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Genetics of α-amylase in Sitophilus oryzae (L.) (Coleoptera: Curculionidae)
J.srored Prod.Rex.Vcsl.26,No. I,pp.7-10,1990
0022-474X/90 $3.00+0.00 PergamonPressplc
Printed in Great Britain
GENEtTICS
Stored-Product
OF a-AMYLASE (COLEOPTERA:
IN SITOPHILUS CURCULIONIDAE)”
ORYZAE
J. E. BAKER, W. R. HALLIDAY and P. T. M. LUM Insects Research and Development Laboratory, U.S. Department Agricultural Research Service, Savannah, GA 3 1403, U.S.A.
(L.)
of Agriculture,
(Received for publication 16 October 1989)
Abstract-Grossing experiments with geographic strains of Sitophilus orytae (L.) that differ in a-amylase isozyme composition were used to determine the gene system for coding these enzymes in this species. Savannah strain is monomorphic and has two isozymes designated RW-I and RW-2. Japan and Tanzania-90 strains are also monomorphic but both lack RW-1. All F, progeny of reciprocal crosses of Savannah with either Japan or Tanzania-90 have both isozymes. Two phenotypes were found in F, progeny from mass F, self crosses. Depending on the cross, there is a 3: I ratio of Savannah phenotype (RW-I + RW-2) to either the Japan or Tanzania-90 phenotype (RW-2). Overall results suggest that two structural genes control synthesis of the two amylase isozymes in this weevil. The gene RW-2 (hereafter referred to as Amy-2) is found in all three strains whereas the gene RW- 1 (hereafter referred to as Amy- I) has an acti,ve allele (Amy-l”) expressed in Savannah and a null allele (Amy-IO) in Japan and Tanzania-90.
INTRODUCTION
Electrophoretic variants of a-amylase are common in the maize weevil, Sitophilus zeamais Motschulsky (Baker, 1987a). The adaptive significance of these differences in isozyme composition of this physiologically-important enzyme in different geographic strains of this granivorous species is unknown. However, this enzyme system has potential as a coleopteran model for studying ecological effects on selection of specific gene arrangements (Baker and Halliday, 1989). In an earlier study, strain differences in isozyme composition were used to determine the gene system responsible for coding ol-amylase in S. zeamais (Baker and Halliday, 1989). At least two structural genes are involved, Amy-l and Amy-2. Among three tested strains of this species, China, Fresno, and Iowa, there were two codominant alleles of Amy-2 whereas a11strains examined were monomorphic for Amy-l. Although some strains of S. zeamais are monomorphic at both loci, other strains are polymorphic, and as many as 12 phenotypes (combinations of electrophoretic variants of a-amylase) were noted in individuals of a strain from Peru (Baker et al., 1989). Less strain to strain variation in isozyme composition is found in the rice weevil, S. oryzae (L.) (Baker, 1987a) Twelve of fourteen strains of this species have identical isozyme patterns as determined by gel electrophoresis. Generally, two major isozymes are present. Nevertheless, two strains, Japan and Tanzania-90, are monomorphic and also lack one isozyme. Crossing experiments of these strains with the Savannah strain of S. oryzae were used to determine the genetic mechanisms involved in amylase synthesis in this species. MATERIALS
AND
METHODS
Strains Savannah, Japan, and Tanzania-90 of S. oryzae were cultured on long grain brown rice at 28°C and 5040% r.h. Infested rice grains containing virgin adults were obtained by use of a light table. F, progeny were obtained from single pair reciprocal crosses of Savannah with Japan or Tanzania-90. Generally, 6-10 pairs of each cross were set up. F, progeny from at least 2-3 families of each cross were combined and allowed to mate inter se to produce an F, generation. Two F, cultures were analyzed from each cross. Amylase phenotypes of individual weevils were determined by dissecting the intestinal tract (foregut plus midgut), homogenizing in 50 ~1 of electrophoresis sample buffer, and analyzing the supernatant (following centrifugation) by anionic polyacrylamide gel electrophoresis (PAGE) (Baker and Halliday, 1989). Gels were analyzed with starch zymograms and by staining with *Mention of a commercial or proprietary product does not constitute a recommendation Agriculture.
