. 3. The bands of homo- and heterozygotes exhibit slightly different inhibition by pyrazole and KCN, the hybrid pattern being of intermediate sensitivity. 4. Thermal inactivation is also different for the enzyme phenotypes, the bands of ADH-S being more heat-resistant than those of ADH-F. Sub-bands are thermally more stable than their related allozyme bands. 5. In early homozygous and reciprocal hybrid embryos ADH activity is due to the existence of a maternal effect through which the females transmit the mRNA to the progeny rather than the active ADH.

INTRODUCTION A hypothesis on the adaptive significance of allele dependent enzyme polymorphisms should primarily be based on different properties of the allozymes. With regard to this, one of the best-known examples is the alcohol dehydrogenase ( A D H ) of Drosophila melanogaster (for reviews see Dickinson and Sullivan, 1975; David, 1977). As far as the activity levels, kinetics, temperature stability and coding region on the one hand and allele frequencies in different environments on the other hand are concerned, this enzyme system can serve as a standard of comparison with other species. In this paper, we report the results of characterization studies of an A D H polymorphism found in the flour moth, Ephestia kf~hniella. Although this species, in contrast to Drosophila, does not have to cope with stress by environmental alcohol, the dcctromorphs of both organisms reveal remarkable similarities. MATERIAL AND METHODS Three Ephestia stocks (+Sbr, a +, and he; M; a) were raised by mass culture on coarsely ground meal at 26°C and 75% relative humidity. Single-pair matings yielded progenies with different enzyme phenotypes. Crude homogenates of staged single animals were made in a drop of gel buffer and then adsorbed onto a 5 x 5 mm filter-paper, which remained in the starch-gel during the run. For heat inactivation studies, extract concentration had to be adjusted for optimal band-staining intensities fitting the linear area of a densitometric standard curve. After centrifugation the supernatant was divided and incubated for either a constant time at different temperatures or exposed to the same temperature for different lengths of time. Horizontal starch-gel electrophoresis and staining for ADH activity were performed according to the methods given by Leibenguth and Steinmetz (1976). In order to test the inhibitory effect of pyrazole and KCN, parts of the same gel were incubated prior to staining in 455

hydrous solutions of the inhibitor and in water as a control. Various alcohols other than ethanol were tested as substrates (with ethanol as a control = 100%). Band-staining intensities were registered by the DD2 densitometer and the BD7 recorder (Kipp & Zonen, Delft, Holland). Fields of extinction curves were measured by a planimeter. RESULTS A total of three A D H electrophoretic phenotypes were found in our Ephestia stocks (Fig. 1), two different three-banded patterns and a seven-banded pattern. On account of the most prominent bands alone, these patterns seemed to be due to an allelic enzyme polymorphism with a s a n d f a l l e l e at the Adh locus. Mating of parents with known phenotype yielded progenies with the expected phenotypes (Table I). Therefore, two types of homozygotes, (Adh "/" = phenotype A D H - S and Adh ~" = phenotype A D H - F ) each comprising either three slow or fast migrating bands and a heterozygous type (Adh//'= phenotype A D H - F S ) with a maximum of seven bands were realized (Fig. 2). Ignoring the weaker staining bands (sub-bands), the three promi-

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Fig. I. Electrophoretic phenotypes (S, FS, and F) of alcohol dehydrogenase in homo- and heterozygous Ephestia kf~hniella. The underlying genotypes at the Adh locus are also given. Allozymes are black, sub-bands dotted or white according to their staining intensity. Band Nos. as used in the text.

456

F. LEIBENGUTHand U. KUTZ

Table I. Segregating progenies of parents with known phenotype are in accordance with the assumption that A D H in Ephestia is controlled by two co-dominantly manifesting alleles at the autosomal Adh locus. (*With Yate's correction) Parental phenotypes F SF SF FS FS F F FS SF SF FS S S

SF FS FS SF SF FS F F S S FS SF

21 22 26 28 44 44 40 45

Progeny FS 49 48 54 49 42 41 44 43 44 51 46 52

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n

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P

23 24 24 23

93 94 104 100 86 85 84 88 90 105 89 102

0.4893 0.1277 0.2308 0.54 0.0581" 0.1176" 0.2024" 0.0568* 0.0556* 0.0857 0.1124" 0.0392

