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No patrigenes required for femaleness in the haplodiploid Beukeboom, L.W.; Kamping, A.

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DOI: 10.1534/genetics.105.044743

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Citation for published version (APA): Beukeboom, L. W., & Kamping, A. (2006). No patrigenes required for femaleness in the haplodiploid wasp Nasonia vitripennis. Genetics, 172(2), 981-989. https://doi.org/10.1534/genetics.105.044743

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No Patrigenes Required for Femaleness in the Haplodiploid Wasp Nasonia vitripennis

Leo W. Beukeboom1 and 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 wasp Nasonia vitripennis is an emerging model organism for developmental and be- havioral genetics. It reproduces by ; 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 determination in Nasonia.

LL (, , and ) have hap- mode of sex determination is typically detected with A lodiploid sex determination. The most common inbreeding crosses, which lead to diploid homozygous mode of reproduction is arrhenotoky; i.e., males develop males (Whiting 1943; Stouthamer et al. 1992; Cook from unfertilized eggs and are haploid, whereas fe- 1993a,b). A sl-CSD mechanism of sex determination males develop from fertilized eggs and are diploid. As a has now been shown for .40 species of Hymenoptera consequence, males inherit genes from their mother (reviewed in Stouthamer et al. 1992; Cook 1993a; only and have no genetic father. also is wide- Periquet et al. 1993; Butcher et al. 2000), covering all spread among Hymenoptera. This reproductive mode major suborders. However, the precise phylogenetic refers to species that produce females parthenogenet- distribution of this mode of sex determination is still ically in the absence of males. is auto- unclear (Beukeboom and Pijnacker 2000). Moreover, mictic; i.e., haploid eggs are produced meiotically but although the sex locus has recently been character- undergo diploidy restoration by a variety of mecha- ized from the honey (Beye et al. 2003), the precise nisms (Suomalainen et al. 1987). Hence, these diploid molecular genetic mechanism of sl-CSD remains to be eggs develop into uniparental females. elucidated. In haplodiploids there are no heteromorphic sex In a number of hymenopteran species inbreeding chromosomes; the only genetic difference between males does not result in diploid males and indicates the ab- and females is the number of chromosome sets. The sence of a sl-CSD mechanism (Skinner and Werren question of how this difference in copy number of the 1980; Cook 1993b; Beukeboom and Pijnacker 2000). genome can lead to the development of either males or sl-CSD is also incompatible with most forms of thelytoky. females can still not be answered satisfactorily. A number Peri-meiotic mechanisms that include union of meiotic of models have been proposed for sex determination in products, such as central and terminal fusion, cause an Hymenoptera and available data indicate that at least increase of homozygosity. Homozygosity of the csd gene two different mechanisms exist (reviewed in Cook 1993a; leads to diploid males rather than females. Therefore, Dobson and Tanouye 1998). CSD mechanisms can be reconciled with thelytoky only Whiting (1943) introduced single locus complemen- if the csd gene resides in a region that remains perma- tary sex determination (sl-CSD) based on his studies of nently heterozygous (Beukeboom and Pijnacker 2000). the parasitoid Bracon hebetor. Sex is determined by a Wolbachia-induced thelytoky (Stouthamer et al. 1990; single locus; heterozygotes develop into females and Braig et al. 2002) causes gamete duplication leading to hemizygotes and homozygotes develop into males. This complete homozygosity and therefore cannot occur in species with CSD. The parasitoid wasp Nasonia vitripennis is one of the 1Corresponding author: Evolutionary Genetics, Center for Ecological and best-studied hymenopterans genetically. It is an emerging Evolutionary Studies, Biological Center, University of Groningen, P.O. eukeboom Box 14, NL-9750 AA Haren, The Netherlands. model organism for studying complex traits (B E-mail: [email protected] and Desplan 2003; Pultz and Leaf 2003; Shuker et al.

