DOI: 10.1534/genetics.105.044743
No Patrigenes Required for Femaleness in the Haplodiploid Wasp
Nasonia vitripennis
Leo W. Beukeboom
1and Albert Kamping
Evolutionary Genetics, Center for Ecological and Evolutionary Studies, University of Groningen, NL-9750 AA Haren, The Netherlands Manuscript received April 22, 2005
Accepted for publication September 29, 2005
ABSTRACT
The parasitoid waspNasonia vitripennis is an emerging model organism for developmental and be-havioral genetics. It reproduces by haplodiploidy; males typically develop parthenogenetically from haploid eggs and females from fertilized diploid eggs. A polyploid mutant strain is available in which females are triploid and lay haploid and diploid eggs that normally develop into males when unfertilized. In contrast to previous reports,2% of triploid females were found to occasionally produce daughters as well as gynandromorphs from diploid unfertilized eggs. Daughter production increased with age and differed among familial lineages. This is the first report of parthenogenetic female development in Nasonia. The results show that a paternally provided genome is not required for femaleness and call for modifications of existing models of sex determination in Nasonia.
A
LL Hymenoptera (ants, bees, and wasps) have hap-lodiploid sex determination. The most common mode of reproduction is arrhenotoky;i.e., males develop from unfertilized eggs and are haploid, whereas fe-males develop from fertilized eggs and are diploid. As a consequence, males inherit genes from their mother only and have no genetic father. Thelytoky also is wide-spread among Hymenoptera. This reproductive mode refers to species that produce females parthenogenet-ically in the absence of males. Parthenogenesis is auto-mictic;i.e., haploid eggs are produced meiotically but undergo diploidy restoration by a variety of mecha-nisms (Suomalainenet al.1987). Hence, these diploideggs develop into uniparental females.
In haplodiploids there are no heteromorphic sex chromosomes; the only genetic difference between males and females is the number of chromosome sets. The question of how this difference in copy number of the genome can lead to the development of either males or females can still not be answered satisfactorily. A number of models have been proposed for sex determination in Hymenoptera and available data indicate that at least two different mechanisms exist (reviewed in Cook1993a;
Dobsonand Tanouye1998).
Whiting(1943) introducedsinglelocusc
omplemen-tarysexdetermination (sl-CSD) based on his studies of the parasitoid Bracon hebetor. Sex is determined by a single locus; heterozygotes develop into females and hemizygotes and homozygotes develop into males. This
mode of sex determination is typically detected with inbreeding crosses, which lead to diploid homozygous males (Whiting1943; Stouthameret al. 1992; Cook
1993a,b). A sl-CSD mechanism of sex determination has now been shown for .40 species of Hymenoptera (reviewed in Stouthamer et al. 1992; Cook 1993a;
Periquetet al. 1993; Butcheret al. 2000), covering all
major suborders. However, the precise phylogenetic distribution of this mode of sex determination is still unclear (Beukeboomand Pijnacker2000). Moreover,
although the sex locus has recently been character-ized from the honey bee (Beyeet al. 2003), the precise
molecular genetic mechanism of sl-CSD remains to be elucidated.
In a number of hymenopteran species inbreeding does not result in diploid males and indicates the ab-sence of a sl-CSD mechanism (Skinner and Werren
1980; Cook1993b; Beukeboomand Pijnacker2000).
sl-CSD is also incompatible with most forms of thelytoky. Peri-meiotic mechanisms that include union of meiotic products, such as central and terminal fusion, cause an increase of homozygosity. Homozygosity of thecsdgene leads to diploid males rather than females. Therefore, CSD mechanisms can be reconciled with thelytoky only if thecsdgene resides in a region that remains perma-nently heterozygous (Beukeboomand Pijnacker2000).
Wolbachia-induced thelytoky (Stouthameret al. 1990;
Braiget al. 2002) causes gamete duplication leading to
complete homozygosity and therefore cannot occur in species with CSD.
