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Mutations in the Anopheles gambiae Pink-Eye and White Genes Define Distinct, Tightly Linked Eye-Color Loci M. Q. Benedict, N. J. Besansky, H. Chang, O. Mukabayire, and F. H. Collins New eye-color mutations were induced in the mosquito Anopheles gambiae by EMS or 7-irradiation treatments. Seven new sex-linked mutations were isolated, five of which were viable and fully fertile. Of those, three were in the previously described pink-eye (p) gene in which two spontaneous mutations have previously been identified. Two other mutations, w1 and w2, were in a gene with no extant mutant alleles that we designate the white gene. One of these, w\ is due to a large deletion in the 5' end of the cloned homolog of the D. melanogaster white gene. The pink-eye and white loci are tightly linked with recombination frequencies of 3.5% and 1.1% between w1 or w2 and the spontaneous mutant allele, pw, respectively. Small samples of F2 larvae were examined for intragenic recombination between various alleles, but none was observed in any experiment. The white mutants, but not the pink-eye, exhibit epistasis over the expression of the larval body pigmentation phenotype collarless* and pigmentation of the male accessory glands and testis sheath. These pleiotropic effects are similar to those of D. melanogaster white mutants and also suggest that white is probably identical to the previously described white-eye gene. From the Division of Parasitic Diseases, Centers for Disease Control and Prevention, Mailstop F22, 4770 Buford Highway, Chamblee, GA 30341, and Department of Biology, Emory University, Atlanta, GA 30322. M.Q.B. and O.M. were supported by grants from the John D. and Catherine T. MacArthur Grant to F.H.C. and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases to N.J.B. We appreciate the exceptional efforts of C. F. Curtis of the London School of Hygiene and Tropical Medicine for supplying A. gambiae strains containing the p and p" alleles. We thank Brian Holloway and the staff of the CDC Biotechnology Core Facility for providing all oligonucleotide primers used in this study. H. Chang performed work contained herein in partial fulfillment of the requirements for his bachelor's degree senior research project at Emory University. Address correspondence to Dr. Benedict at the CDC. Journal of Heredity 1996;87:48-53; 0022-1503/96/S5.00 48 Eye-color mutations are one of the most commonly identified types of mutations in insects. Anopheles gambiae is no exception with the white-eye (w), pink-eye (p; Mason 1967), and red-eye (r, Beard et al. 1995) mutations comprising half of the described mutations. Relatively few eye-color phenotypes are observed in mosquitoes due to the fact that, unlike Drosophila melanogaster, ommochrome pigments are the only major eye pigments present in mosquitoes studied thus far (Beard et al. 1995). In contrast, D. melanogaster has several pteridine pigments in addition to ommochromes. Mason (1967) described the first A. gambiae eye-color mutations, white eye and pink eye. He found that both were sexlinked and later reported that they were nonallelic (Kitzmiller and Mason 1967). Furthermore, white eye, but not pink eye, showed reduced fertility, demonstrated epistatic suppression of the collarless* phenotype, and eliminated male accessory gland and testis sheath pigmentation. However, later reports by Curtis (1976) and Cooper et al. (1983) were in disagreement with the findings of Mason, clearly showing that white eye and pink eye were indeed allelic. Considerable confusion re- garding the complementation groups of several mutants has resulted, and no satisfying conclusion can be reached about their relationships because the original white-eye mutant has been lost (see Discussion). Recently, the A. gambiae homolog of the D. melanogaster white gene has been cloned and extensively characterized primarily for use as a genetic transformation marker (Besansky et al., in press). The cloned white gene has been mapped by in situ hybridization to the X chromosome close to the locus of the only sex-linked gene for which eye-color mutants were extant, pink-eye (Zheng et al. 1993). However, white molecular lesions were not detected in any pink-eye mutants (Besansky et al., in press); therefore, conclusive evidence of a relationship between pink-eye mutants and the cloned white gene was lacking. To identify mutations in the cloned white gene and to isolate new mutations