Download Mutations in the Anopheles gambiae Pink

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.
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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
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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
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SW, 1979. Ebony, a semidominant lethal mutant in the
X chromosome of Anopheles albimanus. Can J Genet
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Seawright JA, Benedict MQ, Suguna SG, and Narang S,
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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
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Benedict et al • Tightly Linked Pink-Eye and White Genes 5 3