The parasitoid waspNasonia vitripennisis one of the best-studied hymenopterans genetically. It is an emerging model organism for studying complex traits (Beukeboom
and Desplan2003; Pultzand Leaf2003; Shukeret al.
1Corresponding author:Evolutionary Genetics, Center for Ecological and
Evolutionary Studies, Biological Center, University of Groningen, P.O. Box 14, NL-9750 AA Haren, The Netherlands.
E-mail: l.w.beukeboom@rug.nl
2003) because genetical analysis is greatly facilitated by haploidy of males. It has been firmly established that Nasonia does not have sl-CSD (Whiting1967; Skinner
and Werren 1980; A. Kamping and L. Beukeboom,
unpublished results). Several alternative models for sex determination in Nasonia have been proposed. They includefertilizationsexdetermination (FSD, following notation of Dobsonand Tanouye1998),maternal-effect
sexdetermination (MESD), andgenomicimprintingsex determination (GISD). Dobson and Tanouye (1998)
tested these alternative models of sex determination using a polyploid mutant strain ofN. vitripennis. Polyploid mutants of N. vitripennis have been known for a long time (Whiting1960; Macyand Whiting1969). They
appeared several times spontaneously in Whiting’s stock cultures and at least one strain has been maintained ever since. Triploid females produce, in addition to unviable aneuploid eggs, haploid and diploid eggs that typically develop into males. Diploid males are fertile and produce diploid sperm. Use of the polyploid strain enables one to vary the number of chromosome sets contributed by either parent to the embryo and to test their effect on offspring sex.
Here, we present additional results of breeding ex-periments with the polyploid mutant of N. vitripennis. We show for the first time that unfertilized diploid eggs of triploid females do not always develop into males, but can yield fertile females or gynandromorphs,i.e., male-female mosaics. Our results differ from previous studies that used this polyploid strain. Moreover, they have important implications for the mode of sex determi-nation inN. vitripennisand for the validity of different models.
MATERIALS AND METHODS
Nasonia strains and culturing: N. vitripennis is a pupal
parasitoid of cyclorraphous flies and is easily cultured in the laboratory on Protocalliphora. It has a generation time of 14 days at 25°. Virgin wasps are obtained by breaking open the host puparia a few days prior to emergence and separating male and female wasp pupae. Both sexes can easily be dis-tinguished because males have small wing buds (short wings as adults) and females have large wing buds (long wings as adults). Mated females typically produce female-biased prog-eny when provided with hosts individually. Virgin wasps lay only unfertilized eggs that develop into all-male broods.
The polyploid mutant strain originates from Whiting’s cul-tures (Whiting1960) and has been maintained ever since by
the late G. Saul (University of Vermont). It is the same strain as used by Dobsonand Tanouye(1998). It carries two recessive
eye-color mutations:oyster (oy) and scarlet(st) (Saul 1990).
Both mutations occur at a single segregation unit (‘‘R-locus’’) in which no recombination occurs (Whiting1965).
Homo-zygous and hemiHomo-zygous oyster individuals have pinkish-gray eyes andscarletindividuals have bright red eyes. Diploid het-erozygousoy1/1stindividuals have dark purple eyes similar to wild type. The females of this polyploid strain are triploid and lay haploid, diploid, and aneuploid eggs. Aneuploid eggs do not develop into adult wasps, which greatly reduces the
number of offspring of triploid females (Whiting1960). Brood
size of females can therefore be diagnostic for ploidy level: triploid females produce 0–3 offspring per fly host, whereas normal diploid females produce 20–30 offspring.
The maintenance scheme of the polyploid mutant strain is shown in Figure 1. Triploidoy1/1st/1stfemales reproduce as virgins and lay haploidoy1and1steggs as well as diploid oy1/1stand1st/1steggs, all of which typically develop into males. Diploidoy1/1stmales are crossed to standard diploid 1st/1stfemales, giving rise to triploid females again. Thus, triploid females receive the full diploid complement from their father (due to mitotic spermatogenesis) in addition to a haploid complement from their mother. Note that haploid and diploidscarletmales cannot be distinguished even though diploid males are typically larger than haploid males, but this difference is not always conclusive.
