Genetica 109: 235–243, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 235 Genetic and biochemical analysis of brown eye mutation in Drosophila nasuta nasuta and Drosophila nasuta albomicans J.S. Ashadevi & S.R. Ramesh∗ Drosophila Stock Centre, Department of Studies in Zoology, University of Mysore, Manasagangotri, Mysore570 006, India; ∗ Author for correspondence (Phone: 0091-821-515525/Ext.49 (Univ.); 0091-821-472218 (Res.); Fax: 0091-821-421263) Received 16 September 1999 Accepted 4 December 2000 Key words: Drosophila, brown eye, eye pigments, fitness, gene localization Abstract By analyzing the progeny of crosses involving brown eye mutants and the wild types in two members of Drosophila nasuta subgroup namely D. n. nasuta and D. n. albomicans we could show that the mutant gene is recessive, located in the chromosome 2 and the alleles of this gene are present at different loci. A study of fitness in the eye color mutants in comparison with the wild types revealed that D. n. nasuta mutant has higher viability at both 25 ± 1◦ C and ambient temperatures; while D. n. albomicans mutant has faster rate of development only at 25 ± 1◦ C. Quantitative analysis of eye pigments in the mutants revealed that there is biosynthesis of both pteridines and xanthommatins unlike in bw/bw of D. melanogaster, where only xanthommatins are synthesized. In both the species, the pteridine quantities in mutants are similar; whereas xanthommatin quantity in bwn /bwn is 10 times higher than that of bwa /bwa . Further, the F1 progeny of intraspecific crosses (wild type X mutant) are found to have high amounts of pteridine, even when compared with parental wild type. Introduction Mutations are rare events. It is generally believed that a vast majority of the mutations are detrimental. Genes were identified and functions of many genes were understood exclusively through the existence of mutant alleles. Due to the availability of numerous genetic markers, the analysis of mutations has been made mostly in Drosophila melanogaster. More than 3000 mutations have been identified and described in D. melanogaster (Lindsley & Grell, 1972). Such studies though limited, have been made in the mutants of a few other species namely D. hydei, D. virilis, D. subobscura, D. pseudoobscura, D. ananassae, D. bipectinata and D. malerkotliana (Lifschytz, 1974; Stursa, 1983; Taylor, 1983; Mohanty et al., 1988; Lozovskaya & Ergener, 1991; Hegde & Krishna, 1995; Krishna & Hegde, 1998; Singh & Sisodia, 1999). The nasuta subgroup of Drosophila immigrans group consists of an assemblage of morphologically almost indistinguishable cluster of closely related spe- cies. Various species of this subgroup have been studied to understand their interrelationships (Ranganath & Krishnamurthy, 1975; Ranganath & Hägele, 1981, 1982; Ranganath et al., 1982; Rajasekarasetty et al., 1979, 1980; Ramesh & Rajasekarasetty, 1980). D. n. nasuta was first described by Lamb (1914) from Seychelles Islands, Africa; while D. n. albomicans by Duda (1923) from Paroe, Formosa. These two species belong to frontal sheen complex of nasuta subgroup and are widely distributed in Southeast Asian region (Nirmala & Krishnamurthy, 1972; Mather & Pope, 1972; Ranganath & Krishnamurthy, 1972; Wakahama & Kitagawa, 1971, 1972; Clyde, 1977; Gai & Krishnamurthy, 1972; Shyamala et al., 1987). Wakahama and Kitagawa (1973) as well as Kalisch and his co-workers (personal communication) isolated few spontaneous and induced mutants of D. n. nasuta and D. n. albomicans. These mutants have not been further analyzed due to the non-availability of genetic markers. In view of this, present investigations were undertaken to study the genetic and biochemical as- 236 pects of brown mutation in these two closely related members of nasuta subgroup. conducted between the brown mutants of D. n. nasuta and D. n. albomicans and the F1 progenies were examined for their phenotype. Further, these F1 flies were inbred to study the phenotypes of F2 progeny. Materials and methods Stocks For the present study, the brown eye colour mutant stocks of D. n. nasuta (spontaneous mutation from wild type of Seychelles island) and D. n. albomicans (spontaneous mutation from wild type of AmamiOshima, Japan) were provided by W.