0.78 0.94 0.89 0.77 0.81 0.74 0.66 0.82 0.82 0.77 0.74 0.85

46 54 43 50

nent bands of the heterozygotes (true allozymes) indicate that the ADH molecule is a dimer and that the alleles manifest codominantly leading to different homodimer types and a hybrid band composed of the heterodimers. The sub-bands staining successively weaker with decreasing migration rate are assumed to be generated as in Drosophila by the dimers binding different amounts of either N A D (Jacobson, 1968) or NADcarbonylic-addition complex (Schwartz and Sofer, 1976). Although it is possible to eliminate sub-bands in Drosophila by keeping cultures at 28"C from the time eggs are laid (Leibenguth and Steinmetz, 1976), this was not successful in Ephestia. Thus, most of the following measurements of ADH levels include at least one sub-band. Nearly all alcohols tested can be used as substrates by the Ephestia ADH. Secondary alcohols give higher activities than ethanol, and methanol results in lowest activities (Table 2). However, since faint bands are seen in the pherogram even in the absence of any alcohol in the staining solution (possibly due to hydroxylic groups in the starch gel), methanol can hardly be considered as a substrate. A D H activities of the three genotypes generally respond to the different substrates in a similar fashion. There are only three exceptions from I:1:1 expectation: n-butanol and isobutanol induce higher

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Fig. 2. Pherogram showing segregating 1.2 progen). Crude homogenate of single adults was applied. Each of the three phenotypes is represented by four patterns.

activities in heterozygotes and with tert-butanol, A D H - F is more active than ADH-FS. The action of the ADH inhibitors pyrazole (Borack and Sorer, 1971) and KCN (Sund and Theorell, 1963) on the patterns of homo- and heterozygotes reveals only minor differences (Table 3), the pattern of hybrids being of intermediate sensitivity. ADH activities as a function of temperature are given in Fig. 3 for the three genotypes. ADH-S bands are significantly more stable than the A D H - F bands and the bands of the heterozygous type inactivate intermediately. Since all visible bands of a given genotype are included in this inactivation study, it was interesting to find out, if allozymes and subbands contribute differently to inactivation. Tests were made with the two homozygous types at different temperatures and different incubation times (Fig. 4). In both types, the sub-bands No. 3 in ADH-S and No. 5 in A D H - F were more stable than

Table 2. Relative band staining intensities of the homo- and heterozygotes with different alcohol~ as substrates referred to a control stained with ethanol in the same gel. Activities of all visible bands of a given phenotype were summarized. Activities of the homo- and heterozygotes were tested for significant deviation from I : I : 1 proportion using ;C2 test. Staining was stopped when controls had reached optimal band intensity for densitometry. Fixation and transparency of the gels were achieved by glycerol ADH-S (band Nos. 5 + 3) n ~ Methanol Ethanol n-Propanol n-Butanol n-Pentanol n-Octanol lsobutanol sec-Propanol see-Butanol Cyclohexanol tert-Butanol

II 9 3 3 6 6 5 4 6 3

26 100 93 41 32 53 77 137 200 53 70

Activities of A D H (7;,) ADH-FS ADH-I(band Nos. 3 7) (band Nos. 7 4- 5 4 3t n ~ n .~' 5 II 3 3 6 4 6 3 3 3

27 100 91 72 36 51 131 131 187 55 53

9 12 3 3 6 5 6 6 9 3

24 100 92 46 44 47 107 123 16g 64 94

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Table 3. Inhibitoryeffect of pyrazole and KCN upon the activity of the three phenotypes.Segmentsof the samegel wereincubatedprior to stainingin a hydrous solution of the inhibitorand in water as a control ~,-inhibitionof band staining Incubation intensitiesof the ADH phenotypes ADH inhibitors time (br) F FS S Pyrazole (0.05 m)

KCN (0.06m)

1

74

76

79

2~

75

80

84

3~

80

84

88

1~

93

96

98

100

homo- and heterozygotes (Fig. 5). No activities were detected in freshly laid eggs, but, as development proceeded, the slow allozyme in ADH-S and the fast allozyme in ADH-F appeared and staining became successively more intense. Whether this is due to embryonic gene activation or to translation of maternal mRNA can be clarified using reciprocal hybrids. Since ADH-FS and ADH-SF hybrids first exhibit the respective maternal band, early ADH activity is considered to be due to maternal predetermination. The three-banded allozyme pattern appeared when larvae were tested just before they hatched, indicating that both parental alleles had now become activated.

