Journal of Heredity 2004:95(1)
© 2004 The American Genetic Association 95:81-84
Brief Communication |
Estimation of Allele Numbers at the Sex-Determining Locus in a Field Population of the Turnip Sawfly (Athalia rosae)
From the Division of Molecular Science, Graduate School of Science and Technology, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan (Fujiwara), and Department of Applied Molecular Biosciences, Graduate School of Agriculture, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan (Akita, Okumura, Kodaka, Tomioka, and Naito).
Address correspondence to Yoshihiro Fujiwara at the address above, or e-mail: fwara{at}scitec.kobe-u.ac.jp.
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Abstract |
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Hymenopteran insects (sawflies, ants, bees, and wasps) have an unusual genetic system called haplodiploidy, where parthenogenetically produced haploid eggs become males, and fertilized, diploid eggs become females. Several hypotheses have been proposed to explain the mechanism of such sex determination, including control at a single polymorphic locus. From experiments of mother-son mating and using a genetic marker, we show that a single multiallele locus controls sex determination in the turnip sawfly (Athalia rosae). We estimated the number of alleles at this single locus in a field population by analyzing the rate of diploid males in the field and the rate of diploid males by random crossing in the laboratory. Only one diploid male was discovered in 1306 diploid larvae collected in the field. However, the number of alleles calculated by random crossing in the laboratory was 45–50. We suggest that the effective population size may be much larger than that from the areas where we collected larvae, and that there are mechanisms for avoiding inbreeding, including protogyny, dispersion, and sperm displacement by second-mated males.
Hymenopteran insects have a genetic determination system called haplodiploidy, where fertilized, diploid eggs become females, and unfertilized, haploid eggs become males. A single-locus complementary sex determination (CSD) system has long been proposed for the wasp Bracon hebetor (Whiting 1943) and extrapolated to explain sex determination in other hymenopterans (Cook 1993). In this system, the single sex locus has multiple alleles; heterozygotes become females and hemi- and homozygotes become males. The production of diploid males by sibmating is the indication of this system. In hymenopterans that produce few or no diploid males by inbreeding, the multilocus CSD system (Crozier 1971) or genetic imprinting theory (Dobson and Tanouye 1998) have been proposed to explain their sex determination mechanism.
Diploid males produce diploid sperm, and crossing with normal diploid females produces triploid progenies. However, triploid females are sterile because they produce aneuploid gametes. Consequently the diploid males are regarded as a genetic load. Therefore mechanisms to avoid the production of diploid males and triploid females have evolved in hymenopterans that have a single-locus CSD. For example, inbreeding was avoided by premating dispersion in Bombus atratus (Plowright and Pallett 1979) and B. hebetor (Ode et al. 1995). The diploid males of some species are reported to have low viability or low fertility (Holloway et al. 1999; Inaba 1939; Smith and Wallace 1971; Whiting 1961).
The turnip saw fly (Athalia rosae) is a potential pest of cruciferous crops and is also attractive as a model insect for the study of egg activation, fertilization, meiotic division, and sex determination (Oishi et al. 1993). The single-locus CSD system has been used to explain the sex determination mechanism of A. rosae (Naito and Suzuki 1991). In this species, fertile diploid males are produced from the half crossing of sibmating. Triploid males and females are further produced from sibmating between diploid males and females (Naito and Suzuki 1991). The viability and fertility of diploid males are high in A. rosae (Naito and Suzuki 1991), so there must be some other mechanism for avoiding the production of diploid males. One possible solution is an increase in the allele numbers for the sex-determining locus in field populations (Cook and Crozier 1995), since the production rate of diploid males will decrease as the number of alleles increases in the single-locus CSD system. Theoretically the equilibrium allele number increases with population size (Cook and Crozier 1995).
Sex allele numbers in field populations have been estimated for several hymenopterans (Cook and Crozier 1995); at least nine for B. hebetor (Whiting 1961), 11–19 for Apis mellifera, and 46 for the bumblebee, Bombus terrestris (Duchateau et al. 1994). In a study of Solenopsis, more than 86 alleles were estimated in a natural Argentinean population and 10–13 were estimated in an American population, introduced by founder effects (Ross et al. 1993).
In this article we confirm that A. rosae fits the single-locus, multiallele model using mother-son mating and marker-gene experiments. We estimate the number of alleles at the sex locus in a field population by determining the frequencies of diploid males from a field population and from random crossings in the laboratory. In this context, the mechanisms for avoiding the production of diploid males are discussed.
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Materials and Methods |
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Collection, Rearing, and Ploidy Determination of A. rosae
We collected 1880 larvae of A. rosae ruficornis Jakovlev from radish fields in Hyogo and Osaka prefectures, Japan, from August 1986 to September 1991. The larvae were reared as described previously (Naito and Suzuki 1991). Ploidy can be estimated from body size and was confirmed by cytological examination, as described previously (Naito and Suzuki 1991).
Mating and Crossing Experiments
Mating methods have also been described previously (Naito and Suzuki 1991). Leaves of a Verbenaceae plant, Clerodendron trichotomum, or Labiatae, Ajuga reptans, were provided to activate mating.
For mother-son mating experiments, 18 virgin females were maintained in a refrigerator at 5°C after laying several eggs. After the offspring eclosed about 1 month later, the mothers were mated with their sons, and the sex and ploidy of the progeny were analyzed.