by the U.S. Department
of
8
J. E. BAKER et (11.
Coomassie Brilliant Blue R. Analyses were performed on 40 weevils from each parent strain, 20 F, progeny from each cross, and a total of 3 19 Fz progeny. In addition, midgut homogenates of 40 individuals of the parent strain from Tanzania were analyzed on linear polyacrylamide gradients of 5-15% and 7.5-22.5% in gels of 0.75 mm thickness. Amylase activity was detected as above. RESULTS
AND
DISCUSSION
a-Amylase phenotypes of parental strains Savannah, Japan, and Tanzania-90, and F, progeny from crosses of Savannah to Tanzania-90 are shown in Fig. 1. Mean mobilities of the isozymes, designated RW-1 and RW-2 (Baker, 1987b) were 0.59 and 0.60, respectively, on the 10% gels. All individuals examined from Japan and Tanzania-90 strains lacked RW-1 as shown by both zymogram analysis and protein stain. There was no evidence for additional amylase isozymes when midgut homogenates of Tanzania-90 were analyzed on either of the two polyacrylamide gradients. Although Japan and Tanzania-90 lack RW- I, all F, progeny examined from single pair reciprocal crosses of Savannah to either Japan or Tanzania-90 had both isozymes. Two phenotypes were expressed in the F, generation resulting from inter se mating of F, (Savannah x Tanzania). A representative sample of the isozyme pattern of these progeny is shown
Fig. 1. Representative oc-amylase phenotypes of adult and 50-60% r.h. Phenotypes of individual weevils Savannah; lane 2. Japan: lane 3, Tanzania-90; (Tanzania x
S. or,~xze reared on long grain brown rice at 28’C were determined by PAGE on 10% gels. Lane I, lane 4, F, (Savannah x Tanzania); lane 5, F, Savannah).
Genetics
of d(-amylase
in S. oryzrre
Fig. 2. Representative F, phenotypes among 20 female progeny of the F, (Savannah x Tanzania) mass self cross. Gel shows the two amylase bands of S. oriole (Band 1 = RW-I = Amy-l: Band 2 = RW2 = Amy-2). Five individuals (lanes marked with arrows) do not have RW-1 (Amy-l), an expression of Amy- 1”.
in Fig. 2. On this gel, 15 individuals expressed the Savannah-type pattern (RW-1 + RW-2) while 5 individuals expressed the Tanzania-type pattern (RW-2 only). Among all Savannah crosses with Tanzania, 125 tested F, individuals had the Savannah phenotype and 34 individuals had the Tanzania phenotype (Table 1). There was no significant difference in phenotype expression with regard to sex of progeny. The same pattern was observed in progeny of Fz crosses involving Japan and Savannah. Fz crosses (F, (Savannah x Japan) x F, (Savannah x Japan)) produced 56 individuals with Savannah phenotype and 24 individuals with Japan phenotype. The reciprocal cross of F, (Japan x Savannah) x F, (Japan x Savannah) produced 65 individuals with Savannah phenotype Table I. Phenotypes of F, progeny produced by inter se mating of F, weevils from reciprocal crosses of S. orww strains Savannah (S) with Tanzania-90 (T) or Japzan (J). Weevils were cultured on long grain brown rice at 28 C and 50-60% r.h. Phenotype*
crosst SXT
SXT TxS TxS SXJ SXJ JXS JxS Total
Sex
RW-I + RW-2
RW-2
M F M F M F M F
30 32 29 34 29 27 33 32 246
9 8 II 6 II 13 7 8 13
Y3
0.01 0.30 0.03 I .63 0.03 0.83 0.83 0.30 0.65
*Phenotypes determined by PAGE analysis of intestinal tract homogenates of individual weevils (with the change of nomenclature. RWI = Amy-l and RW-2 = Amy-?). tBy convention. females are listed first in all crosses. :r- (0.05 level, I d.f.) = 3.84.