on the X chromosome, we embarked on a mutation screen specifically designed to recover X chromosome eye-color mutations. This is facilitated by the sex determination system of A. gambiae in which, like D. melanogaster, XX individuals are fe- males and XY males. We also hoped to resolve some of the apparent conflicts in the literature regarding the sex-linked eye-color complementation groups and epistatic interactions with collarless* in A. gambiae. We show in the present report that five new induced mutations fall into two complementation groups. One of the complementation groups that we designate white includes a mutation consisting of a large deletion in the 5' end of the cloned white gene. The second group of mutations are all in the pink-eye complementation group. We show that these two loci are tightly linked. Materials and Methods Mosquito Culture and Mutagenesis With the exception of the new mutations, three mosquito strains were used for all experiments: G3 is a wild-eye strain isolated in The Gambia; WE is pure-breeding for an undescribed recessive mutation conferring white eyes and was obtained from the London School of Medicine and Tropical Hygiene; PE (homozygous for the pink-eye allele p) was obtained from the same source and has been described elsewhere (Beard et al. 1995). The white-eyed mutation of the WE strain has been confirmed to be an allele of pink-eye (Beard et al. 1995), and we have named the WE allele pw. The original hypomorphic p allele confers a pink eye-color, whereas the pw allele confers white eye-color and thus appears to be null. All of the above strains are polymorphic for collarless (c recessive; Mason 1967) with the c* phenotype predominating. Mosquito larvae were reared at 27°C and fed 2:1 TetraMin Baby-E Fish Food/brewers yeast mixed with water as a 2% w/v slurry (Benedict et al. 1979). Adults were held at 27°C, -80% RH, and fed on human blood and 10% Karo syrup. Matings to establish mutant lines were performed in 1-pt paper cups with screen lids, or, for subsequent inheritance crosses, in — 1 -gal cages. Hatchability was determined on isofemale lines 3 days after eggs were collected, and the phenotypes of mosquitoes was determined in the pupal stage. Mutagenesis was by standard methods either by feeding 0.1% EMS in 10% Karo syrup containing a trace of green food-coloring, or by ^Co irradiation of <48-h-old males. Further details of the mutagenesis and screening method have been described elsewhere (Benedict and Chang, in press). Briefly, larval anopheline mosquitoes undergo a background-color-in- duced morphological color change called homochromy; i.e., larvae reared on a black background become extremely dark, whereas those reared on a white background are pale. This response depends on normal eye-color (Benedict and Seawright 1987); therefore, eye-color mutants are easily isolated by rearing larvae in black containers and scanning third or fourth stage larvae for exceptional pale individuals. All mutants were isolated from the F2 generation and were established by outcrossing to G3. Crosses After the initial outcrosses to propagate the mutant alleles, F, progeny were inbred, and F2 mutant-eye males were backcrossed to wild-eye sib females. In the F3, mutant types were inbred to establish pure-breeding stocks. To determine complementation groups, males of all strains were crossed to the WE strain. To determine the rate of intragenic recombination, F, progeny from this cross were inbred, and the F2 progeny were collected both as isofemale lines and en masse. These were screened by either microscopic examination of individuals (in the case of isofemale lines), or by the color-change response for en masse collections of eggs. Mutants that were not in the pink-eye gene were crossed to one another for further complementation and intragenic recombination analysis as above. In order to determine if an epistatic interaction between the eye-color mutations and collarless existed, female mutants from uniformly collarless phenotype strains were crossed to G3 collarless males and the F, scored for sex, eye color, and collarless. All crosses were analyzed by standard x2 analysis, and significance levels were P < .05. Cytogenetic Analysis Heterozygous females were obtained from the F, of mutant males crossed to G3 females. Ovarian nurse-cell polytene chromosomes were prepared from half-gravid mated adult females —24 h after bloodfeeding. Ovaries were removed and placed in 4°C modified