Journal of Heredity 2004:95(1)
© 2004 The American Genetic Association 95:35-45
Identification of Quantitative Trait Loci Affecting Sex Determination in the Eastern Treehole Mosquito (Ochlerotatus triseriatus)
From the Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523. Funding for this research was provided by National Institutes of Health grant ROl (AI)41041 (to W.C.B.) and by the Colorado State University College Research Council.
Address correspondence to William C. Black IV at the address above, or e-mail: wcb4{at}lamar.colostate.edu.
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Abstract |
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Laboratory colonies of the eastern treehole mosquito (Ochlerotatus triseriatus (Say)) exhibit a consistent female-biased sex ratio. This is unusual among mosquito species, in which heritable sex ratio distortion is usually male biased and mediated by meiotic drive. Quantitative trait loci (QTL) affecting sex were mapped in an F1 intercross to better understand the genetics underlying this female bias. In P1 and F1 parents and in 146 F2 individuals with a female-biased sex ratio (106 females:40 males), regions of seven cDNA loci were analyzed with single-strand conformation polymorphism (SSCP) analysis to identify and orient linkage groups. Genotypes were also scored at 73 random amplified polymorphic DNA (RAPD)-SSCP loci. In addition to the sex locus, at least four QTL affecting sex determination were detected with interval mapping on linkage groups I and II. Alleles at the sex locus cumulatively accounted for approximately 61–77% of the genetic variance in sex. Alleles at QTL adjacent to the sex locus and at a QTL on the opposite end of linkage group I increased the proportion of females, but alleles at a QTL on linkage group I and a second QTL on linkage group II increased the proportion of males. The female-biased sex ratio observed in laboratory colonies of O. triseriatus is most easily explained by the existence of multiple female biased distorter loci, as have been observed in other Diptera.
The eastern treehole mosquito (Ochlerotatus triseriatus) is the principal vector of La Crosse (LAC) virus, the leading cause of pediatric arboviral encephalitis in the United States (McJunkin et al. 2001). Variation in traits related to the vector competence of LAC virus has been described for different strains of O. triseriatus. Studies have documented variation in (1) adult susceptibility and ability to orally transmit LAC virus (Grimstad et al. 1977), (2) permeability of anatomical barriers to LAC dissemination (Paulson et al. 1989), (3) survival of LAC-infected embryos during diapause (McGaw et al. 1998), and (4) ability to transovarially transmit LAC virus (Graham et al. 1999). Most recently we mapped quantitative trait loci (QTL) affecting the ability of O. triseriatus to transovarially transmit LAC virus (Graham et al. 2003). Over the course of this and other experiments, we consistently observed significant female-biased sex ratios in the O. triseriatus strains in our insectary. The sex ratios recorded in 64 families in two laboratory strains are shown in Figure 1.
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The mosquito family Culicidae contains three subfamilies: Anophelinae, Toxorhychitinae, and Culicinae. The Culicinae contains the well-known genera Culex and Aedes. O. triseriatus was formerly in the genus Aedes (Reinert 2000). All members of Culicinae have 2N = 6 chromosomes and all lack heterogametic sex chromosomes. Instead, in Culicinae, sex is determined by genes at a single locus; females are homozygous recessive (mm), while males are heterozygous for a dominant allele (Mm) (Gilchrist and Haldane 1947). Thus the male parent determines sex in the progeny and, given normal segregation, roughly equal numbers of males and females should result. Genetic mechanisms causing a departure from this theoretical expectation have been documented in several culicine species. In some strains of the mosquito Aedes aegypti, male-biased sex ratios are caused by interaction of a Distorter (D) gene, linked to M, and variants of m that are susceptible to D (Hickey and Craig 1966). An additional locus, t, has been shown to confer tolerance to D (Wood and Ouda 1987). In Culex pipiens, male-biased sex ratios have been ascribed to a similar interplay of dominant (MD) and susceptible recessive (ms) alleles (Clements 1992; Sweeney and Barr 1978).