-E. Kalisch, Institut für Genetik, Ruhr Universität, Bochum, Germany. These mutant stocks are being maintained in our laboratory since 1995 and are found to be true breeding, having full penetrance and expressivity. The wild stocks of D. n. nasuta (Coorg, Mysore, India) and D. n. albomicans (Okinawa) were obtained from Drosophila Stock Centre, Department of Zoology, University of Mysore, Mysore. Genetic crosses Unmated males and virgin females of bw/bw mutants (brown eye) of both species and wild type stocks (red eye) were isolated every 4 h after their eclosion as imagines from the pupae. They were maintained at 22 ± 1◦ C on standard wheat cream agar medium for 5 days before using them for conducting crosses. All the crosses were conducted in 8×2.5 cm vials containing equal quantity of the medium and all the experimental cultures were maintained at 22 ± 1◦ C to obtain the progeny. Crosses were conducted between mutant and wild type flies of the same species, to determine the dominant or recessive nature of the mutant alleles and the phenotypes of the F1 progenies were recorded. For chromosomal localization of mutant genes, interspecific reciprocal crosses were conducted (see Kalisch & Zajonz, 1995). To localize mutant genes in case of D. n. nasuta, the bwn /bwn females of D. n. nasuta were crossed with wild type males of D. n. albomicans and vice versa. Further, the males of F1 were backcrossed with the females of parental mutant stocks to study the back cross progeny. In all these crosses, different phenotypes of the F1 and backcross progenies were recorded. The details of these crosses are depicted in Figures 1 and 2. To determine whether the bw alleles in D. n. nasuta and D. n. albomicans are situated at the same locus or at different loci, interspecific reciprocal crosses were Fitness studies The procedure of Ramachandra and Ranganath (1986a) was followed to determine the fecundity, rate of development and viability. The experimental cultures of different strains set up for the present investigations were divided into two batches. Batch-I was raised at a constant temperature of 25 ± 1◦ C and the cultures of Batch-II were maintained at ambient temperature, where the fluctuation was recorded to be between 25◦ C and 30◦C. We used 30 replicates for every batch for the analysis of fecundity and 12 replicates for the determination of rate of development and viability. The results of fecundity and viability were subjected to one way analysis of variance with Duncan multiple range test (DMRT) and student ‘t’test for rate of development for comparison of fitness components among the strains analyzed. Quantification of red and brown pigments Red and brown pigment content was determined in the eyes of 5-day-old adults of wild type, brown mutants of both the members as well as in the eyes of F1 progeny of wild type and brown mutants. We followed the procedure of Real et al. (1985) to estimate the red pigment content. Five milligram of decapitated heads of each sex were homogenized in 3 ml of 30% AEA (30% alcohol acidified with HCl, of pH 2.0). These extracts were kept for 24 h, filtered through glass fiber and the absorbance was measured at 480 nm by using Schimadzu Spectrophometer UV 1601. Modified procedure of Ferre et al. (1986) was followed to estimate the brown pigment content. For this purpose 12 mg of decapitated heads of adult males and females were separately homogenized in 3 ml of 2 M HCl. Twenty milligram of sodium metabisulfite as well as 4 ml of n-butanol were added and the mixture was tumbled for 30 min. Then the samples were centrifuged for 5 min at 4000 rpm, so that the organic layer containing the brown pigment was separated. An aliquot of 3.4 ml of organic layer was mixed with 20 mg of sodium metabisulfite and 3 ml of distilled water. The mixture was tumbled again for 30 min and centrifuged. The procedure was repeated with 2.4 ml of organic layer. After centrifugation, the absorbance was 237 Figure 1. Schematic illustration of the interspecific crosses showing the karyotypes in F1 and back cross progeny as well as their phenotypes, if the mutation is recessive and the gene is located in chromosome 2 in case of D. n. nasuta. The black/gray dot on the chromosome represents the ‘bw gene’. P = Karyotype of parents; F1 = Karyotype of first filial generation; Xn = X-chromosome of D. n. nasuta.; Yn = Y-chromosome of D. n. nasuta.; Xa = X-chromosome of D. n. albomicans; Ya = Y-chromosome of D. n. albomicans. 238 Figure 2. Schematic illustration of the interspecific crosses showing the karyotypes in F1 and back cross progeny as well as their phenotypes, if the mutation is recessive and the gene is located in chromosome 2 in case of D. n. albomicans. 