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Fig. 3. Phenotype specific decreasing band staining intensities as a function of temperature. Equal amounts of supernatants incubated for 10 rain prior to e]ectrophoresis were applied to the same starch-geL (O) phenotype ADH-S (band Nos. 5 + 3), (O) phenotype ADH-F (band Nos. 7 ÷ 5), ( × ) phenotype ADH-SF (band Nos. ]-7). M ± s,

5- l0 repeats. their corresponding allozyme band. As already shown, the ADH-F band system inactivates faster than the slower migrating system of the ADH-S phenotype. Finally, we studied the emergence of ADH activity during embryonic development of Ephestia in both

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D~CU~ION There is an earlier report on ADH polymorphism in Ephestia kahniella. In a preliminary note Imberski (1972) concludes that "the traits no activity, one isozyme and two isozymes are allelic". Our patterns correspond better with Drosophila ADH in as much as homozygotes exhibit three bands of different mobilities and heterozygotes show maximally seven bands. ADH-negative mutants reported by Imberski and induced in considerable number by ethyl methane sulfonate in Drosophila (Schwartz and Sofer, 1976) were not found in our stocks. In both species ADH is apparently not essential for survival. Curiously this is contradictory to sequencing studies of

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Time (rain.) Fig. 4. Differential inactivation of ADH allozymes (O band No. 5 of ADH-S phenotype, • band No. 7 ofADH-F phenotype) and their nearest sub-band (O band No. 3 of ADH-S, A band No. 5 of ADH-F) in homozygotes as a function of temperature and incubation time. 4-10 repeats.

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A D H activity in eggs and embryos is due to predetermination in Ephestia as well as in Drosophila, but whereas eggs of Drosophila contain remarkable amounts of the active enzyme (Wright and Shaw, 1970; Leibenguth et al., 1979) only the maternal type gradually develops in embryos of Fphestia. This may be due either to the successive activation of preformed A D H or, as wc assume, more probably to translation of maternally inherited m R N A . The latc activation of both parental alleles in heterozygous embryos just before hatching of egg larvae coincides with the tirst appearance of the hybrid pattern of the csterasc alleles (Lcibenguth et al., 1979). In Drosophila embryos, synchronous activation o1" the parental alleles occurs 16hr after egg laying, in both species early inactivity of the embryonic Adh alleles is bridged by maternal storage products.

Acknowledgements--I'he authors wish to thank Ms I1. Porschke for technical assistance and Ms W. Pattullo tor correcting the English version of this paper. REFERENCES

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Fig. 5. Schematic presentation of ADI-'I allozyme bands (Nos. 5-7, ordinate) in homo- and heterozygous embryos of different age (hr). At least 150 eggs of a given age were applied. Hachure indicates different band staining intensities. Only in AI)H-F a sub-band is seen from 92 hr onward.

eleven cloned Drosophila Adh genes. Only one of the previously hidden 43 polymorphisms is responsible for the fast and slow electromorphs (Kreitman, 1983). Therefore, the author's implication is that most amino acid changes in A D H would be selectively deleterious. A remarkable inter-individual variance of A D H allozyme activities in both homo- and heterozygous Fphestia is obvious. Especially the allozymes of the hybrid pattern exhibit variable deviations from the expected 1:2:1 proportion concerning their relative staining intensities. This may be due, as in Drosophila ( M c D o n a l d and Ayala, 1978), to regulatory loci on other chromosomes. As far as A D H inactivation as a function of temperature and incubation time is concerned, Ephestia and Drosophila behave quite identically (Gibson, 1970; Day et al., 1974: Day and Needham, 1974). Even the shoulder at 3 min of incubation at 4 2 C (Fig. 4) is present and may be due to changes in the conformation of A D H as it is heated. Subbands are more heat stable than their corresponding allozymes probably by N A D binding. Their biological significance remains unknown. It is possible that they are artifacts of the extraction process and that in vivo only the true allozymes exist. Ephestia A D H is an enzyme with low specificity, it reacts with a number of alcohols. Conforming with Drosophila A D H (Vigue and Johnson, 1973) it exhibits highest activity with short chained secondary alcohols.

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ADH in Ephestia

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