Experimental design: Previous culturing of the polyploid
mutant strain (Beukeboom 1992) suggested that unmated
triploid females very rarely produce daughters. After confirm-ing this findconfirm-ing in a pilot experiment, we systematically tested the sex of all offspring in progeny of 1200 virgin triploid fe-males. Three diploid males each were collected from eight broods of the stock culture (designated the grand paternal generation). Each male was individually mated to fivescarlet females (see Figure 1), yielding 120 progeny containing triploid females. Next, 10 virgin triploid females (designated the parental generation) were collected from each progeny and individually given four hosts at three consecutive ages, Figure1.—The polyploid mutant strain of Nasonia.oyster
0–1, 7–8, and 20–21 days, respectively. All experimental pro-cedures, including matings, culturing, and testing, were car-ried out at 25° to minimize variation in phenotypic and epigenetic effects. The number, sex, and eye color of the F1 offspring were scored.
Females and males can easily be recognized inN. vitripennis (Figure 2, A and B). Females have dark brown antennae and legs, large wings, and an ovipositor. Males have yellow antennae and legs, short wings, and no ovipositor. Close observation of the offspring was performed to identify gynandromorphs, de-fined here as having both female and male body parts (Figure 2, C and D). In gynandromorphic individuals the distribution of male and female tissues was also determined. The design of this experiment additionally allowed us to track rare female production back to the grand paternal generation and to mea-sure the effects of female age on daughter production.
If daughters were found among the F1progeny, they were bred further. Note that due to the low fecundity of triploid females leading to incomplete digestion of the fly hosts, the usual separation of male and female offspring a few days prior to emergence was not feasible. Moreover, developmental times among progeny of triploid females are slightly longer and more variable than standard progeny. Therefore, it was not always possible to obtain virgin daughters from broods of trip-loid females. If F1 females had emerged in the absence of brothers, they were hosted as virgins (three hosts for life). If they had emerged simultaneously with one or more of their haploid or diploid brothers, they were likely to be mated already. Their ploidy level (diploid or triploid) and genotype was inferred from the number, eye color, and sex of their offspring.
RESULTS
Segregation of eye-color markers:A random sample of 3805 offspring of 245 triploid virgin females was screened for eye color and ploidy level (haploid or diploid). The observed numbers of oyster, scarlet, and purple were 903 (24%), 2136 (56%), and 766 (20%), respectively (Table 1). These breeding data are broadly
consistent with previously reported ones (Conner1966;
Dobson and Tanouye 1998). Note that haploid and
diploidscarletmales cannot be distinguished unequivo-cally. Theoysterandscarletmutant eye-color markers can be treated as a single segregation unit because recom-bination does not occur between these loci. If one assumes Mendelian inheritance and equal numbers of haploid and diploid males, the expected ratios ofoyster, scarlet, and wild type are 1:3:2 (scenario 1 in Table 1; see also Figure 1). The observed numbers deviate signifi-cantly from this ratio; there is a great shortage of wild-type males. A previous study (Conner 1966) showed
that triploid females produce on average 40% diploid sons. If the expected ratios are adjusted for 40% diploids and 60% haploids, the observed numbers still deviate significantly (scenario 2 in Table 1). Conner(1966) also
reported preferential transmission of the maternal allele at a frequency of 40%vs.30% for each of the paternal alleles. If the expected ratios are also adjusted for pref-erential transmission, the observed numbers do not fit better (scenario 3 in Table 1). However, an alternative scenario based on 70% haploids and 30% diploids with-out preferential segregation fits the data well (scenario 4 in Table 1).
A decrease by age of the triploid female in the pro-portion of diploids in the offspring was observed in a random sample of 166 families and ranges from 0.225 (0–1 days) to 0.195 (7–8 days) to 0.161 (20–21 days). Pairedt-tests on the diploid frequencies of the different paternal lineages show that this decrease is significant for 0–1 daysvs.7–8 days (P,0.01) and for 0–1 daysvs. 20–21 days (P,0.05). The frequency ofpurplediploids in the offspring is highly different for the eight paternal lineages and ranges from 0.151 to 0.267 (x2-test, Yates
corrected,P,0.001).
