Inducing Factors O F Amylase and Fitness

“Inducibility” of amylase in Drosophila melanogaster was defined and investigated in a natural population from Japan. Inducibility represents the effects of factors remote from the structural gene that control the amount of enzyme produced. Inducibility of an isogenic line is measured as the ratio of the enzyme’s specific activity in two different inducing environments. There was considerable genetic variability with respect to inducibility of amylase in 44 isogenic lines derived from a natural population of D. melanogaster. Net fitness and its components in these isogenic lines were also measured. The results indicated that, although the inducibility of the enzyme was positively correlated with the net fitness (r, = 0.63 f 0.2), the enzyme activities in the normal medium were not (r, = 0.12 & 0.37). The analysis of the data shows that the differences in inducing factors are mainly responsible for the differences in the fitness of lines and are the genetic materials for the adaptive evolution of organisms. HE raw material for the adaptive evolution of organisms is polymorphic T genetic variation. The existence of polygenic variation related to the fitness of individuals is known from extensive studies of natural populations (DOBZHANSKY and SPASSKY 1968; TRACEY and AYALA 1974; MUKAI and YAMAGUCHI 1974). This genetic variation must have a significant role in adaptive evolution. Understanding the molecular basis of fitness-related genetic variation is essential. Since the discovery of allozyme polymorphisms in natural populations of many organisms (HARRIS 1966; LEWONTIN and HUBBY 1966; AYALA, POWELL and DOBZHANSKY 197 1 ; SELANDER, HUNT and YANG 1969; YAMAZAKI 198 la), population geneticists have been trying to elucidate the maintenance mechanisms for these polymorphisms and their evolutionary significance, hoping that these enzyme polymorphisms are the materials for adaptive evolution (YAMAZAKI and MARUYAMA 1972; MUKAI, WATANABE and YAMAGUCHI 1974; LEWONTIN and KRAKAUER 1973; AYALA and GILPIN 1973; YAMAZAKI et al. 1984). ’ To whom correspondence should be addressed. Genetics 108 223-235 September, 1984 224 T. YAMAZAKI AND Y. MATSUO Recent findings in experimental studies of polymorphisms and molecular evolution seem to indicate the selective neutrality of these allozyme polymorphisms in natural populations (KIMURA and OHTA 1971; YAMAZAKI 1977; MUKAI, TACHIDA and ICHINOSE 1980; MIYATA and YASUNACA 1981). In this study we show that there is indeed genetic variation in the inducibility as well as the specific activities of amylase in a natural population of D. melanogaster. Also, differences in fitness result from differences in the inducibility of this enzyme. On the other hand, differences in the specific activity of amylase have little effect on the fitness of individuals. These results indicate the importance of regulatory gene polymorphisms for the fitness differences of individuals. A preliminary report of this work was published elsewhere (YAMAZAKI 198 1 b; YAMAZAKI and MATSUO 1983). MATERIALS AND METHODS Materials: D. melanogaster collected from Akayu in northern Japan in 1977 were used in this study; 430 male flies collected from nature were each mated to Cy/Pm;Sb Ser/Pr (or Zn(2LR)SMZI In(2LR)bw"'; Zn(3LR) TM3/Pr ) females. A single male progeny with the Cy; Sb Ser phenotype was again mated with Cy/F"; Sb Ser/Pr females. The resulting Cy; Sb Ser F2 progeny were mated with each other. Wild-type Fs progeny from this mating were maintained as an isogenic line. In this way, 200 isogenic lines homozygous for both chromosome 2 and 3 were established. These lines had neither lethal nor sterile genes on any of their chromosomes [see YAMAZAKI et al. (1984) for details of these materials]. T h e sex chromosome and chromosome 4 were likely to be homozygous since inbreeding for ten generations was done by single brother-sister mating. However, the origin of these two chromosomes cannot be determined because no marker was used on them. Of the 200 isogenic lines established, 44 were randomly selected for this experiment. Each line had the amylase phenotype of 1.00 [equivalent to type 1 of