Sex determination in the Diptera is best understood in the housefly (Musca domestica) and the fruit fly (Drosophila melanogaster) (Dubendorfer et al. 2002). The bifunctional switch gene doublesex is common to the sex regulatory cascades of both species, and so is transformer-2, a genetic element required for the sex-specific regulation of doublesex (Schutt and Nothiger 2000). However, the upstream regulators are different: Drosophila utilizes Sex-lethal to control sex determination and to condition the process that equilibrates the difference of two X chromosomes in females versus one X chromosome in males (i.e., dosage compensation) (Schutt and Nothiger 2000). In the housefly, Sex-lethal is not involved in sex determination, and there is little evidence for a mechanism of dosage compensation. A male-determining factor M can be located on either the X or the Y chromosome, or on four of the five autosomes, depending on the strain (Dubendorfer et al. 2002; Inoue and Hiroyoshi 1982; Inoue et al. 1986). There are also strains in which males and females are homozygous for M and females are heterozygous for a dominant female-determining factor FD, on autosome IV, that is epistatic to M (Hilfiker-Kleiner et al. 1993; McDonald et al. 1978). This allows for more adaptive plasticity in the housefly system, and natural housefly populations vary greatly in their mechanisms of sex determination (Cakir and Kence 2000).
Female-biased sex ratios in culicines have been described in the context of thermal stress during embryogenesis (Horsfall and Anderson 1963, 1964, 1965) and differential larval mortality (Danks and Corbet 1973). Also, in several Aedes species, feminization of genetic males was observed to result from high temperatures (Brust 1968; Horsfall 1974) and developmental abnormalities associated with interspecific crosses (Hilburn and Rai 1982). However, heritable female bias is highly unusual and genetic analyses of mechanisms underlying female-biased primary sex ratios in the Culicidae are lacking.
In this study we treat sex as a threshold trait in O. triseriatus to ascertain the presence and location of any QTL with a significant effect on sex determination. The presence of a single QTL (associated with the sex locus) would suggest that random environmental factors in the laboratory cause the consistent differential survival or development of females. Alternatively, identification of multiple independent loci would indicate that sex determination is conditioned by genes that differentially affect survival and possibly development of the sexes.
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Materials and Methods |
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Ochlerotatus triseriatus strains
The AIDL and Holmen strains of O. triseriatus originated from eggs collected at different sites near La Crosse, WI. The AIDL strain originated in 1983, and at the time of these experiments had been in colony for approximately 30 generations, whereas the Holmen strain, established in 1992, had been through approximately 15 generations. Each colony was maintained at 20–23°C, 75% relative humidity, and a photocycle of 16 h:8 h (light:dark) with a 60 min crepuscular period at dawn and dusk. Larvae were fed Tetra-Min fish food. Adults were housed in 2 ft3 flight cages and consisted of 500–1000 individuals per strain.
Females from the AIDL and Holmen strains were allowed to mate, blood fed on mice, and removed to individual 1 pint oviposition containers. Eggs were oviposited on strips of moist paper towel. Sex ratios in 64 families were calculated among adults (average number of individuals/family = 36) (Figure 1).
Mating Scheme
Ochlerotatus triseriatus is a swarm-breeding mosquito (Wright et al. 1966). Copulation and insemination occur only in the presence of numerous males, thereby precluding single-pair mating. Our attempts to artificially induce copulation were unsuccessful, therefore paternal random amplified polymorphic DNA (RAPD) profiles were inferred from the maternal genotype and marker segregation ratios in the offspring. Female mosquitoes mate only once (Craig 1967), thus precluding the possibility of multiple paternal genotypes among progeny. Experiments aimed at maximizing insemination rates in O. triseriatus females with a minimum number of males revealed an optimum of 10–20 males per 5 females and a minimum cage size of 1 ft x 1 ft x 1 ft.
Linkage mapping was performed on the AIDL strain using RAPD single-strand conformation polymorphism (SSCP) and cDNA markers segregating in an F1 intercross. Five virgin females were placed in a 1 gallon carton with 20 males, allowed to mate over 5 days, and then fed on a mouse. Males were collected and frozen. Females were removed from the carton and individually placed in 1 pint oviposition containers with mesh fabric lids. Eggs were oviposited on strips of moist paper towel. Following oviposition, each female was collected and frozen. Eggs from each female were hatched separately to produce five separate F1 families. Each F1 family was allowed to interbreed and females were fed on a mouse. Males were collected and frozen. Each F1 female was removed to an oviposition carton. Multiple gonotrophic cycles were induced by blood-feeding each F1 female multiple times over several weeks to obtain a large F2 family for mapping. Each female fed on hanging droplets consisting of equal parts 10% sucrose solution and defibrinated sheep blood (Colorado Serum Co., Denver, CO). Several candidate F2 families were hatched, reared, and frozen. The family chosen for mapping consisted of 146 individuals and had a significantly female-biased sex ratio (106/146 = 73%; 
2 = 29.8, df = 1, P <.001).