Figure 2.—The distinction between females
Daughter and gynandromorph production: Three subsequent broods of 1200 triploid virgin females de-rived from 24 families (eight times three diploid males crossed with fivescarlet females each) were scored for female and gynandromorph production. A total of 13 females (from 11 triploid mothers: 0.92%,N¼1200) and 16 gynandromorphs (from 16 triploid mothers: 1.33%) were found among the offspring. Table 2 lists all progeny of triploid females that contained daughters or gynandromorphs. Two progeny (4a3#7 and 7b3#3) con-tained more than one daughter. All progeny with females or gynandromorphs also contained one or more diploid males that could unequivocally be determined by their purpleeye color, with two exceptions. These two prog-eny contained onlyoysterandscarletindividuals, of which the latter could be haploid or diploid.
The frequency of females and gynandromorphs dif-fered greatly among consecutive hostings (Table 3). In the first broods (0- to 1-day-old females), no females and gynandromorphs were found. In the second broods (7–8 days), 9 females and 16 gynandromorphs were found. In the third broods (20–21 days), 4 females were found, of which 2 had a mother that also produced a female in the second brood. Note that the number of third broods is lower due to premature death or aging of females. When the number of females and gynandro-morphs in each age class are corrected for the number of offspring per triploid female at different ages (see Table 2), the expected numbers are significantly differ-ent from the observed (x2-test, Yates corrected, 10.00,
d.f.¼2,P,0.01). These data show that later eggs in the oviposition sequence have a higher probability of be-coming female.
The production of females and gynandromorphs is not random. Paternal line 3 (see Table 3) has a signif-icantly higher number of gynandromorphs than the other lineages (x2-test,P,0.001). Paternal line 7 has a
significantly higher number of females than the other lineages (P,0.01). These two lineages produce a sig-nificantly higher (P , 0.001) number of females and
gynandromorphs than the others, probably indicating a genetic basis for the production of females and gynan-dromorphs from unfertilized eggs.
Table 4 compares the composition of broods (all three consecutive broods pooled) containing at least one female or gynandromorph with broods lacking any ab-errant individuals. Most females and gynandromorphs had either scarlet or purple eyes. These ratios are sig-nificantly different from the distribution among their male brothers (x2 ¼ 25.97, P , 0.001, broods with
females and gynandromorphs pooled). The overall fre-quencies (males, gynandromorphs, and females pooled) of the different eye colors in the families containing females or gynandromorphs do not differ significantly from those in the families with only males (x2¼0.59,P¼
0.74). These results indicate that females and gynan-dromorphs develop predominantly from diploid eggs. However, there were also two gynandromorphs with oystereyes reminiscent of haploidy. Note again that the ploidy level of the scarlet individuals cannot be un-equivocally determined (see Figure 1).
Of 13 uniparentally produced females, 12 were bred. Eight produced normal-sized broods with Mendelian seg-regation of the eye-color markers among sons and daugh-ters, indicating that they were diploid and that they had mated with one of their brothers. Two females produced normal-sized broods consisting entirely of males, indicat-ing that these females were unmated. Two other females did not produce progeny, which may be due to gynan-dromorphism that was not apparent from the external morphology, and one female was dead prior to the test.
None of the 16 individuals scored as gynandro-morphs produced offspring, indicating that they were sterile. This is not surprising since 15 of 16 appeared to have a male abdomen (Table 5). The phenotype of gyn-andromorphs showed a clear pattern. They shifted from being female in the anterior body parts to being pre-dominantly male in the posterior parts. All individuals had female antennae, but only one had a partial female abdomen.