KIKKAWA (1964)l. It is known that the amylase loci are inducible by particular substances that are related to the starch metabolism of the fruit fly. Therefore, each isogenic line was raised under three different media: (1) normal (or cornmeal) medium, (2) starch medium, and (3) sucrose medium. Compositions of three inducing media: Normal medium: agar, 0.6 g, molasses, 2.8 ml; sugar, 1.4 g; Ebios, 2.0 g; cornmeal, 11 g; propionic acid, 0.4 ml; distilled water, 100 ml. Starch medium: agar, 0.6 g; starch (Electrostarch Company), 15.2 g; Ebios, 2.0 g; propionic acid, 0.4 ml; distilled water, 100 ml. Sucrose medium: agar, 0.6 g; sucrose, 15.2 g; Ebios, 2.0 g; propionic acid, 0.4 ml; distilled water, 100 ml. Cornmeal medium is the normal food used in our laboratory. Starch is known for its inducing effects on amylase loci (ABE 1958), since it is the substrate of the amylase enzyme. Sucrose is considered to be a suppressor of that locus (DICKINSON and SULLIVAN 1975). Measurement of enzyme activity: A Simazu UV 240 spectrophotometer was used throughout the experiment for the measurement of amylase activity. T h e wavelength used was 550 nm. Ten female adult flies of about 60 h r posthatching were used for each measurement. T h e ten flies were ground together with a glass rod with 50 PI of 0.1 M Tris-HCI (pH 8.9) and 0.1 M MgC12; then 1 ml of additional buffer was added to the mixture and centrifuged with an Eppendorf microcentrifuge for 8 min. A portion of the supernatant (0.15 ml) was kept in a -70" deep freeze until the protein content could be measured by the LOWRY method (LOWRY et al. 1951). Another 0.5 ml of supernatant from the mixture was used for the measurement of enzyme activity: it was mixed with 3 ml of 0.025% 12-Kl, 0.1 M Tris-HCI (pH 8.9) and 1% starch. Every 30 sec the transmission rate was measured for 1 min by the spectrophotometer. As a control, a similar quantity of buffer and starch without the supernatant was measured. T h e increase of transmission rate of the sample (as a percentage) was subtracted from that of the control. This value was defined as the amount of enzyme activity. T h e approximate linearity of this value was confirmed by measuring it in different amounts of enzyme. T h e amount of enzyme was changed by putting different amounts of supernatant of the same line in a test tube to be examined. Values for specific activity were obtained INDUCIBILITY, AMYLASE, FITNESS 225 by measuring the enzyme activity per unit amount of protein. Similar values for specific activity were obtained in other experiments (Y. MATSUO and T . YAMAZAKI, unpublished data) in which measurements were made by a different method (NELSON 1944), indicating that the amount of specific activity in this experiment properly reflects the amount of enzyme present. Detection of genetic variability with respect to inducibility: The term “inducing factors” is used to denote factors at all levels (i.e., transcriptional, translational and posttranslational) that control the amount of amylase produced. “Inducibility” measures the effects of the inducing factors. We are proposing a new method of analysis to isolate the inducibility, which is free of the effects of the structural gene for amylase. In this analysis it is assumed that the catalytic efficiency of the enzyme does not depend on the environment in which the fly was reared and that there is no interaction between the structural gene and the inducing factors in regard to the expression of the structural gene. Specific activity (SA) is expressed by the catalytic efficiency of the structural gene (STE) times the amount of enzyme produced (PRE) as follows. SA(i,k) = STE(k) X PRE(i,k) where SA(i,k) is the specific activity of line k at the structural gene under environment i. STE(k) indicates the catalytic efficiency of the enzyme product of line k at the structural gene. PRE(i,k) indicates the amount of the enzyme produced in line k under environment i. The effect of the structural gene can be eliminated by taking the ratio of specific activity (SA(j,k)/SA(i,k)) in two different inducing environments (e.g., starch and normal foods). SA(j,k)/SA(i,k) = (STE(k) X PRE(j,k))/(STE(k) X PRE(i,k))