DNA Isolation and Polymerase Chain Reaction (PCR)
DNA was extracted following the salt extraction method of Coen et al. (1982). DNA was resuspended in 1 ml TE (10 mM Tris-HCl, 1 mM EDTA pH 8.0). The majority of the DNA was archived at -80°C; DNA to be used immediately for PCRs was stored at 4°C. PCR was performed using each of the eight RAPD primers and each of the eight pairs of targeted PCR primers for cDNA loci listed in Table 1. All primers were synthesized by Operon Technologies (Alameda, CA).
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For mapping, PCR was performed in 96 well plates. A single, large PCR mixture sufficient to perform one hundred 50 µl reactions was made by mixing 4350 µl dd H2O, 500 µl 10x Taq buffer (500 mM KCl, 100 mM Tris-HCL pH 9.0), 50 µl of 20 mM dNTPs, and 5 nm of the primer. This reaction mixture was set under an ultraviolet (UV) light source (302 nm) for 10 min, after which 10 µl of Taq polymerase were added. The mixture was then dispensed into a 96 well plate. Two microliters of template DNA were added to each well, followed by a drop of sterilized mineral oil. Each set of PCRs (i.e., one 96 well plate) was checked for contamination with a negative control containing all reagents except template DNA. For RAPD-PCR, each plate was subjected to 45 cycles of 95°C for 1 min (denaturation), 35°C for 1 min (annealing), ramp to 72°C at a rate of 1°C every 8 s, and 72°C for 2 min (extension). A final 72°C extension was carried out for 7 min and the temperature was held at 4°C. For targeted PCR of cDNAs, each plate was subjected to 30 cycles of 95°C for 1 min (denaturation), Ta listed in Table 1 for 1 min, and 72°C for 2 min (extension). A final 72°C extension was carried out for 7 min and the temperature was held at 4°C. Samples were stored at 4°C until electrophoresis. The contents of each well were tested for the presence of amplified products by loading 5 µl from each well onto a 1.5% TBE agarose gel, performing electrophoresis for 15–20 min at 112 V, staining with ethidium bromide, and viewing the gel over a UV transilluminator.
Electrophoresis to Detect SSCPs
SSCP analysis and silver staining were performed according to Black and DuTeau (1997). Briefly, PCR products (2.5 µl out of 50 µl) were mixed with 4.5 µl of denaturing loading buffer and electrophoresed on large (40 cm x 50 cm), thin (0.4 mm) glycerol (5%) polyacrylamide (5%, 2% cross-linking) gels. Electrophoresis proceeded at constant current (16 mA) at room temperature for 16 h (overnight). DNA fragments were detected with silver staining.
Scoring of SSCP Gels
Amplified markers were scored directly from dried gels by measuring band mobility relative to known size markers (1 kb ladder; BRL Laboratories). To estimate sizes of amplified DNA fragments, the reciprocal of fragment size was regressed on the reciprocal of mobility (Schaffer and Sederoff 1981). RAPD markers were named by the primer designation followed by a period and the estimated size of the fragment.
The RAPD-SSCP method generates numerous bands, not all of which are repeatable. Spurious bands that appeared in a few individuals were disregarded. Mendelian segregation was tested in the remainder of the RAPD loci with the JMSLA procedure in JoinMap 2.0 (Stam and Ooijen 1995). Loci that failed to meet Mendelian expectations were discarded.
Linkage Analysis
F2 offspring genotypes were analyzed using JoinMap 2.0 (Stam and Ooijen 1995). Recombination fractions were converted to map units (cM) by the Kosambi mapping function (Kosambi 1944) using the JMREC procedure of JoinMap 2.0. Initially a minimal logarithm of odds (LOD) score of 3.0 was used to group markers. DRAWMAP 1.1 (van Ooijen 1994) was used to plot a linkage map.
Mapping Sex as a Quantitative Trait
Sex was treated as a binary, threshold trait and scored as zero (female) or one (male). Associations between genotypes at each locus and sex were initially assessed by a contingency chi-square analysis. The null hypothesis was that sexes were equal in each genotype class. Thus marginal probabilities were the frequencies of each genotype at a locus and the overall rate of each sex. The inheritance of the alleles at that locus were examined when a significant chi-square was detected.