TABLE 1
Observed and expected genotypes and phenotypes (eye color) among progeny of 245 virgin triploid females
Phenotype oyster scarlet purple Total
Genotype Haploidoy1 Haploid1stor
diploid1st/1st
Diploid oy1/1st
Observed no. 903 2136 766 3805
Expected ratio under scenario 1 1 3 2 x2¼341.57, d.f.¼2,P,0.001
Expected ratio under scenario 2 1.2 3.2 1.6 x2¼93.05, d.f.¼2,P,0.001
Expected ratio under scenario 3 1.07 3.42 1.51 x2¼113.07, d.f.¼2,P,0.001
Expected ratio under scenario 4 1.40 3.40 1.20 x2¼0.48, d.f.¼2, not significant
Four scenarios are used for the expected numbers in each class. Scenario 1 is based on equal ratios of haploid and diploid males and Mendelian segregation of the eye-color markers. Scenario 2 is based on 40% diploid malesvs.60% haploid males (Conner
1966) and Mendelian segregation. Scenario 3 is based on 40% diploid malesvs.60% haploid males and preferential transmission of the maternalvs.both paternal alleles in the ratio of 4:3:3 (Conner1966). Scenario 4 is based on 30% diploid malesvs.70%
These results document for the first time develop-ment of females from unfertilized diploid eggs in N. vitripennisand confirm our previous loose observations during many years of culturing of this polyploid strain. Furthermore, these parthenogenetically produced fe-males appear diploid and fully fertile, but they themselves in turn did not produce daughters parthenogenetically.
DISCUSSION
Segregation in the triploid strain:Like all Hymenop-tera,N. vitripennishas haplodiploid sex determination;
unfertilized haploid eggs develop into males and fer-tilized diploid eggs develop into females. Mutations to polyploidy have arisen spontaneously in laboratory cultures several times (Whiting1960). Triploid females
produce haploid and diploid eggs, in addition to a large proportion of aneuploid eggs resulting in a low off-spring number. Results of this study are consistent with previous reports that the proportion of diploids among the adult offspring is lower than that of haploids, but we find 30% diploids instead of 40% as previously reported (Conner1966; Dobsonand Tanouye1998). Moreover,
preferential transmission or segregation (Conner1966)
TABLE 2
Progeny of individual virgin triploid females that produce at least one female (A) or one gynandromorph (B) during life (three hostings)
Triploid mother
Males Females
oyster scarlet purple oyster scarlet purple Total
A. Progeny of virgin triploid females producing at least one female
2a5#6a 1 7 1 — — 1 10
2c3#1 2 5 3 — — 1 11
3c3#3 3 8 — — — 1 12
4a3#7 1 7 3 — — 2 13
5b3#2 3 4 3 — — 1 11
7a2#6 — 5 3 — 1 — 9
7a5#5 6 7 1 — 1 — 15
7b3#3 9 15 2 — 1 1 28
7c4#3 4 4 3 — — 1 12
Totalb 29 62 19 — 3 8 121
Triploid mother
Males Gynandromorphs
oyster scarlet purple oyster scarlet purple Total
B. Progeny of virgin triploid females producing at least one gynandromorph
1c4#7 6 21 6 — 1 — 34
2a1#1 4 2 4 — — 1 11
3a1#4 3 7 — — — 1 11
3a2#3 3 4 5 — — 1 13
3a3#2 1 3 2 — 1 — 7
3b2#5 4 9 1 — 1 — 15
3b5#5 2 11 5 1 — — 19
3c1#3 6 18 4 — 1 — 29
3c2#7 7 19 4 — — 1 31
3c3#8 7 9 3 — — 1 20
4a2#10 1 10 1 — — 1 13
4b1#3 3 6 1 — — 1 11
4b4#1 4 10 3 — 1 — 18
5b2#6 6 10 3 — — 1 20
7a2#5 1 8 2 1 — — 12
7c2#8 1 9 5 — 1 — 16
Total 59 156 49 2 6 8 280
Eye color is informative for ploidy level:oysteris haploid,scarletis haploid or diploid, andpurpleis diploid (see Figure 1).
aFemale code 2a5#6 indicates paternal line 2 (#1–8), father a (a, b, or c), mother 5 (#1–5), female number 6
(#1–10).
bTwo additional females, a wild type and ascarlet, respectively, were found in the progeny of mothers 3a3#7
needs not be invoked to explain our results. Molecular marker studies indicate that recombination rates in triploid females are comparable to diploid females (L. W. Beukeboom, unpublished results).