Carnoy's solution (1:3 glacial acetic acid/ethanol) and fixed at least 24 h. Ovarioles were squashed in 50% propionic acid and examined by phase-contrast microscopy. Southern Blot Analysis Total genomic DNA was prepared from two adult female mosquitoes, using an adaptation of a Drosophila protocol (Collins et al. 1987). This was double-digested with 5 units each of Hin&\\\ and HindW (Boehringer Mannheim), electrophoresed through a 1% agarose gel, and transferred to Magna nylon membrane (MSI). The insert of pP[Agw]B, which contains the A. gambiae white gene (Besansky et al., in press), was ^P-labeled with the Random Primers DNA Labeling System (Gibco/BRL) and added without purification to hybridization buffer (6 x SSPE, 5 x Denhardt's, 1% SDS, 50 n.g/ ml denatured salmon testes DNA, 5% dextran sulfate). After overnight hybridization at 65°C, the membrane was washed at high stringency according to the manufacturer and autoradiographed for 24-48 h. PCR Reactions PCR was performed using a Perkin-Elmer GeneAmp PCR System 9600, with the GeneAmp PCR Reagent Kit and AmpliTaq DNA polymerase (Perkin Elmer). Each 50 \y\ reaction contained 1.5 mM MgCl2, 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 0.001% gelatin, 200 \iM each dNTP, 2.5 U Amplitaq, 50 pmoles each primer, and 1 |JL1 template DNA (l/100th of the DNA extracted from a single mosquito). PCR amplification conditions were 60 s at 94°C, 35 X (15 s at 94°C, 15 s at 60°C, and 60 s at 72°C). Following PCR, 10 |xl of the reactions were electrophoresed through 2% agarose gels stained with EtBr and photographed. Results Mutant Isolation By exploiting the inability of eye-color mutants to undergo background-color-induced larval color change, seven new Xlinked eye-color mutations were isolated. Mutant strain ml resulted from EMS mutagenesis; all others were isolated from -yirradiated males. All mutant individuals originally identified were males and were established by outcrossing to G3. As expected, all F, individuals were wild type, and mutant males appeared in the F2 (Table 1). F3 mutants were inbred to establish pure-breeding strains. All showed typical sex-linkage and assorted in expected Mendelian ratios. Since the complementation groups were initially unknown, the strains were named ml-m7. All m4 individuals died as pupae, and m6 males had testes with underdeveloped sperm and were infertile. These two lines therefore could not be established or studied in detail. Of the remaining five, all were fully fertile and viable at all stages, and exhibited no semisterility. All strains were nearly white-eyed except m7, which has a red eye-color that darkens to almost wild-type in adults. Benedict et al • Tightly Linked Pink-Eye and White Genes 4 9 the consistency of the observed difference. Table 1. Inheritance patterns of mutants Phenotypes + White Cross (female x male) No. fams. Female Male Female Male Total (G3 (G3 (G3 (G3 (G3 (G3 5 9 7 4 5 8 5 4 4 3 5 7 5 4 4 260 265 319 226 241 350 120 127 105 149 162 341 94 111 30 135 134 153 141 113 372 83 116 121 147 141 354 95 103 42 0 0 0 0 0 0 98 0 100 0 164 0 95 0 52° 131 126 159 109 526 525 631 476 491 722 421 243 429 296 605 695 403 214 x x X X x X ml) m2) m3) m5) m7) ml) F2 F2 F2 F2 F2 F2 X ml (G3 X m2) F2 X m2 (G3 x m3) F2 x m3 (G3 X m5) F2 X m5 (G3 X m7) F2 X m7 137a 0 120 0 103 0 138 0 119 0 47° 171' ° Phenotype is red-eyed rather than white. " Eye-color deviation for all crosses P < .05. Complementation Groups Complementation groups were determined for all viable mutants isolated (Table 2). Crosses to the WE strain carrying the pw allele demonstrated that the ml, m3, and m7 mutations were alleles of pinkeye. In contrast, m2 and m5 crosses to WE females produced F, wild-eyed females and white-eyed males showing that the mutations were not alleles of pink-eye. However, when m2 and m5 were crossed to each other, they produced only whiteeyed progeny. Therefore, all of the new mutations fell into two complementation groups: (1) ml, m3, and m7 are included in pink-eye, and (2) m2 and m5 in a second complementation group. Tests were conducted to detect intragenic recombination between the pink-eye allele, pw, and all new pink-eye mutations. No recombinant progeny were observed in the F2 generation in any cross (Table 2) nor in —15,000 additional progeny obtained from en masse egg collections. Crosses were performed to determine the recombination frequency between the pink-eye and m2/m5 loci. Homozygous pw females were