The JoinMap linkage map and genotype/phenotype datasets were next translated into the format used for standard interval mapping (Lander and Botstein 1989). BINARYQTL (Xu et al. 1998) is a FORTRAN program for interval mapping of QTL associated with binary traits. This algorithm assumes the presence of an unobservable continuous variable (often referred to as "liability" or "threshold") that underlies the binary expression of sex. Most quantitative genetic models can be applied to threshold traits even though the threshold is distributed as an unobservable continuous quantitative trait. A probit model is used when the threshold is assumed to be normally distributed. In using this model for sex determination, we assume that below a certain liability threshold, a mosquito will develop into a female, but above that threshold it will develop into a male. BINARYQTL estimates the threshold using a single linear model with a heterogeneous residual variance and then uses standard interval mapping to determine the most probable location of QTL on the linkage map generated by JoinMap 2.0. Use of the heterogeneous residual variance model allows for calculation of the sampling variance surrounding each estimated parameter. BINARYQTL was modified to calculate 90%, 95%, and 99% comparison-wise thresholds at each centiMorgan interval and 90%, 95%, and 99% experiment-wise thresholds (Churchill and Doerge 1994).
Estimation of Variance Components
Marker genotypes were numerically scored as 0, 0.5, 1, 1.5, or 2 according to the average number of alleles inherited from the male P1 parent. Pearson correlation coefficients between sex and marker genotypes were computed using the PROC CORR procedure in SAS 8.0e. Sex (zero or one) was regressed on marker genotypes. The RSQDELTA macro in SAS 8.0e combines the information from PROC REG and PROC GLM to compute the change in R2 and the associated F statistics and P values as genotypes are added to a linear regression model. The F statistics and P values represent a partial F statistic for the general linear model.
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Results |
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Linkage Mapping
A total of 72 polymorphic RAPD-SSCP and 8 cDNA markers were identified that segregated in a Mendelian fashion. At a LOD of 4.3, JoinMap split the markers into three linkage groups. Linkage group I contained 50 markers distributed over 112 cM, while linkage group II contained 17 markers over 48 cM, and linkage group III contained 13 markers on 57 cM (Figure 2). At a LOD of 4.6, markers began to split from linkage group I. Linkage groups in Figure 2 are oriented following Anderson et al. (2001). Those authors used restriction fragment length polymorphisms (RFLPs) in 19 conserved cDNAs to orient linkage groups in six evolutionarily disparate culicine mosquito species. One of those species was O. triseriatus. Anderson et al. (2001) showed that the primary locus conditioning sex determination segregates at the top of a linkage group that, based on conservation with other culicine linkage groups, they labeled linkage group I. Furthermore, linkage group II is the shortest linkage group in Anderson et al. (2001). Linkage groups in Figure 2 are also oriented with respect to the map presented in Graham et al. (2003), in which the primary locus conditioning sex determination segregated at the top of linkage group I and linkage group II was the shortest linkage group.
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Sex-Marker Genotype Associations
Contingency chi-square analysis identified 10 marker loci in which sexes were unequally distributed in each genotype class. The locations of these markers are indicated by asterisks in Figure 2. Table 2 lists the observed numbers of each sex in each genotypic class at each of these 10 markers. An "aa" genotype indicates that an F2 inherited no alleles from the P1 father, an "ab" genotype indicates that an F2 inherited one allele from the P1 father, and a "bb" genotype indicates that an F2 inherited both alleles from the P1 father. Some genotypes were not completely informative. An "a–" indicates that the F2 individual can be either "ab" or "aa" and indicates that on average an F2 inherited 0.5 alleles from the P1 father. Similarly, a "b–" indicates that the F2 individual can be either "ab" or "bb" and indicates that on average an F2 inherited 1.5 alleles from the P1 father.
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Figure 3 plots the percentage of females as a function of the average number of alleles inherited from the P1 father. Alleles inherited through the P1 father increased the proportion of F2 females in 7 of the 10 markers. Six of these loci are located toward the top 57 cM of linkage group I (Figure 2). These patterns are unexpected based on the Gilchrist and Haldane (1947) model in which males are heterozygous for a dominant M allele that conditions male development. In the remaining three markers, alleles inherited through the P1 father decreased the proportion of F2 females. For the remainder of the analyses we assume that the QTL in the vicinity of B20.274 is the major sex-determining locus. This assumption is based on its high chi-square value (Table 2) and the observation that the dominant B20.274 allele inherited from the P1 father (genotype bb) is associated with a large excess of F2 males (30 observed, 11.5 expected; Table 2), while the recessive allele inherited from the P1 mother (genotype a–) is associated with an excess of F2 females (90 observed, 70.5 expected; Table 2).