10
J. E. BAKER et al.
and 15 individuals with Japan phenotype. Total F2 results indicated that the ratio of individuals with the Savannah phenotype to those with the Japan or Tanzania phenotype was 246:73. This ratio is not significantly different from 3: 1 by chi square analysis with a correction for continuity (x2 = 0.65) (Zar, 1984). Overall results of parental strain, F,, and F? analyses are in agreement with what we expect for a system in which two genes code for RW-1 and RW-2, and RW-1 has a null allele in the Tanzania and Japan strains. To standardize nomenclature for structural genes in Sitophilus that code a-amylases, RW-1 and RW-2 in S. oryzae will henceforth be termed Amy-l and Amy-2, respectively. If we assume Amy- 1 and Amy-2 are products of separate genes and that in Japan and Tanzania a null allele exists, we would expect a 3: 1 segregation of progeny expressing the Amy-1”“” allele (Amy-l*) to those with the null (Amy-l’). Had Amy-l and Amy-2 been alleles of the same gene, the F, progeny would not have been monomorphic. We would have seen individuals lacking the Amy-l (RW-1) isozyme as well as individuals with both Amy-l and Amy-2. In addition, this conclusion is supported by the fact that had Amy-l and Amy-2 been alleles, we would see a segregation of phenotypes in Savannah, the strain with both Amy-l and Amy-2. However, the parent Savannah strain was monomorphic at both loci. Differential mortality could probably not account for the lack of both Amy-l and Amy-2 homozygotes since it is apparent that Amy-2 homozygotes, at least in Japan and Tanzania-90, are able to survive. Although to confirm this latter point, we would have to positively identify Amy-2 in Japan and Tanzania-90 as complementing alleles. Amy-l in S. oryzae and Amy-l in S. zeamais have identical mobilities when examined by PAGE (Baker, 1987a). The Amy-l gene is apparently common to both species and present in all strains of S. zeamais that have been examined and in 12 of 14 tested strains of S. oryzae. In S. oryzae kinetic properties of purified Amy-l differ from those of Amy-2 (Baker, 1987b). Since Amy-l in S. zeamais has not been purified we do not know if properties of Amy-l are identical in the two species. Similarly, properties of Amy-2 in S. zeamais have not been determined, although multiple alleles are present. It is known that, regardless of diet, total amylase activity is about 2-fold higher in S. oryzae compared with that found in S. zeamais (Baker, 1988). Differences in activity levels may be a reflection of different properties of individual isozymes in the two species or that more copies of the isozymes are produced in S. oryzue. Genes that control expression of a-amylase have not yet been demonstrated in either of these weevils although they are well documented in Drosophila (Abraham and Doane, 1978; Klarenberg et al., 1987; Klarenberg et al., 1988). Acknowledgemenfs-We thank Dr T. Yoshida. Laboratory of Applied Entomology, Okayama University, Okayama 700, Japan, and Dr P. Dobie. Tropical Development and Research Institute, Slough SL3 7HL, England, for kindly providing S. oryzae strains Japan and Tanzania-90, respectively. We also thank S. M. Woo for excellent technical assistance during this study.
REFERENCES Abraham I. and Doane W. W. (1978) Genetic regulation of tissue-specific expression of amylase structural genes in Drosophila melanogaster. Proc. nam. Acad. Sci. U.S.A. 75, 444&4450. Baker J. E. (I 987a) Electrophoretic analysis of amylase isozymes in geographical strains of Sirophilus oryzae (L.), S. zeamais Motsch.. and S. granarius (L.) (Coleoptera: Curculionidae). J. stared Prod. Res. 23, 1255131. Baker J. E. (1987b) Purification of isoamylases from the rice weevil, Sitophitus oryzae (L.) (Coleoptera: Curculionidae), by high-performance liquid chromatography and their interaction with partially-purified amylase inhibitors from wheat. Insect Biochem. 17, 37-44. Baker J. E. (1988) Dietary modulation of a-amylase activity in eight geographical strains of Sitophilus oryzae and Sitophilus zeamais. Entomologia exp. appl. 46, 47-54. Baker J. E. and Halliday W. R. (1989) Gene system coding a-amylases in Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae). J. stored Prod. Res. 25, 17-24. Baker J. E., Lum P. T. M. and Halliday W. R. (1989) Phenotypic variants and total a-amylase activity in the maize weevil (Coleoptera: Curculionidae). J. Kans. em. Sot. 62, 430434. Klarenberg A. J., Sikkema K. and Scharloo W. (1987) Functional significance of regulatory map and structural Amy variants in Drosophila melanogaster. Heredity 58, 383-389. Klarenberg A. J., Vermeulen J. W. C., Jacobs P. J. M. and Scharloo W. (1988) Genetic and dietary regulation of tissue-specific expression patterns of or-amylase in larvae of Drosophila melanogaster. Camp. Biochem. Physiol. 89B, 143-146. Zar J. H. (1984) Biosfatistical Analysis. 718 pp. Prentice Hall, Inc., Englewood Cliffs, N.J., U.S.A.