Genetics 172: 981–989 (February 2006) 982 L. W. Beukeboom and A. Kamping

2003) because genetical analysis is greatly facilitated by haploidy of males. It has been firmly established that Nasonia does not have sl-CSD (Whiting 1967; Skinner and Werren 1980; A. Kamping and L. Beukeboom, unpublished results). Several alternative models for sex determination in Nasonia have been proposed. They include fertilization sex determination (FSD, following notation of Dobson and Tanouye 1998), maternal-effect sex determination (MESD), and sex determination (GISD). Dobson and Tanouye (1998) tested these alternative models of sex determination using a polyploid mutant strain of N. vitripennis.Polyploid mutants of N. vitripennis have been known for a long time (Whiting 1960; Macy and Whiting 1969). 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. Figure 1.—The polyploid mutant strain of Nasonia. oyster Here, we present additional results of breeding ex- (pink eyes) and scarlet (red eyes) are recessive complementing periments with the polyploid mutant of N. vitripennis. eye-color mutations that occur at a single segregation unit We show for the first time that unfertilized diploid eggs (‘‘R-locus’’) in which no recombination occurs. Virgin triploid of triploid females do not always develop into males, but oy 1/1 st/1 st females are designated as the parental gener- ation in the experiments. They lay haploid oy 1 and 1 st eggs can yield fertile females or gynandromorphs, i.e., male- as well as diploid oy 1/1 st and 1 st/1 st eggs, which typically female mosaics. Our results differ from previous studies develop into males. Diploid oy 1/1 st males can be distin- that used this polyploid strain. Moreover, they have guished by their purple (wild type) eyes and produce fertile important implications for the mode of sex determi- diploid sperm. They are crossed with 1 st/1 st females of a nation in N. vitripennis and for the validity of different standard stock, resulting in triploid females again. models. number of offspring of triploid females (Whiting 1960). Brood size of females can therefore be diagnostic for level: MATERIALS AND METHODS triploid females produce 0–3 offspring per fly host, whereas normal diploid females produce 20–30 offspring. Nasonia strains and culturing: N. vitripennis is a pupal The maintenance scheme of the polyploid mutant strain is parasitoid of cyclorraphous flies and is easily cultured in the shown in Figure 1. Triploid oy 1/1 st/1 st females reproduce laboratory on Protocalliphora. It has a generation time of 14 as virgins and lay haploid oy 1 and 1 st eggs as well as diploid days at 25°. Virgin wasps are obtained by breaking open the oy 1/1 st and 1 st/1 st eggs, all of which typically develop into host puparia a few days prior to emergence and separating males. Diploid oy 1/1 st males are crossed to standard diploid male and female wasp pupae. Both can easily be dis- 1 st/1 st females, giving rise to triploid females again. Thus, tinguished because males have small wing buds (short wings triploid females receive the full diploid complement from as adults) and females have large wing buds (long wings as their father (due to mitotic spermatogenesis) in addition to a adults). Mated females typically produce female-biased prog- haploid complement from their mother. Note that haploid eny when provided with hosts individually. Virgin wasps lay and diploid scarlet males cannot be distinguished even though only unfertilized eggs that develop into all-male broods. diploid males are typically larger than haploid males, but this The polyploid mutant strain originates from Whiting’s cul- difference is not always conclusive. tures (Whiting 1960) and has been maintained ever since by Experimental design: Previous culturing of the polyploid the late G. Saul (University of Vermont). It is the same strain as mutant strain (Beukeboom 1992) suggested that unmated used by Dobson and Tanouye (1998). It carries two recessive triploid females very rarely produce daughters. After confirm- eye-color mutations: oyster (oy) and scarlet (st)(Saul 1990). ing this finding in a pilot experiment, we systematically tested Both mutations occur at a single segregation unit (‘‘R-locus’’) the sex of all offspring in progeny of 1200 virgin triploid fe- in which no recombination occurs (Whiting 1965). Homo- males. Three diploid males each were collected from eight zygous and hemizygous oyster individuals have pinkish-gray broods of the stock culture (designated the grand paternal eyes and scarlet individuals have bright red eyes. Diploid het- generation). Each male was individually mated to five scarlet erozygous oy 1/1 st individuals have dark purple eyes similar females (see Figure 1), yielding 120 progeny containing to wild type. The females of this polyploid strain are triploid triploid females. Next, 10 virgin triploid females (designated and lay haploid, diploid, and aneuploid eggs. Aneuploid eggs the parental generation) were collected from each progeny do not develop into adult wasps, which greatly reduces the and individually given four hosts at three consecutive ages, Females From Unfertilized Diploid Eggs in Nasonia 983

Figure 2.—The distinction between females and males in N. vitripennis. (A) Females have dark-brown antennae and legs, large wings, and an ovipositor. (B) Males have yellow antennae and legs, short wings, and no ovipositor. (C and D) Gynandromorphs have features of both sexes. Black arrows indicate femaleness; red arrows, maleness.