239 Table 1. Results of interspecific crosses and backcrosses between brown eye and wild type strains of D. n. nasuta and D. n. albomicans Crosses +a / +a ♂♂ X bwn /bwn ♀♀ +n / +n ♂♂ X bwa /bwa ♀♀ F1 wild type wild type Backcross bwn /bwn ♀♀ X F1 ♂♂ bwa /bwa ♀♀ X F1 ♂♂ bw♂♂ Backcross progeny bw♀♀ wild ♂♂ wild ♀♀ 44 46 51 48 60 62 58 56 bwn =brown eye mutant of D. n. nasuta; bwa =brown eye mutant of D. n. albomicans. +n = D. n. nasuta wild type; +a = D. n. albomicans wild type. measured at 492 nm. The estimations were repeated thrice to confirm the results obtained. Results and discussion Drosophila nasuta subgroup consists of an assemblage of closely related species. Morphological and interspecific hybridization studies have revealed that D. n. nasuta and D. n. albomicans belong to ‘Frontal sheen complex’ and are cross-fertile, irrespective of their geographic origin (Wilson et al., 1969; Nirmala & Krishnamurthy, 1974; Ranganath et al., 1974; Rajasekarasetty et al., 1980). Intraspecific reciprocal crosses involving wild type and mutant strains of the same species were conducted to find out the dominant or recessive nature of the bw mutation. The phenotypes of the F1 progeny of such crosses were all found to be of only wild type. Further, red eyed and brown eyed individuals appeared in the F2 generation in a typical 3:1 ratio (3 wild type: 1 mutant), when the F1 progeny was allowed to inbreed. With these results, we could infer that in both species the bw alleles are recessive to the wild type and the bw gene is not X-linked, but autosomal. The nature and the number of chromosomes in the two species under study differ. D. n. nasuta has 2 n = 8, consisting of two pairs of acrocentrics (representing chromosomes 3 & X), one pair of metacentrics (chromosome 2) and a pair of dots (chromosome 4). The Y-chromosome in case of male is submetacentric. D. n. albomicans has 2 n = 6, consisting of two pairs of metacentrics, one of the pair representing the fused products of chromosome 3 and X or Y (in male) and the other pair, chromosome 2. The chromosome 4 in this species is represented by two long dots (Wilson et al., 1969; Ramachandra & Ranganath, 1986b). We exploited the cross fertility of D. n. nasuta and D. n. albomicans to conduct interspecific crosses for the autosomal localization of bw mutation. To find out on which of the two major autosomes namely, chromosome 2 or 3, the bw gene is located, crosses involving mutant and wild type strains of D. n. nasuta and D. n. albomicans were conducted. The possible phenotypes of F1 and back cross progeny of such interspecific crosses could be predicted based on differences in the segregation patterns arising from variation in chromosomal composition in the parents and location of the mutant gene. Figures 1 and 2 illustrate the phenotypes encountered in the F1 and backcross progeny of interspecific crosses, when the mutant gene is recessive and is located in chromosome 2. A scrutiny of these figures reveal that if the mutant gene is recessive and is located in chromosome 2, the back cross progeny will be consisting of males and females of both mutant as well as wild type phenotypes (Figures 1 and 2). Table 1 includes the data from the crosses, involving bw females of D. n. nasuta and wild type males of D. n. albomicans as well as the reciprocal crosses. A study of which reveals that all the F1 individuals had wild type (red eye) phenotype, while in the backcross progeny, both the sexes with red as well as brown eyed phenotypes occur. These results are in confirmity with the scheme Table 2. Results of reciprocal crosses between brown eye mutants of D. n. nasuta and D. n. albomicans Crosses F1 F2 bw♂♂ bw♀♀ wild ♂♂ wild ♀♀ bwn /bwn ♂♂ Wild type 82 X bwa /bwa ♀♀ bwa /bwa ♂♂ Wild type 98 X bwn /bwn ♀♀ 77 83 81 94 89 91 240 Table 3. Fecundity∗ in brown eye mutants and wild type strains of D. n. nasuta and D. n. albomicans Strains A D. n. nasuta bwn /bwn D. n. albomicans bwa /bwa F value 7304 8387 7512 6416 13.39 25 ± 1◦ C B 243.00(ab) 279.57(a) 250.40(ab) 213.96(b) Ambient temperature B C C A 16.20 18.64 16.69 14.26 9993 6519 6801 5998 105.86 333.10(b) 217.30(a) 226.70(a) 199.90(a) 22.20 14.49 15.11 13.32 ∗ Based on daily egg production (counted for 15 days). d.f.