Female and gynandromorph production: Until now
unfertilized haploid and diploid eggs ofN. vitripennis were reported to always develop into males (Conner
1966; Beukeboom1992; Dobsonand Tanouye 1998).
Using one of Whiting’s original strains, we now have shown that unfertilized diploid eggs occasionally de-velop into females or gynandromorphs. These unipa-rentally produced females are diploid, have normal fertility, and appear in no way different from normally produced females from fertilized eggs.
A number of important observations require expla-nation: (1)2% of all females produced one or more daughters or a gynandromorph; (2) all females and almost all gynandromorphs have the purple or scarlet phenotype, indicating that they are diploid; (3) the frequency of diploid eggs decreases with female age; (4) the frequency of females and gynandromorphs in-creases with age; (5) the frequency of females and gynandromorphs differs significantly between lineages; (6) gynandromorphs show an anterior-posterior gra-dient in femaleness; and (7) progeny with females or gynandromorphs also contained one or more diploid males. The rare production of daughters is discussed below in relation to the mode of sex determination.
TABLE 3
Females and gynandromorphs in the offspring of 1200 virgin triploid females at different ages in eight paternal lines (150 females/line)
Age of female
0–1 day 7–8 days 20–21 days Total
Paternal line F G F G F G F G
1 0 0 0 1 0 0 0 1
2 0 0 2 1 0 0 2 1
3 0 0 1 8 2 0 3 8
4 0 0 1 3 1 0 2 3
5 0 0 1 1 0 0 1 1
6 0 0 0 0 0 0 0 0
7 0 0 4 2 1 0 5 2
8 0 0 0 0 0 0 0 0
Total 0 0 9 16 4 0 13 16
Average progeny no.a(no. of females) 4.67 (n¼166) 1.01 (n¼166) 3.0 (n¼48)b
F, females; G, gynandromorphs.
aBased on a subsample of the 1200 females. bBased on 48 of 166 females that were still alive.
TABLE 4
Offspring of virgin triploid females per family type of only males, males and females, or males and gynandromorphs
Offspring type
Total
Family type oyster scarlet purple
Only malesa
No. of males (proportion) 664 (0.242) 1528 (0.556) 555 (0.202) 2747
Females and males
No. of females (proportion) 0 4 (0.308) 9 (0.692) 13
No. of males (proportion) 29 (0.264) 62 (0.563) 19 (0.173) 110
Total no. of offspring (proportion) 29 (0.236) 66 (0.536) 28 (0.228) 123
Gynandromorphs and males
No. of gynandromorphs (proportion) 2 (0.125) 6 (0.375) 8 (0.5) 16
No. of males (proportion) 59 (0.223) 156 (0.591) 49 (0.186) 264
Total no. of offspring (proportion) 61 (0.217) 162 (0.579) 57 (0.203) 280
Proportions refer to the relative frequencies of each eye color within a sex.
a
Daughter production increases with the mother’s age, but the frequency of diploid eggs decreases with age. Therefore, daughter production not only is the result of an increase in the production of diploid eggs by age, but also implies that these two phenomena are at least partially independent. It suggests that daughter produc-tion is partially under maternal control and/or under control of genes that have age-dependent expression. The low frequency of virgin triploid females that produce females can be increased by changing culture conditions. In a pilot experiment we found a strong increase for female and gynandromorph production at high temper-ature, providing further evidence for a maternal effect.