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Sex determination was next examined with respect to the model presented in Figure 4. This model assumes that females are homozygous recessive (mm) at the putative sex locus, while males are heterozygous for a dominant allele (Mm) (Gilchrist and Haldane 1947). In this model, a and b alleles at marker loci are inherited through the P1 female and male parents, respectively. The marker locus is r cM from the sex locus at 27 cM. The expected number of marker genotypes in the female and male F2 (listed in the box at the bottom of Figure 4) are therefore a function of expected Mendelian proportions and the recombination distance between the marker and the sex locus. Expected numbers of individuals in this model are listed by genotype for each locus in Table 2. Genotypes deviated significantly (P <.001) from expectations at all 10 loci. Loci A06.2461, A20.409, A09.409, C14.404, C04.592, and C04.1286 are all located at the top of linkage group I. Loci A06.2461 and A20.409 are located above the sex locus, while A09.409, C14.404, C04.592, and C04.1286 occur below the sex locus (Figure 2). Among these six loci, 60–70 males were expected in genotypes inherited from the P1 male (b- or ab). Instead, 13–24 males were actually seen. Similarly 1–12 males were expected in genotypes inherited from the P1 female (aa) and from 16–25 males were observed. Alleles at loci A20.712, C13.1385, and C04.914 are unlinked to sex (r = 0.5). Among these loci, approximately 54 males were expected in genotypes inherited from the P1 male, but 26 males were actually seen. Similarly approximately 18 males were expected in genotypes inherited from the P1 female (aa) and from 3–11 males were actually observed. Notice, however, that female-determining alleles at A20.712 and C04.914 were inherited from the P1 female.
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QTL Mapping
The most probable locations of QTL conditioning sex were estimated with standard interval mapping using BINARYQTL (Xu et al. 1998) (Figure 5). Comparison-wise 95% thresholds were also estimated (Churchill and Doerge 1994).
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LOD values estimated for sex by standard interval mapping exceeded the comparison-wise 95% threshold in six locations at 5–21 cM, 21–33 cM, 35–41 cM, 51–56 cM, 85 cM, and 96 cM on linkage group I and at 44–48 cM on linkage group II (Figure 5). In general, the locations of QTL identified through standard interval mapping agree with locus-wise chi-square contingency tests (Figure 2). Based on the magnitude of its effect and the distribution of sexes among P1 parental genotypes, we assume that the major sex locus is in the 21–33 cM interval.
Variance Components
Pearson correlation coefficients estimated between marker genotypes and sex were significant (P 
.05) at the same loci that were statistically associated with sex in the contingency chi-square tests (Figure 2). Sex phenotypes were regressed on numbers of alleles inherited from the P1 male at the 10 marker loci (Table 3). Genotypes at these marker loci collectively accounted for 52% of the overall phenotypic variance (
2p) for sex. Considering each marker locus separately, it appears that the sex locus near B20.274 alone accounts for 41.7% of 2p for sex.
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The six QTL identified by standard interval mapping accounted for 51% of
2p for sex and B20.274 near the sex locus accounted for 74% of the overall genetic component (2g) for sex. Significant additional contributions were identified at the QTL located at 5–21 cM, 21–33 cM, 35–41 cM, and 96 cM on linkage group I. Removing B20.274 from the analysis, the remaining loci accounted for 23.7% of 2p for sex with five of the marker loci making significant additional contributions. |
Discussion |
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The results of this mapping experiment suggest that, aside from the sex locus located at
27 cM on linkage group 1, there are six loci in O. triseriatus that condition the differential survival or development of females. It is difficult to separate these two forces because the sex of O. triseriatus cannot be determined earlier than the pharate adult stage. Alleles at the one to three QTL distributed from 6–57 cM on linkage group I and proximal and distal to the sex locus, caused a marked deficiency of males (Figure 3). Deleterious recessive alleles at loci linked to the M allele at the sex locus could explain this pattern. However, it is likely that recombination over a 57 cM interval would have disrupted cosegregation between deleterious recessive alleles at these loci and the M allele at the sex locus. Furthermore, genotypes at all of the marker loci in this 57 cM interval fit Mendelian expectations. There was no differential survival among mosquitoes with alternate genotypes that would suggest the existence of recessive deleterious or lethal alleles.