crossed to m2 or m5 males, the F, progeny inbred, and F2 progeny scored. Using this scheme, only one half of the recombinants could be detected as wild-eye males; the other half of the recombinants would be double-mutant males that would presumably be indistinguishable from other white-eyed males. The recombination frequency between the pink-eye and m2 allele was estimated at 3.5% and with the m5 allele at 1.1%. Contingency x2 analysis demonstrates significant differences (P < .05) in recombination frequencies between pink eye and m2 or m5. This difference is possibly due to an undetected rearrangement; however, further crosses are needed to substantiate Table 2. Allelism and recombination crosses Phenotypes + Cross (female x male) No. fams. Female (PE x ml) F, (WExml)F, (WE X m2) F, (WE x m3) F, (WE X m5) F, (WE X m7) F, (m2 X m5) F, (WE X ml) F2 (WE X m3) F2 (WE X m7) F2 (m2 X m5) F2 (WE X m2) F2 (WE X m5) F2 4 7 5 12 8 6 4 8 6 4 5 14 11 0 0 101 0 290 0 0 0 0 87" 0 287 180 Female phenotype red-eyed. Red-eyed. 5 0 The Journal of Heredity 1996:87(1) White Male 0 0 0 0 0 0 0 0 0 104" 0 12 2 Female Male 150 270 0 183 0 151 302 98 193 325 171 220 404 302 76 224 677 394 167° 217 432 282 94 220 272 174 Total 301 572 199 376 615 338 437 836 584 361 444 1,248 750 Phenotypic Interaction With collarless All mutant lines were examined for expression of the collarless* phenotype which is due to uric acid deposition on the dorsum of the abdomen and thorax of larvae (Benedict et al., in press). This produces a prominent white speckling that is especially dense on the thorax. The collarless* phenotype is due to a dominant allele and is the predominant phenotype in most laboratory strains. All mutant strains except m2 and m5 contained collarless* individuals; m2 and m5 were uniformly of the collarless phenotype, having no white pigment. Because the parental G3 strain is polymorphic for collarless, the collarless condition of m2 and m5 could have resulted by chance isolation from collarless parents, by pseudo-linkage to the autosomal collarless locus via a translocation, or by an epistatic interaction between the m2/ m5 alleles and the collarless gene, preventing expression of the dominant collarless* allele. To test these possibilities, we outcrossed m2 and m5 females (of unknown collarless genotype) to G3 collarless males. Because collarless* is dominant, if F, progeny contained collarless* only among the wild-eye individuals, the only explanation would be epistatic interaction. In F, progeny of outcrosses of both m2 and m5, families were of two types (Table 3): those with only collarless individuals, and families in which collarless* appeared in wild-eye, but not in any whiteeyed mosquitoes. This experiment confirmed that the uniform collarless phenotype of m2 and m5 is due to epistatic suppression of the collarless* phenotype and that those strains are polymorphic for collarless alleles. Microscopic examination of the testis sheath and male accessory gland of the m2/m5 and the pink-eye mutants revealed that all pink-eye mutants had normal pigmentation (brown testis sheath, bright yellow accessory glands), but m2 and m5 had unpigmented testis sheaths and the accessory glands were very pale yellow (Table 4). Cytogenetic Analysis All new mutations were examined for cytologically detectable aberrations in heterozygous females produced by outcrossing mutant males to G3 females. F, females were examined for lesions on the X chromosome particularly in the vicinity of white. None of the pink-eye mutants con- 9.6 kb. Although we have observed no lesion in m5 by these molecular analyses, a small mutation cannot be ruled out without DNA sequence analysis. Table 3. Demonstration of epistasis over collarless* Phenotypes Collarless Cross (female x male) (m2 X G3cc) F, (m5 x G3cc) F, No. fams. 4 2 2 1 White White Female Male Female Male Female Male 127 0 14 0 0 0 0 0 102 117 27 26 0 0 0 0 tained any detectable lesion on the X chromosome; however, a possible alteration was detectable in m2 (data not shown). Heterozygotes appeared to have a small deletion in region 2A in a puff immediately proximal to the band that hybridizes to white probes by in situ hybridization (Besansky et al., in press). This puff contains four very faint bands, and definite identification of the region included in the deletion was not possible. Efforts are currently underway to isolate new aberrations affecting the pink-eye gene since the location of this gene remains