0022-474X/90 $3.00+0.00 PergamonPressplc
Printed in Great Britain
GENEtTICS
Stored-Product
OF a-AMYLASE (COLEOPTERA:
IN SITOPHILUS CURCULIONIDAE)”
ORYZAE
J. E. BAKER, W. R. HALLIDAY and P. T. M. LUM Insects Research and Development Laboratory, U.S. Department Agricultural Research Service, Savannah, GA 3 1403, U.S.A.
(L.)
of Agriculture,
(Received for publication 16 October 1989)
Abstract-Grossing experiments with geographic strains of Sitophilus orytae (L.) that differ in a-amylase isozyme composition were used to determine the gene system for coding these enzymes in this species. Savannah strain is monomorphic and has two isozymes designated RW-I and RW-2. Japan and Tanzania-90 strains are also monomorphic but both lack RW-1. All F, progeny of reciprocal crosses of Savannah with either Japan or Tanzania-90 have both isozymes. Two phenotypes were found in F, progeny from mass F, self crosses. Depending on the cross, there is a 3: I ratio of Savannah phenotype (RW-I + RW-2) to either the Japan or Tanzania-90 phenotype (RW-2). Overall results suggest that two structural genes control synthesis of the two amylase isozymes in this weevil. The gene RW-2 (hereafter referred to as Amy-2) is found in all three strains whereas the gene RW- 1 (hereafter referred to as Amy- I) has an acti,ve allele (Amy-l”) expressed in Savannah and a null allele (Amy-IO) in Japan and Tanzania-90.
INTRODUCTION
Electrophoretic variants of a-amylase are common in the maize weevil, Sitophilus zeamais Motschulsky (Baker, 1987a). The adaptive significance of these differences in isozyme composition of this physiologically-important enzyme in different geographic strains of this granivorous species is unknown. However, this enzyme system has potential as a coleopteran model for studying ecological effects on selection of specific gene arrangements (Baker and Halliday, 1989). In an earlier study, strain differences in isozyme composition were used to determine the gene system responsible for coding ol-amylase in S. zeamais (Baker and Halliday, 1989). At least two structural genes are involved, Amy-l and Amy-2. Among three tested strains of this species, China, Fresno, and Iowa, there were two codominant alleles of Amy-2 whereas a11strains examined were monomorphic for Amy-l. Although some strains of S. zeamais are monomorphic at both loci, other strains are polymorphic, and as many as 12 phenotypes (combinations of electrophoretic variants of a-amylase) were noted in individuals of a strain from Peru (Baker et al., 1989). Less strain to strain variation in isozyme composition is found in the rice weevil, S. oryzae (L.) (Baker, 1987a) Twelve of fourteen strains of this species have identical isozyme patterns as determined by gel electrophoresis. Generally, two major isozymes are present. Nevertheless, two strains, Japan and Tanzania-90, are monomorphic and also lack one isozyme. Crossing experiments of these strains with the Savannah strain of S. oryzae were used to determine the genetic mechanisms involved in amylase synthesis in this species. MATERIALS
AND
METHODS
Strains Savannah, Japan, and Tanzania-90 of S. oryzae were cultured on long grain brown rice at 28°C and 5040% r.h. Infested rice grains containing virgin adults were obtained by use of a light table. F, progeny were obtained from single pair reciprocal crosses of Savannah with Japan or Tanzania-90. Generally, 6-10 pairs of each cross were set up. F, progeny from at least 2-3 families of each cross were combined and allowed to mate inter se to produce an F, generation. Two F, cultures were analyzed from each cross. Amylase phenotypes of individual weevils were determined by dissecting the intestinal tract (foregut plus midgut), homogenizing in 50 ~1 of electrophoresis sample buffer, and analyzing the supernatant (following centrifugation) by anionic polyacrylamide gel electrophoresis (PAGE) (Baker and Halliday, 1989). Gels were analyzed with starch zymograms and by staining with *Mention of a commercial or proprietary product does not constitute a recommendation Agriculture.