0–1, 7–8, and 20–21 days, respectively. All experimental pro- consistent with previously reported ones (Conner 1966; cedures, including matings, culturing, and testing, were car- Dobson and Tanouye 1998). Note that haploid and ried out at 25° to minimize variation in phenotypic and diploid scarlet males cannot be distinguished unequivo- epigenetic effects. The number, sex, and eye color of the F1 offspring were scored. cally. The oyster and scarlet mutant eye-color markers can Females and males can easily be recognized in N. vitripennis be treated as a single segregation unit because recom- (Figure 2, A and B). Females have dark brown antennae and bination does not occur between these loci. If one legs, large wings, and an ovipositor. Males have yellow antennae assumes Mendelian inheritance and equal numbers of and legs, short wings, and no ovipositor. Close observation of haploid and diploid males, the expected ratios of oyster, the offspring was performed to identify gynandromorphs, de- fined here as having both female and male body parts (Figure scarlet, and wild type are 1:3:2 (scenario 1 in Table 1; see 2, C and D). In gynandromorphic individuals the distribution also Figure 1). The observed numbers deviate signifi- of male and female tissues was also determined. The design of cantly from this ratio; there is a great shortage of wild- this experiment additionally allowed us to track rare female type males. A previous study (Conner 1966) showed production back to the grand paternal generation and to mea- that triploid females produce on average 40% diploid sure the effects of female age on daughter production. sons. If the expected ratios are adjusted for 40% diploids If daughters were found among the F1 progeny, they were bred further. Note that due to the low fecundity of triploid and 60% haploids, the observed numbers still deviate females leading to incomplete digestion of the fly hosts, the significantly (scenario 2 in Table 1). Conner (1966) also usual separation of male and female offspring a few days prior reported preferential transmission of the maternal allele to emergence was not feasible. Moreover, developmental times at a frequency of 40% vs. 30% for each of the paternal among progeny of triploid females are slightly longer and more variable than standard progeny. Therefore, it was not alleles. If the expected ratios are also adjusted for pref- always possible to obtain virgin daughters from broods of trip- erential transmission, the observed numbers do not fit loid females. If F1 females had emerged in the absence of better (scenario 3 in Table 1). However, an alternative brothers, they were hosted as virgins (three hosts for life). If they scenario based on 70% haploids and 30% diploids with- had emerged simultaneously with one or more of their haploid out preferential segregation fits the data well (scenario 4 or diploid brothers, they were likely to be mated already. Their ploidy level (diploid or triploid) and genotype was inferred in Table 1). from the number, eye color, and sex of their offspring. 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). RESULTS Paired t-tests on the diploid frequencies of the different Segregation of eye-color markers: A random sample paternal lineages show that this decrease is significant of 3805 offspring of 245 triploid virgin females was for 0–1 days vs. 7–8 days (P , 0.01) and for 0–1 days vs. screened for eye color and ploidy level (haploid or 20–21 days (P , 0.05). The frequency of purple diploids diploid). The observed numbers of oyster, scarlet, and in the offspring is highly different for the eight paternal purple were 903 (24%), 2136 (56%), and 766 (20%), lineages and ranges from 0.151 to 0.267 (x2-test, Yates respectively (Table 1). These breeding data are broadly corrected, P , 0.001). 984 L. W. Beukeboom and A. Kamping

TABLE 1 Observed and expected genotypes and phenotypes (eye color) among progeny of 245 virgin triploid females

Phenotype oyster scarlet purple Total Genotype Haploid oy 1 Haploid 1 st or Diploid diploid 1 st / 1 st oy 1 / 1 st Observed no. 903 2136 766 3805 Expected ratio under scenario 1 1 3 2 x2 ¼ 341.57, d.f. ¼ 2, P , 0.001 Expected ratio under scenario 2 1.2 3.2 1.6 x2 ¼ 93.05, d.f. ¼ 2, P , 0.001 Expected ratio under scenario 3 1.07 3.42 1.51 x2 ¼ 113.07, d.f. ¼ 2, P , 0.001 Expected ratio under scenario 4 1.40 3.40 1.20 x2 ¼ 0.48, d.f. ¼ 2, 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 males vs. 60% haploid males (Conner 1966) and Mendelian segregation. Scenario 3 is based on 40% diploid males vs. 60% haploid males and preferential transmission of the maternal vs. both paternal alleles in the ratio of 4:3:3 (Conner 1966). Scenario 4 is based on 30% diploid males vs. 70% haploid males and Mendelian segregation. Scenario 4 fits the data the best by far.