Independently segregating alleles at the QTL at 95 cM at the opposite end of linkage group I also caused a significant decrease in the proportion of males. In contrast, alleles at the independently segregating loci at the QTL at 85 cM on linkage group I and at 45 cM on linkage group II caused a significant increase in the proportion of males. In all three cases it is difficult to explain these patterns based on deleterious recessive alleles associated epistatically with the M or m alleles at the sex locus.
Genetic mechanisms causing a male-biased sex ratio have previously been described in Aedes aegypti (Hickey and Craig 1966) and Culex pipiens (Sweeney and Barr 1978). In each case, a meiotic drive-based genetic model was postulated in which a single dominant distorter gene linked to the male-determining allele M rendered that linkage group more likely to participate in fertilization. However, to explain a female-biased sex ratio, a reciprocal scenario in which a distorter gene is linked to m cannot be invoked, as this would affect both sexes equally. In theory, for a "distorting" gene to bias sex ratio in favor of females in culicine mosquitoes, it would have to be unlinked to the sex locus. However, in our study, six of the markers linked to QTL affecting sex determination were located in proximity to the sex locus.
The observed distribution of genetic factors affecting sex determination instead suggests that the developmental mechanism in Aedes triseriatus may be analogous to sex determination systems that have been described in other Diptera. In houseflies, and in some other Cyclorrhaphous Diptera, male development is determined by a dominant factor M that acts as the primary sex-determining signal to prevent activity of F, a gene needed for female sexual differentiation (Hilfiker-Kleiner et al. 1993; Inoue and Hiroyoshi 1986; Nothiger and Steinmann-Zwicky 1985). In the absence of M, zygotic F is activated by maternally provided F product and is continuously required throughout development to maintain the cells on the female pathway (Dubendorfer and Hediger 1998; Hilfiker-Kleiner et al. 1993, 1994; Schmidt et al. 1997b). F alleles always map to autosome IV. In contrast, M factors are found throughout the housefly genome including the Y (Hiroyoshi 1964) and X linkage groups (Denholm et al. 1983), or on any of the five autosomes (Cakir 1996; Hiroyoshi and Inoue 1979; Inoue and Hiroyoshi 1986; Wagoner 1969). The location of the M factors on different linkage groups could represent separately evolved sex-controlling elements randomly scattered over the genome, as proposed for Chironomus thummi (Kraemer and Schmidt 1993). Alternatively, they could be of common origin and have become dispersed through transpositions.
If an analogous F/M system exists in O. triseriatus, our results suggest that genes analogous to M would be located at the sex locus on linkage group I and control the majority of sex determination. However, we would have to propose that F factors rather than M factors, as in other Diptera, have become dispersed in the O. triseriatus genome. Under this model, in the absence of M (mm genotype), zygotic F would be activated by maternal F product and female development would occur. Alleles at the various dispersed F loci would interact epistatically with M alleles to determine the ultimate sex ratio. Constitutive alleles at the F loci might cause a female-biased sex ratio. Alternatively, recessive alleles at the F loci would cause a male-biased sex ratio. In fact, M in the housefly can be overruled by an epistatic factor, FD (FDominant) (Dubendorfer et al. 1992; Rubini and Palenzona 1967), that maintains female development even in the presence of up to three M factors (Rubini et al. 1972). FD is believed to be a constitutive allele of F that escapes the repressing action of M. In addition, two recessive mutations Ftra (transformer) (Inoue et al. 1986) and Fman (masculinizer) (Schmidt et al. 1997a) lead to male development in the absence of M and are assumed to be hypomorphic alleles of F.
Ultimately sex determination in mosquitoes can be determined by many variables, from genetic mechanisms acting—sometimes in concert with environmental effects—early in development, to such late-acting factors as differential larval and pupal survival. At this time we cannot draw any conclusions as to the precise mechanism(s) causing the occurrence of excess females in O. triseriatus other than to state that the female-biased sex ratios observed in this study were not limited to a few families (Figure 1), and there appears to be a significant genetic component involved. We do not yet know whether this occurs in nature because of the difficulty in rearing O. triseriatus families from the field.
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Footnotes |
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Corresponding Editor: R. C. Woodruff
Received October 20, 2003
Accepted November 5, 2003
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