by the U.S. Department
of
8
J. E. BAKER et (11.
Coomassie Brilliant Blue R. Analyses were performed on 40 weevils from each parent strain, 20 F, progeny from each cross, and a total of 3 19 Fz progeny. In addition, midgut homogenates of 40 individuals of the parent strain from Tanzania were analyzed on linear polyacrylamide gradients of 5-15% and 7.5-22.5% in gels of 0.75 mm thickness. Amylase activity was detected as above. RESULTS
AND
DISCUSSION
a-Amylase phenotypes of parental strains Savannah, Japan, and Tanzania-90, and F, progeny from crosses of Savannah to Tanzania-90 are shown in Fig. 1. Mean mobilities of the isozymes, designated RW-1 and RW-2 (Baker, 1987b) were 0.59 and 0.60, respectively, on the 10% gels. All individuals examined from Japan and Tanzania-90 strains lacked RW-1 as shown by both zymogram analysis and protein stain. There was no evidence for additional amylase isozymes when midgut homogenates of Tanzania-90 were analyzed on either of the two polyacrylamide gradients. Although Japan and Tanzania-90 lack RW- I, all F, progeny examined from single pair reciprocal crosses of Savannah to either Japan or Tanzania-90 had both isozymes. Two phenotypes were expressed in the F, generation resulting from inter se mating of F, (Savannah x Tanzania). A representative sample of the isozyme pattern of these progeny is shown
Fig. 1. Representative oc-amylase phenotypes of adult and 50-60% r.h. Phenotypes of individual weevils Savannah; lane 2. Japan: lane 3, Tanzania-90; (Tanzania x
S. or,~xze reared on long grain brown rice at 28’C were determined by PAGE on 10% gels. Lane I, lane 4, F, (Savannah x Tanzania); lane 5, F, Savannah).
Genetics
of d(-amylase
in S. oryzrre
Fig. 2. Representative F, phenotypes among 20 female progeny of the F, (Savannah x Tanzania) mass self cross. Gel shows the two amylase bands of S. oriole (Band 1 = RW-I = Amy-l: Band 2 = RW2 = Amy-2). Five individuals (lanes marked with arrows) do not have RW-1 (Amy-l), an expression of Amy- 1”.
in Fig. 2. On this gel, 15 individuals expressed the Savannah-type pattern (RW-1 + RW-2) while 5 individuals expressed the Tanzania-type pattern (RW-2 only). Among all Savannah crosses with Tanzania, 125 tested F, individuals had the Savannah phenotype and 34 individuals had the Tanzania phenotype (Table 1). There was no significant difference in phenotype expression with regard to sex of progeny. The same pattern was observed in progeny of Fz crosses involving Japan and Savannah. Fz crosses (F, (Savannah x Japan) x F, (Savannah x Japan)) produced 56 individuals with Savannah phenotype and 24 individuals with Japan phenotype. The reciprocal cross of F, (Japan x Savannah) x F, (Japan x Savannah) produced 65 individuals with Savannah phenotype Table I. Phenotypes of F, progeny produced by inter se mating of F, weevils from reciprocal crosses of S. orww strains Savannah (S) with Tanzania-90 (T) or Japzan (J). Weevils were cultured on long grain brown rice at 28 C and 50-60% r.h. Phenotype*
crosst SXT
SXT TxS TxS SXJ SXJ JXS JxS Total
Sex
RW-I + RW-2
RW-2
M F M F M F M F
30 32 29 34 29 27 33 32 246
9 8 II 6 II 13 7 8 13
Y3
0.01 0.30 0.03 I .63 0.03 0.83 0.83 0.30 0.65
*Phenotypes determined by PAGE analysis of intestinal tract homogenates of individual weevils (with the change of nomenclature. RWI = Amy-l and RW-2 = Amy-?). tBy convention. females are listed first in all crosses. :r- (0.05 level, I d.f.) = 3.84.