unknown. Molecular Analysis We used two approaches to determine whether any of the mutations were associated with molecular lesions in the white gene. The first was genomic Southern analysis, using a subclone containing the entire white gene as a hybridization probe (Figure 1A, B). The resulting pattern of hy- 0 0 0 0 0 0 0 0 Female Male Total 0 0 0 0 474 237 86 47 245 120 45 21 Discussion bridization from all mutants except m2 matched that of wild-type individuals, although fragments A and H were polymorphic. In contrast, fragments A through E were not detected in m2, demonstrating a large deletion at the 5' end of white. This was confirmed by a second approach— PCR amplification of white alleles from exon 2 through the polyadenylation site. Pairs of sequencing primers were chosen whose products ranged from 350 to 950 bp. Numerous primer pairs failed to amplify the 5' region of the white gene from m2, whereas the expected products were produced from all other mutants (Figure 1C). We attempted to define the 5' end of the m2 deletion by "PCR-walking," in which an upstream-directed primer from exon 3 was coupled with alternative downstream-directed primers; the last primer tested annealed -400 bp inside the first restriction site of Figure IB. No white-specific products were amplified upstream of exon 3, thus the deletion spanned at least Table 4. Comparison of phenotypes of select eye-color mutants Species/strain A. gambiae Wild (wp*) Ml(p') M2 (w1) M3 (p«) M5(w*) M7(p=) WE (p-) Pink-eye (p) White-eye A. albimanus Wild Snow White eye Vermilion A. quadrimaculatus Wild Pink eye Rose eye D. melanogaster Wild White Location Eye color Urate null Testis sheath Accessory gland Reddish-black White White White White Brown Brown nc» Brown nc Brown Brown Brown Bright yellow Bright yellow Pale Bright yellow Pale Bright yellow Bright yellow Bright yellow nc nc X X X X X X X X White Pink White No" No Yes No Yes No No No Yes Reddish-black White White No No Yes nc nc X X nc nc nc nc A Red Yes No data No data Reddish-black Pink Dark pink No No Yes nc nc nc nc X X Bright red White No data Brown nc Yellow X Red No nc nc nc • Urate null phenotype is inferred in some cases by complete absence of white pigmentation consisting of uric acid (Benedict et al., in press). 6 nc = no color. 'As described by Mason (1967). We have presented in this report descriptions of three new mutations in the pinkeye gene, and two new mutations in a second X-linked gene. One of the latter mutations, m2, is due to a large deletion in the cloned white gene confirmed by PCR and Southern hybridization. Taken together with complementation tests, our results confirm that the m2 and m5 mutations are in the A. gambiae homolog of the D. melanogaster white gene and are therefore properly designated white mutants. We believe that the new mutations in the white gene are probably alleles of Mason's (1967) white-eye. This conclusion clarifies a long-standing confusion regarding sex-linked eye-color complementation groups. Mason isolated a mutation, whiteeye, that had epistatic interaction with the collarless* allele, and eliminated the brown and yellow color of the testis sheath and male accessory gland respectively (Mason 1967). He later showed that this gene was not allelic to pink eye (Kitzmiller and Mason 1967). Curtis (1976), however, crossed a white-eyed strain that was supposed to be Mason's white eye to pink eye and found that they were indeed allelic, and determined that the intragenic recombination frequency was, by our calculations, -2.34 X 10-" (Cooper et al. 1983). We have obtained the white-eyed strain of Curtis (WE) and confirm that its mutation is indeed an allele of pink eye (Beard et al., 1995; and this report). However, like pink-eye, the WE mutation does not have an epistatic interaction with collarless* and has no effect on testes sheath or male accessory gland pigmentation (Table 4). Thus, the WE strain we obtained from Curtis cannot be Mason's white eye, but rather must be an undescribed mutation of pink eye. Furthermore, Mason's white eye strain is no longer in existence (Curtis CF, personal communication), preventing a complementation test with m2 or m5. Therefore, to eliminate further confusion, we propose to call the m2 and m5 strain alleles wl and w2, respectively. This nomenclature is reasonably consistent with both Mason and D. melanogaster white (Ashburner 1989). Because all other extant sex-linked eyecolor mutations in A. gambiae fall into