= (3, 116) The strains with the same letter in the parenthesis are not significantly different at 5% level according to DMRT. A = Total number of eggs deposited by 30 pairs of flies; B = Number of eggs per individual; C = Number of eggs per individual per day. Table 4. Mean developmental time (in days) and summary of the student ‘t’-test in brown eye mutants and wild type strains of D. n. nasuta and D. n. albomicans 25 ± 1◦ C a. D. n. nasuta b. bwn /bwn c. D. n. albomicans d. bwa /bwa 12.00 ± 0.55 12.40 ± 0.46 15.86 ± 0.10 13.34 ± 0.09 13.31 ± 0.64 13.56 ± 0.55 11.84 ± 0.06 10.92 ± 0.09 t t 0.78 2.24∗ Strains 25 ± 1◦ C No. of % Viability adults emerged Ambient temperature No. of % Viability adults emerged D. n. nasuta bwn /bwn D. n. albomicans bwa /bwa F value 341 432 274 211 34.50 346 425 323 162 74.34 Mean ± SE Ambient temperature Strains a/b c/d Table 5. Viability in brown eye mutants and wild type strains of D. n. nasuta and D. n. albomicans 0.55 1.86 ∗ Significant at 5% level. d.f. = 22. depicted in the form of Figure 1, for bw mutation in D. n. nasuta and Figure 2 for bw mutation in D. n. albomicans. Hence, it could be inferred that bw gene in both species under investigation is recessive and is located in chromosome 2. Interspecific crosses were conducted between the mutant strains to determine whether the bw gene is present at the same locus or at different loci in the two species under study. The phenotypes of the F1 and F2 progeny in such crosses depend on the location of mutant alleles. If the bw genes are isolocus in both species, all the F1 individuals would have brown eyes. However, if the mutant genes are located at different loci, only red-eyed flies would appear in the F1 generation, which upon inbreeding yields red and brown-eyed F2 individuals in the ratio of 1:1. Table 2 embodies the results of the crosses involving only bw mutants of D. n. nasuta and D. n. albomicans as parents. A scrutiny of the data reveals that all the F1 56.83(a) 72.00(b) 45.66(c) 35.16(d) 57.67(a) 70.83(b) 53.83(a) 27.00(c) Total No. of eggs placed in culture vials = 600. d.f. = (3, 44), The strains with the same letter in the parenthesis are not significantly different at 5% level according to DMRT. individuals of the interspecific progeny had red eye colour and the F2 generation consisted of both red and brown phenotypes in 1:1 ratio. Thus, it could be inferred that the bw gene in the two species under study are not isolocus. Fitness of an individual is the outcome of an interaction between its genotype, environment and competing individuals in a population (Bonnier, 1960; Gowda, 1979). Fecundity, rate of development and viability are the three important parameters employed to assess fitness in Drosophila. Perusal of the literature reveals that mutants of D. melanogaster have reduced fitness when compared with that of wild type (Lewontin, 1955; Ribo & Prevosti, 1969; Prout, 1971; Batabyal & Sidhu, 1972). In the present study, we have analyzed fitness in the brown mutants of D. n. nasuta and D. n. albomicans and compared with the data obtained from respective wild type strains. A scrutiny of Tables 3–5 reveals that in contrast to 241 Table 6. Results of quantitative estimation of eye pigments in D. n. nasuta, D. n. albomicans and their brown eye mutants Stocks Absorbance at 480 nm for red pigment (per 1 mg tissue) Male Female Absorbance at 492 nm for brown pigment (per 1 mg tissue) Male Female a. D. n. nasuta b. bwn /bwn c. F1 A d. F1 B e. D. n. albomicans f. bwa /bwa g. F1 C h. F1 D 0.261 ± 0.003 0.207 ± 0.007 0.607 ± 0.002 0.555 ± 0.013 0.282 ± 0.004 0.228 ± 0.003 0.478 ± 0.008 0.427 ± 0.021 0.397 ± 0.001 0.255 ± 0.004 0.619 ± 0.004 0.523 ± 0.002 0.355 ± 0.016 0.237 ± 0.0003 0.518 ± 0.014 0.400 ± 0.008 0.0148 ± 0.0001 0.131 ± 0.003 0.0209 ± 0.0001 0.0187 ± 0.0014 0.0272 ± 0.0001 0.0165 ± 0.0001 0.0291 ± 0.00001 0.0205 ± 0.0002 0.0185 ± 0.002 0.215 ± 0.0026 0.0276 ± 0.0001 0.0171 ± 0.0004 0.0213 ± 0.0001 0.0164 ± 0.0003 0.0201 ± 0.00002 0.0192 ± 0.0002 a/b a/c a/d b/c b/d c/d e/f e/g e/h f/g f/h b/f g/h t 8.03∗ 17.03∗ 9.98∗ 20.48∗ 11.39∗ 1.35 6.03∗ 14.71∗ 5.22∗ 25.28∗ 7.56∗ 1.89 1.82 t 28.03∗ 41.68∗ 41.80∗ 46.20∗ 50.63∗ 15.94∗ 7.15∗ 6.79∗ 2.77 16.08∗ 14.74∗ 3.58 6.29∗ t 30.91∗ 2.81∗ 59.25∗ 28.05∗ 29.29∗ 1.58 54.57∗ 4.15∗ 22.78∗ 22.78∗ 132.95∗ 30.46∗ 17.84∗ t 178.16∗ 2.48 28.80∗ 166.72∗ 172.46∗ 19.77∗ 27.26∗ 6.67∗ 3.77 6.59∗ 79.45∗ 186.98∗ 1.62 ∗ Significant at 5% level by student ‘t’-test. d.f. = 4. F1 A = +n / +n ♂♂ X bwn /bwn ♀♀; F1 C = +a / +a ♂♂ X bwa /bwa ♀♀; F1 B = +n / +n ♀♀ X bwn /bwn ♂♂; F1 D = +a / +a ♀♀ X bwa /bwa ♂♂. D. melanogaster, some fitness components in the mutants under study show