Only diploid eggs appear to undergo a sex switch. This indicates that the ploidy level of the egg plays an important role in sex determination. In this respect it would be informative to know the ploidy level of the gynandromorphs. They could be ordered haplodiploid mosaics (i.e., consisting of haploid male and diploid female tissues), as described for other hymenopterans (Whitinget al. 1934; Grosch1988; Halstead1988),
or intersexes (i.e., with incompletely differentiated gen-der). The fact that our gynandromorphs descend from uninseminated females rules out any mechanism in-volving fertilization, such as polyspermy or fertilization of binucleate eggs (Stern1968; Pettersand Grosch
1976). Our results suggest that most gynandromorphs are diploid. We found only two gynandromorphs with oystereyes, which is a recessive mutation, indicating that (at least) their eye tissue was haploid. Moreover, our breeding results show that all fertile uniparental females are diploid (not haploid). Haploidy of females has never been described in the Hymenoptera although Whiting
et al. (1934) reported a type of sex intergrade, consisting of two types of genetically male tissue that interact to produce feminization. The higher frequency of femini-zation in anteriorvs.posterior body parts in our gynan-dromorphs suggests that some sort of gradient exists in female induction. We are currently investigating ploidy levels of the gynandromorphs to reveal the mechanism of their origin.
Another finding requiring explanation is the signif-icantly higher proportions of females and gynandro-morphs in some maternal and grand-paternal lineages as well as the fact that some females produce more than one daughter in addition to diploid sons. Uniparentally produced females, however, do not themselves produce females or diploid males parthenogenetically. This rules out a dominant mutation that is transmitted over gener-ations. The data rather suggest the occurrence of a rare event in the germline of some males that is partially manifested (e.g., semidominant) in some of their daugh-ters. The expression of the trait varies within the triploid females as well, given that not all of their diploid off-spring were transformed into females. Our data there-fore appear most easily explained by invoking a dosage effect and/or an epigenetic mutation. Epigenetic in-heritance of traits has been invoked for Nasonia before (Poirie´ et al. 1992; Beukeboom1995). Epigenetic
con-trol over inheritance is now well established although much of the underlying mechanisms remain to be uncovered (Constancia et al. 1998; Lyko and Paro
1999; Vuand Hoffman2000). Genomic imprints usually
undergo cyclic application and erasure within single generations, but Morganet al. (1999) showed that the
epigenetically inherited agouti locus in mice can retain its imprint for several generations through incomplete erasure of the modification in the female germline. Al-though purely speculative at this moment, it is not in-conceivable that a similar process is responsible for the results found in this study. Such a scenario would im-plicate that one may be able to artificially select for the trait ‘‘occasional parthenogenetic female production.’’ This is what we are currently attempting.
Implications for sex determination:Our recovery of diploid daughters and gynandromorphs from unfertil-ized triploid females indicates that the sex-determining mechanism in Nasonia is most likely more complicated than the existing models predict. Different models have been reviewed by Cook(1993a) and Dobsonand
Tanouye(1998). According to the FSD model (Whiting
1960), sex is determined by fertilization, not by ploidy
TABLE 5
Phenotypes of gynandromorphs
Eye color
Body part oyster scarlet purple
Antennae F F F F F F F F F F F F F F F F F F F F F F F F
Fore Wings F F F F F F F F F F F F F F F F/M F F F F F F F F
Hind wings F F F F F F F F M M F F F F F/M F/M F F F F F F M M
Front legs M M F F F F F F F F M M M M F/M F/M F F F F F F F M
Middle legs M M M M F F F M M F/M M M M M M M F F F F F M M M
Hind legs M M M M F F/M M F/M M F/M M M M M M M F F F M F M M M
Abdomen M M M M M F M M M M F/M M
No. of individuals 1 1 1 1 1 1 1 1 5 1 1 1
level;i.e., fertilized eggs develop into females and un-fertilized eggs into males. Our results now show that female development can also occur without fertilization inN. vitripennis. Together with female development under thelytokous reproduction, which is known from many Hymenoptera, these data discard the FSD model. This model also had previously been rejected on the basis of studies with the paternal sex ratio chromosome, which showed that fertilized eggs can develop into males (Werrenet al. 1987; Nur et al. 1988; Beukeboomand
Werren1993; Dobsonand Tanouye1998).