The yellow fat body (yfb) mutation (Sawa and Oishi 1989b) of fat body pigment, the color of which can be distinguished between hemi/homozygous and heterozygous individuals at the pupal stage, was used as a marker gene for a crossing experiment. A +/yfb female and a wild-type male were mated to produce yfb males and +/yfb females. The F1 yfb males and their sister +/yfb females were crossed, and the phenotypes and ploidy of their progeny were analyzed.
Sex Allele Number Determination
To estimate the number of sex alleles and identify the number of diploid males in a field population, a total of 1880 larvae were collected and reared in our laboratory and their sex and ploidy were analyzed. Allele number in the field was estimated according to Adams et al. (1977).
For allele number determination by crossings, four independent experiments were performed from 1986 to 1991. In each experiment, larvae were collected in isolated radish fields, males and females were crossed randomly, and the sex and ploidy of progeny and the number of alleles were analyzed for each field. Allele number determination by crossing experiments in the laboratory was calculated according the equation of Laidlaw et al. (1956), n = 2(N + 1)/H + 1, where n is the number of sex alleles, H is the number of crosses to yield diploid males, and N is the total number of crosses.
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Results and Discussion |
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Confirmation of Single-Locus CSD System in A. rosae
In a previous study, diploid males were produced in half of the crossings of sibling single-pair matings (Naito and Suzuki 1991), indicating that A. rosae has a single-locus, multiallele sex determination system. If Naito and Suzuki's (1991) findings hold, then all mother-son crossings should produce approximately equal proportions of diploid males and females. Of the 18 female sawflies kept for 1 month at 5°C, 7 survived and 4 produced more than 10 diploid progenies. The latter four females all produced diploid males in approximate parity with the number of diploid females (Table 1).
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Virgin A. rosae females produce diploid progeny under some unfavorable conditions, but only rarely (Sawa and Oishi 1989a). In order to reject the possibility that sibmating activates such automictic parthenogenesis, and to confirm that diploid individuals have two sets of chromosomes, one from the mother and one from the father, the yfb marker gene was used in crossing experiments. This approach has been used to confirm the biparental nature of diploid males in A. mellifera (Woyke 1965) and B. hebetor (Whiting 1961). The data from the only F1 crosses between a yfb male and a +/yfb female to produce diploid males are presented in Table 2; yfb/yfb diploid progeny were produced and almost half of them were diploid males. This result indicates that diploid males are not the result of automictic parthenogenesis (Sawa and Oishi 1989a) and that they have genes from both parents.
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Occurrence of Diploid Males in a Field Population
To estimate the number of sex alleles and identify the number of diploid males in a field population, of the 1880 larvae collected from radish fields and reared in our laboratory, 1306 were diploid and only one was male (Table 3). Although the number of diploid males is too small for significance testing, we tentatively estimated the number of sex alleles in the field population (Adams et al. 1977). Assuming that the viability of diploid males is comparable to that of diploid females (Naito and Suzuki 1991), the number of sex alleles is 1306.
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Estimation of Sex Allele Numbers by Random Crossing
In the analysis of 73–157 crosses from four different random crossings of field-collected individuals, two to six diploid male-producing lines were obtained (Table 4). Allele numbers were calculated as ranging from 45.1 to 49.3, with a standard deviation of 16.6 to 27.7 (Laidlaw et al. 1956).
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The Mechanisms for Avoiding Production of Diploid Males
Based on laboratory experiments, we estimated that there are about 50 sex alleles in A. rosae. If this estimate is accurate, 1 in 50 diploids should be male in the field population. However, we observed only one diploid male among 1306 diploid larvae in the field populations (Table 3). This discrepancy may be caused by unique characteristics of diploid males: significantly lower viability in the field than for females, or differential behavior leading to lower collection success of diploid males. Although we cannot deny these possibilities, the viability of diploid males in the laboratory is only slightly lower than for females (Naito 1989), and we did not observe diploid male-specific behavior.
One possible explanation for the low occurrence of diploid males in the field is that the effective population size far exceeds the area of the field in which we collected larvae and therefore the allele number in the field would be much higher than 50. Even if A. rosae has many more alleles and a much larger effective population size than we estimated here, it seems inevitable that some diploid males would be produced by sibmating close to the emergence site. Possible strategies for inbreeding avoidance are dispersal before mating and kin recognition (Cook and Crozier 1995). In some other hymenopteran species, inbreeding is avoided by premating dispersion (Ode et al. 1995; Plowright and Pallett 1979). In fact, A. rosae deposit female eggs earlier than male eggs (data not shown) and the total developmental period for A. rosae haploid males is longer than for females (protogyny), in spite of their small size (Naito 1989). In addition, in contrast with other insects, adult female A. rosae have a greater tendency to disperse than males, particularly in summer (Nagasaka 1992). Furthermore, sperm displacement by second-mated males (Shigemura and Naito 1999) allows the potential removal of sperm from a sibling mating at the emergence site and puts in place another safeguard against sibmating in this species.
We need to collect larger numbers of larvae from the field and conduct more detailed field studies to precisely estimate the actual population size and sex allele number over the range of A. rosae.
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Footnotes |
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Corresponding Editor: Rob DeSalle
Received March 23, 2003
Accepted August 30, 2003
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References |
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