superiority. The brown of D. n. nasuta shows higher viability at both temperatures when compared with its wild type; while brown of D. n. albomicans develops faster at ambient temperature than its wild type. Similar results of superiority in some fitness components were also obtained in other mutants such as sepia and crossveinless of D. n. nasuta; purple and carmine of D. n. albomicans (unpublished data). With these results we suspect that the mutants may dominate over the wild type populations with respect to some fitness parameters even under competitive conditions in a specified environment. Investigations on these lines are in progress. “Eye colour mutants of Drosophila have played an important role in the development of biochemical genetics” (Ferre et al., 1986). The eye in Drosophila contains two major groups of pigments namely ommochromes (brown) and pteridines (red). These two are naturally occurring compounds whose structure, biosynthesis and physiological interrelationships might be elicited by study of mutants affecting them (Ziegler, 1961). Pteridines are a group of fluorescent compounds consisting of drosopterin, pterin, biopterin, sepiapterin and xanthopterin. The eyes of all wild type flies are dull red because of the presence of both pteridines and xanthommatins, while the eyes containing only xanthommatin (ommochromes) appear brown in colour (Ziegler & Harmsen, 1969; Ferre et al., 1986). Wilson and Jacobson (1977) have reported that brown mutant of D. melanogaster fails to biosynthesize pteridines. Further, Ferre et al. (1986) have analyzed the pteridine and xanthommatin quant- 242 ities in brown eye mutations of D. melanogaster with different bw alleles. They have found that only in case of bwV 1 /bwV 1 and bwV 32g /bwV 32g alleles the xanthommatin levels are comparable to that of the wild type (Oregon-R) but not the quantity of pteridines. However, in case of bw/bw only xanthommatins were found to be synthesized. Present study revealed that in the bw mutants of D. n. nasuta and D. n. albomicans, both pteridines and xanthommatins are biosynthesized. In a sex-wise comparison of the eye pigments between wild type and mutant strains (Table 6), we have found that the quantity of red pigment production is higher in the wild type strains of both the species. Surprisingly, the quantity of brown pigment production in brown mutant of D. n. albomicans was found to be lesser than that of its wild type; while in D. n. nasuta mutant, it is found to be about 10-fold higher than bwa /bwa . The composition of red and brown pigments in the bw mutants of nasuta subgroup have similarity with bwV 1 /bwV 1 & bwV 32g /bwV 32g but not with bw/bw alleles of D. melanogaster. Further, red and brown pigment quantities were also estimated in the F1 progeny of the reciprocal crosses made between D. n. nasuta wild type and its brown mutant as well as between D. n. albomicans wild type and its brown mutant. A critical analysis of the data thus obtained (Table 6) reveals that the quantity of red pigments in the F1 individuals is higher than that of the quantity present in respective parents. Similar trend does not exist with regard to quantity of brown pigment. The F1 individuals of D. n. nasuta crosses are found to possess higher quantity of brown pigments when compared with wild type parent. However, the F1 individuals of D. n. albomicans crosses were found to have higher quantity of brown pigments when compared with parental mutant. Thus, though all the F1 individuals have similar eye colour when compared to their wild type parent, they however differ in the red and brown pigment contents. When mutants of both the members are considered, even though the bw alleles are present at different loci, the quantity of pteridine production or accumulation is not drastically altered, but only the xanthommatin levels are altered. There is something in common among brown eye mutants of D. melanogaster, D. n. nasuta and D. n. albomicans that is, the phenotype and location (chromosome 2) of gene. However, they differ from one another in the composition of red and brown pigments. Thus it is possible that the brown mutants of these species are simply the mutants of three dif- ferent genes concerned with pigment synthesis or biochemical pathways. Acknowledgements We thank the Chairman of our department for the facilities. We are grateful to Prof H.A. Ranganath of our department and Prof W.-E. 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