10
J. E. BAKER et al.
and 15 individuals with Japan phenotype. Total F2 results indicated that the ratio of individuals with the Savannah phenotype to those with the Japan or Tanzania phenotype was 246:73. This ratio is not significantly different from 3: 1 by chi square analysis with a correction for continuity (x2 = 0.65) (Zar, 1984). Overall results of parental strain, F,, and F? analyses are in agreement with what we expect for a system in which two genes code for RW-1 and RW-2, and RW-1 has a null allele in the Tanzania and Japan strains. To standardize nomenclature for structural genes in Sitophilus that code a-amylases, RW-1 and RW-2 in S. oryzae will henceforth be termed Amy-l and Amy-2, respectively. If we assume Amy- 1 and Amy-2 are products of separate genes and that in Japan and Tanzania a null allele exists, we would expect a 3: 1 segregation of progeny expressing the Amy-1”“” allele (Amy-l*) to those with the null (Amy-l’). Had Amy-l and Amy-2 been alleles of the same gene, the F, progeny would not have been monomorphic. We would have seen individuals lacking the Amy-l (RW-1) isozyme as well as individuals with both Amy-l and Amy-2. In addition, this conclusion is supported by the fact that had Amy-l and Amy-2 been alleles, we would see a segregation of phenotypes in Savannah, the strain with both Amy-l and Amy-2. However, the parent Savannah strain was monomorphic at both loci. Differential mortality could probably not account for the lack of both Amy-l and Amy-2 homozygotes since it is apparent that Amy-2 homozygotes, at least in Japan and Tanzania-90, are able to survive. Although to confirm this latter point, we would have to positively identify Amy-2 in Japan and Tanzania-90 as complementing alleles. Amy-l in S. oryzae and Amy-l in S. zeamais have identical mobilities when examined by PAGE (Baker, 1987a). The Amy-l gene is apparently common to both species and present in all strains of S. zeamais that have been examined and in 12 of 14 tested strains of S. oryzae. In S. oryzae kinetic properties of purified Amy-l differ from those of Amy-2 (Baker, 1987b). Since Amy-l in S. zeamais has not been purified we do not know if properties of Amy-l are identical in the two species. Similarly, properties of Amy-2 in S. zeamais have not been determined, although multiple alleles are present. It is known that, regardless of diet, total amylase activity is about 2-fold higher in S. oryzae compared with that found in S. zeamais (Baker, 1988). Differences in activity levels may be a reflection of different properties of individual isozymes in the two species or that more copies of the isozymes are produced in S. oryzue. Genes that control expression of a-amylase have not yet been demonstrated in either of these weevils although they are well documented in Drosophila (Abraham and Doane, 1978; Klarenberg et al., 1987; Klarenberg et al., 1988). Acknowledgemenfs-We thank Dr T. Yoshida. Laboratory of Applied Entomology, Okayama University, Okayama 700, Japan, and Dr P. Dobie. Tropical Development and Research Institute, Slough SL3 7HL, England, for kindly providing S. oryzae strains Japan and Tanzania-90, respectively. We also thank S. M. Woo for excellent technical assistance during this study.
REFERENCES Abraham I. and Doane W. W. (1978) Genetic regulation of tissue-specific expression of amylase structural genes in Drosophila melanogaster. Proc. nam. Acad. Sci. U.S.A. 75, 444&4450. Baker J. E. (I 987a) Electrophoretic analysis of amylase isozymes in geographical strains of Sirophilus oryzae (L.), S. zeamais Motsch.. and S. granarius (L.) (Coleoptera: Curculionidae). J. stared Prod. Res. 23, 1255131. Baker J. E. (1987b) Purification of isoamylases from the rice weevil, Sitophitus oryzae (L.) (Coleoptera: Curculionidae), by high-performance liquid chromatography and their interaction with partially-purified amylase inhibitors from wheat. Insect Biochem. 17, 37-44. Baker J. E. (1988) Dietary modulation of a-amylase activity in eight geographical strains of Sitophilus oryzae and Sitophilus zeamais. Entomologia exp. appl. 46, 47-54. Baker J. E. and Halliday W. R. (1989) Gene system coding a-amylases in Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae). J. stored Prod. Res. 25, 17-24. Baker J. E., Lum P. T. M. and Halliday W. R. (1989) Phenotypic variants and total a-amylase activity in the maize weevil (Coleoptera: Curculionidae). J. Kans. em. Sot. 62, 430434. Klarenberg A. J., Sikkema K. and Scharloo W. (1987) Functional significance of regulatory map and structural Amy variants in Drosophila melanogaster. Heredity 58, 383-389. Klarenberg A. J., Vermeulen J. W. C., Jacobs P. J. M. and Scharloo W. (1988) Genetic and dietary regulation of tissue-specific expression patterns of or-amylase in larvae of Drosophila melanogaster. Camp. Biochem. Physiol. 89B, 143-146. Zar J. H. (1984) Biosfatistical Analysis. 718 pp. Prentice Hall, Inc., Englewood Cliffs, N.J., U.S.A.