the pink-eye complementation group, we re- Benedict et al • Tightly Linked Pink-Eye and White Genes 5 1 A. 1 2 3 4 5 6 7 8 — 4.3 A— E eye pigmentation in which mutations are viable. Among 16 anopheline X-linked eyecolor mutants we located by an extensive literature search, no more than two complementation groups have been identified in any one species. In the robust A. stephensi data set, six sex-linked eye-color mutations have been identified and all were assigned to two complementation groups 5.7 cM apart (Akhtar and Sakai 1985). Similarly in A. quadrimaculatus, three mutations have been found, and these also fell into two complementation groups (Mitchell and Seawright 1990). Finally, both A. albimanus and A. culicifaces have two sex-linked eye-color genes, although few mutations have been identified. 9 = —2.3 —2.0 D_ c— F — G— B— F B. a b G H c pP[Agw]B[; C l I I II 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 600— 100— Figure 1. Molecular analysis of the white alleles. (A) Southern blot of DNA from wild-type A. gambiae, G3 (lane 1) and SUA (lane 2), and eye-color mutants, WE (lane 3), PE (lane 4), ml (lane 5), m2 (lane 6), m3 (lane 7), m5 (lane 8), and m7 (lane 9) digested with ///ndlll + W/ndll and probed with a white probe, pP[Aguj]B, containing the entire coding region. Letters at left show eight restriction fragments as indicated in (B); numbers at right show molecular weight standards in kb. (B) Schematic of white locus showing exons (white boxes) and introns (black boxes) with //mdlll + W/ndll sites (arrows) above and PCR products (brackets) below. Restriction fragments (uppercase letters) and PCR products (lowercase letters) correspond to those in (A) and (C), respectively. Probe used for blot is shown beneath. (C) PCR analysis of white alleles. Lanes 1-6 are SUA, ml, m2, m3, m5, and m7, respectively. Shown are PCR products a, b, and c as indicated in (B). Flanking lanes contain molecular weight standard (100-bp ladder, Gibco-BRL) with 100-bp and 600-bp bands indicated. PCR products below 600 bp are artifacts. named the WE strain pink-eye allele of Curtis, pw, to identify it with the phenotype of the strain received from the London School of Hygiene and Tropical Medicine. Likewise, we have named the mutant alleles of strains ml, m3, and m7, p3, p4, and p5, respectively. It should be noted that the gene mapped by microsatellite analysis to regions ID to 2C by Zheng et al. (1993) 5 2 The Journal of Heredity 1996:87(1) referred to as white eye is actually the pink-eye gene. Due to the tight linkage, our conclusions contradict neither the mapping of Zheng et al. (1993) nor in situ hybridization of a cloned white probe (Besansky et al., in press). All viable mutations we isolated fell into only two complementation groups; therefore, we suggest that there may be only two genes on the X chromosome affecting Though the number of sex-linked genes affecting eye-color is uncertain, we can state with certainty that several sex-linked eye-color mutations isolated in Anopheles mosquitoes are in white homologs based on three lines of evidence: chromosomal gene conservation (Hunt 1987; and Cornel AJ, personal communication), phenotypic similarity (Table 4), and evidence of molecular lesions in their white genes. These are the presently reported A. gambiae white mutants, A. albimanus white eye (Seawright et al. 1982a; Kez P, personal communication), and A. quadrimaculatus rose eye (Mitchell and Seawright 1990; and Besansky NJ, personal communication). Where phenotypic comparisons can be made, this group of mutants share suppression of deposition of uric acid in the cuticle, colorless accessory glands and testes sheaths. Unfortunately, these phenotypic observations aside from eye color have not been reported for the large number A. stephensi eye-color mutants. Only one other anopheline eye-color mutation has been isolated that suppressed uric acid deposition, the autosomal vermilion gene (Seawright et al. 1982b). The white mutants of D. melanogaster have pleiotropic effects similar to the A. gambiae white mutants we have isolated (Table 4). These pleiotropic effects result from white gene membership in a family of ATP-dependent membrane transport proteins (Mount 1987). A. gambiae white mutants share with D. melanogaster the loss of yellow pigmentation in the male accessory gland. A. gambiae white mutants are urate null (Benedict et al., in press), likely due to failure of purine transport, particularly guanine