Daughter and gynandromorph production: Three gynandromorphs than the others, probably indicating a subsequent broods of 1200 triploid virgin females de- genetic basis for the production of females and gynan- rived from 24 families (eight times three diploid males dromorphs from unfertilized eggs. crossed with five scarlet females each) were scored for Table 4 compares the composition of broods (all three female and gynandromorph production. A total of 13 consecutive broods pooled) containing at least one females (from 11 triploid mothers: 0.92%, N ¼ 1200) female or gynandromorph with broods lacking any ab- and 16 gynandromorphs (from 16 triploid mothers: errant individuals. Most females and gynandromorphs 1.33%) were found among the offspring. Table 2 lists all had either scarlet or purple eyes. These ratios are sig- progeny of triploid females that contained daughters or nificantly different from the distribution among their gynandromorphs. Two progeny (4a3#7 and 7b3#3) con- male brothers (x2 ¼ 25.97, P , 0.001, broods with tained more than one daughter. All progeny with females females and gynandromorphs pooled). The overall fre- or gynandromorphs also contained one or more diploid quencies (males, gynandromorphs, and females pooled) males that could unequivocally be determined by their of the different eye colors in the families containing purple eye color, with two exceptions. These two prog- females or gynandromorphs do not differ significantly eny contained only oyster and scarlet individuals, of which from those in the families with only males (x2 ¼ 0.59, P ¼ the latter could be haploid or diploid. 0.74). These results indicate that females and gynan- The frequency of females and gynandromorphs dif- dromorphs develop predominantly from diploid eggs. fered greatly among consecutive hostings (Table 3). In However, there were also two gynandromorphs with the first broods (0- to 1-day-old females), no females and oyster eyes reminiscent of haploidy. Note again that the gynandromorphs were found. In the second broods ploidy level of the scarlet individuals cannot be un- (7–8 days), 9 females and 16 gynandromorphs were equivocally determined (see Figure 1). found. In the third broods (20–21 days), 4 females were Of 13 uniparentally produced females, 12 were bred. found, of which 2 had a mother that also produced a Eight produced normal-sized broods with Mendelian seg- female in the second brood. Note that the number of regation of the eye-color markers among sons and daugh- third broods is lower due to premature death or aging of ters, indicating that they were diploid and that they had females. When the number of females and gynandro- mated with one of their brothers. Two females produced morphs in each age class are corrected for the number normal-sized broods consisting entirely of males, indicat- of offspring per triploid female at different ages (see ing that these females were unmated. Two other females Table 2), the expected numbers are significantly differ- did not produce progeny, which may be due to gynan- ent from the observed (x2-test, Yates corrected, 10.00, dromorphismthatwasnotapparentfromtheexternal d.f. ¼ 2, P , 0.01). These data show that later eggs in the morphology, and one female was dead prior to the test. oviposition sequence have a higher probability of be- None of the 16 individuals scored as gynandro- coming female. morphs produced offspring, indicating that they were The production of females and gynandromorphs is sterile. This is not surprising since 15 of 16 appeared to not random. Paternal line 3 (see Table 3) has a signif- have a male abdomen (Table 5). The phenotype of gyn- icantly higher number of gynandromorphs than the andromorphs showed a clear pattern. They shifted from other lineages (x2-test, P , 0.001). Paternal line 7 has a being female in the anterior body parts to being pre- significantly higher number of females than the other dominantly male in the posterior parts. All individuals lineages (P , 0.01). These two lineages produce a sig- had female antennae, but only one had a partial female nificantly higher (P , 0.001) number of females and abdomen. Females From Unfertilized Diploid Eggs in Nasonia 985

TABLE 2 Progeny of individual virgin triploid females that produce at least one female (A) or one gynandromorph (B) during life (three hostings)

Males Females Triploid mother oyster scarlet purple oyster scarlet purple Total A. Progeny of virgin triploid females producing at least one female 2a5#6a 171——110 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

Males Gynandromorphs Triploid mother 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: oyster is haploid, scarlet is haploid or diploid, and purple is diploid (see Figure 1). a Female code 2a5#6 indicates paternal line 2 (#1–8), father a (a, b, or c), mother 5 (#1–5), female number 6 (#1–10). b Two additional females, a wild type and a scarlet, respectively, were found in the progeny of mothers 3a3#7 and 3a4#7. These progeny were not scored for numbers and phenotypes of males.