The GISD model (Poirie´ et al. 1992; Beukeboom
1995) proposes that paternally and maternally derived genes function differently in sex determination and that at least one paternally inherited set of chromosomes is required for female development. This model was prompted by breeding data of the polyploid strain used in this study. Diploid eggs containing two maternally derived chromosome sets were found to develop into males, whereas diploid eggs with one maternal and one paternal set (derived from fertilization of haploid eggs of triploid females) resulted in females. This led Dobson
and Tanouye(1998) to consider GISD as the only model
consistent with all available data. However, we now have shown that diploid eggs without patrigenes can also develop into females that cannot be accounted for easily by GISD. We therefore conclude that sex determination in Nasonia is more complicated than any of the existing models predict.
Stouthamerand Kazmer(1994) proposed that
trip-loid females carry a nonfunctional variant (mutation) of a locus involved in sex determination;i.e., triploid females are functionally diploid and diploid males are functionally haploid. Assuming Mendelian segregation of this mutation in the polyploid strain, two-thirds of diploid eggs are expected to carry one mutated and one intact copy of the sex locus and develop into diploid males. The other one-third are expected to receive two intact copies of the sex locus, resulting in female devel-opment. Our data do not support these predictions because only a very small fraction (,1%) of triploid daughters of a particular diploid father produces females or gynandromorphs. Preferential segregation (Conner
1966) of the mutation to diploid eggs can also be ex-cluded since the ratio of haploid and diploid individuals in the progeny of these ‘‘exceptional’’ triploid females is similar to that of a random sample of virgin triploid females.
Although the proportion of virgin triploid females that produce daughters is very low, the presence of a diploid daughter in the offspring of an ‘‘affected’’ mother is not a sporadic event. The frequency of daughters among their diploid offspring does not significantly deviate from one-third (Table 2). This could be ex-plained simply by a single dominant mutation that oc-curred in the germline of these females. However, our data also show a difference between paternal lineages in
production of diploid females and gynandromorphs through their virgin triploid daughters. Moreover, the data show that female and gynandromorph production are largely independent events. These observations in-dicate that sex-determining factors are polymorphic in the polyploid strain and suggest that at least two seg-regating genes are involved,e.g., a sex-determining gene and a gene that modifies its expression. Recombination in triploid females appears to be completely random (L. Beukeboom, unpublished results). The absence of
females and gynandromorphs in the offspring of virgin diploid F2daughters in turn indicates that the ploidy level of the mother also plays a role in the production of daughters from unfertilized eggs.
The mode of reproduction ofN. vitripennisis called arrhenotoky, which is the common form among hap-lodiploid Hymenoptera. Some species are thelytokous and produce only daughters from haploid eggs that have undergone a diploidy restoration process. Although we have now shown uniparental production of females in N. vitripennis, the mechanism by which these females arise is different from thelytokous species. They develop from unfertilized diploid eggs that result from a reduc-tion division without any subsequent ploidy increase. This is still the consequence of arrhenotokous repro-duction, where it is typically the males that arise by automictic parthenogenesis. Polyploid Hymenoptera with this mode of reproduction are not known from nature. The mechanism is most reminiscent of pre-meiotic doubling thelytoky, whereby the diploid chro-mosome complement is doubled before meiosis and the meiotic products are diploid (Suomalainenet al.
1987). This has been reported for a few hymenop-terans (Doncaster1916; Dodds 1939; Peacock and
Sanderson1939; Ledoux1954).
In conclusion, for the first time we showed unipa-rental development of females in N. vitripennis. The used polyploid mutant strain proves very useful for manipulating the number of paternally and maternally transmitted chromosome sets. Occasional daughter de-velopment from eggs without a paternal chromosome complement poses a new challenge for the existing models of sex determination and additional modifica-tions seem to be required. Our results will contribute to the long-term goal of elucidating the genetic regulation of haplodiploid sex determination. Further study of gynandromorphic individuals, such as obtaining data on ploidy level and dosage compensation, appears par-ticularly promising.
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