and xanthine, a characteristic shared with Drosophila white mutants (Sullivan et al. 1979). In mosquitoes, this defect results in a visible phenotypic loss of white larval and pupal pigment because collarless* pigment consists of uric acid. This same transport defect may be responsible for white epistasis over the Redstripe character observed by Mason (1967), a character that we believe to be due to deposition of pteridines. The mutants we have isolated provide important tools for progress toward genetic transformation of A. gambiae. The wl and w* strains are potential recipients for genetic transformation using the white gene as a marker. Furthermore, these strains will allow simpler isolation of new white-eye deletions enabling more precise definition of the functional promoter and determination of gene orientation. The new pink-eye mutants should assist cloning that gene since its locus can be identified by cytological examination of deletion heterozygotes. This information, combined with in situ hybridization, could serve as the basis for a walk or library screen. These methods should allow cloning of the two spontaneous pink-eye mutations, which may contain a transposable element that could be used as a transformation vector. intra-cistronic recombination in Anopheles gambiae. Genetica 162:161-162. Curtis CF, 1976. Allelism test on two eye colour mutants in Anopheles gambiae species A. Trans Royal Soc Trop Med Hyg 70:281. Ashburner MA, 1989. Drosophila: a laboratory handHunt RH, 1987. Location of genes on chromosome arms book. Cold Spring Harbor, New York: Cold Spring Harin the Anopheles gambiae group of species and their bor Laboratory Press. correlation to linkage data for other anopheline mosquitoes. Med Vet Ent 1:81-88. Beard CB, Benedict MQ, Primus JP, Finnerty V, and ColKitzmiller JB and Mason GF, 1967. Formal genetics of lins FH, 1995. Eye pigments in wild-type and eye-color mutant strains of the African malaria vector Anopheles anophelines. In: Genetics of insect vectors of disease gambiae. J Hered 86:375-380. (Wright JW and Pal R, eds). New York: Elsevier; 3-15. Mason GF, 1967. Genetic studies on mutations in speBenedict MQ and Chang H, in press. Rapid isolation of cies A and B of the Anopheles gambiae complex. Genet anopheline eye-colour mutants based on larval colour Res Camb 10:205-217. change. Med Vet Entomol. Mitchell SE and Seawright JA, 1990. EMS-induced muBenedict MQ, Cohen A, Cornel AJ, and Brummett DL, tations in Anopheles quadrimaculatus (Say), species A. in press. Uric acid in Anopheles mosquitoes (Diptera: J Hered 80:58-61. Culicidae): effects of collarless, stripe, and white mutaMount SM, 1987. Sequence similarity. Nature 325:487. tions. Ann Entomol Soc Am. Seawright JA, Benedict MQ, Narang S, and Kaiser PE, Benedict MQ, Seawright JA, Anthony DW, and Avery 1982a. White eye and curled, recessive mutants on the SW, 1979. Ebony, a semidominant lethal mutant in the X chromosome of Anopheles albimanus. Can J Genet mosquito Anopheles albimanus. Can J Genet Cytol 21: Cytol 24:661-666. 193-200. Seawright JA, Benedict MQ, Suguna SG, and Narang S, Benedict MQ and Seawright JA, 1987. Changes in pig1982b. Red eye and vermilion eye, recessive mutants mentation in mosquitoes (Diptera: Culicidae) in reon the right arm of chromosome 2 in Anopheles albisponse to the color of the environment. Ann Ent Soc manus. Mosq News 42:590-593. Am 80:55-61. Sullivan DT, Bell LA, Duncan RP, and Sullivan MC, 1979. Purine transport by malpighian tubules of pteridine-deBesansky NJ, Bedell JA, Benedict MQ, Mukabayive 0, ficient eye color mutants of Drosophila melanogaster. Hilfiker D, and Collins FH, in press. Cloning and characterization of the white gene from Anopheles gambiae. Biochem Genet 17:565-573. Insect Mol Biol. Zheng L, Collins FH, Kumar V, and Kafatos FC, 1993. A detailed genetic map for the X chromosome of the maCollins FH, Mendez MA, Rasmussen MO, Mehaffey PC, laria vector, Anopheles gambiae. Science 261:605-608. and Besansky NJ, 1987. A ribosomal RNA gene probe differentiates member species of the Anopheles gamReceived February 2, 1995 biae complex. Am J Trop Med Hyg 37:37-41. Accepted July 7, 1995 Cooper PJ, Curtis CF, and Sawyer B, 1983. Evidence for Corresponding Editor: Ross Maclntyre References Akhtar K and Sakai RK, 1985. Genetic analysis of three new eye colour mutations in the mosquito, Anopheles stephensi. Ann Trop Med Parasit 79:449-455. Benedict et al • Tightly Linked Pink-Eye and White Genes 5 3