These results document for the first time develop- unfertilized haploid eggs develop into males and fer- ment of females from unfertilized diploid eggs in N. tilized diploid eggs develop into females. Mutations to vitripennis and confirm our previous loose observations polyploidy have arisen spontaneously in laboratory during many years of culturing of this polyploid strain. cultures several times (Whiting 1960). Triploid females Furthermore, these parthenogenetically produced fe- produce haploid and diploid eggs, in addition to a large males appear diploid and fully fertile, but they themselves proportion of aneuploid eggs resulting in a low off- in turn did not produce daughters parthenogenetically. 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 DISCUSSION find 30% diploids instead of 40% as previously reported Segregation in the triploid strain: Like all Hymenop- (Conner 1966; Dobson and Tanouye 1998). Moreover, tera, N. vitripennis has haplodiploid sex determination; preferential transmission or segregation (Conner 1966) 986 L. W. Beukeboom and A. Kamping

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 100010001 200210021 300182038 400131023 500110011 600000000 700421052 800000000

Total 0 0 9 16 4 0 13 16

Average progeny no.a (no. of females) 4.67 (n ¼ 166) 1.01 (n ¼ 166) 3.0 (n ¼ 48)b F, females; G, gynandromorphs. a Based on a subsample of the 1200 females. b Based on 48 of 166 females that were still alive. needs not be invoked to explain our results. Molecular A number of important observations require expla- marker studies indicate that recombination rates in nation: (1) 2% of all females produced one or more triploid females are comparable to diploid females daughters or a gynandromorph; (2) all females and (L. W. Beukeboom, unpublished results). almost all gynandromorphs have the purple or scarlet Female and gynandromorph production: Until now phenotype, indicating that they are diploid; (3) the unfertilized haploid and diploid eggs of N. vitripennis frequency of diploid eggs decreases with female age; were reported to always develop into males (Conner (4) the frequency of females and gynandromorphs in- 1966; Beukeboom 1992; Dobson and Tanouye 1998). creases with age; (5) the frequency of females and Using one of Whiting’s original strains, we now have gynandromorphs differs significantly between lineages; shown that unfertilized diploid eggs occasionally de- (6) gynandromorphs show an anterior-posterior gra- velop into females or gynandromorphs. These unipa- dient in femaleness; and (7) progeny with females or rentally produced females are diploid, have normal gynandromorphs also contained one or more diploid fertility, and appear in no way different from normally males. The rare production of daughters is discussed produced females from fertilized eggs. below in relation to the mode of sex determination.

TABLE 4 Offspring of virgin triploid females per family type of only males, males and females, or males and gynandromorphs

Offspring type Family type oyster scarlet purple Total 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 Based on a random sample of 166 triploid females. Females From Unfertilized Diploid Eggs in Nasonia 987

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/MFFFFFFFF Hind wings F F F F F F F F M M F F F F F/MF/MFFFFFFMM Front legs M M F F F F F F F F M M M M F/MF/MFFFFFFFM Middle legs M M M M F F F M M F/MMMMMMM FFFFFMMM Hind legs M M M M F F/MMF/MMF/MMMMMMM FFFMFMMM 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 Double symbols refer to, respectively, the left and the right side of the body. F, female; M, male.

Daughter production increases with the mother’s age, Another finding requiring explanation is the signif- but the frequency of diploid eggs decreases with age. icantly higher proportions of females and gynandro- Therefore, daughter production not only is the result morphs in some maternal and grand-paternal lineages of an increase in the production of diploid eggs by age, as well as the fact that some females produce more than but also implies that these two phenomena are at least one daughter in addition to diploid sons. Uniparentally partially independent. It suggests that daughter produc- produced females, however, do not themselves produce tion is partially under maternal control and/or under females or diploid males parthenogenetically. This rules control of genes that have age-dependent expression. The out a dominant mutation that is transmitted over gener- low frequency of virgin triploid females that produce ations. The data rather suggest the occurrence of a rare females can be increased by changing culture conditions. event in the germline of some males that is partially In a pilot experiment we found a strong increase for manifested (e.g., semidominant) in some of their daugh- female and gynandromorph production at high temper- ters. The expression of the trait varies within the triploid ature, providing further evidence for a maternal effect. females as well, given that not all of their diploid off- Only diploid eggs appear to undergo a sex switch. spring were transformed into females. Our data there- This indicates that the ploidy level of the egg plays an fore appear most easily explained by invoking a dosage important role in sex determination. In this respect it effect and/or an epigenetic mutation. Epigenetic in- would be informative to know the ploidy level of the heritance of traits has been invoked for Nasonia before gynandromorphs. They could be ordered haplodiploid (Poirie´ et al. 1992; Beukeboom 1995). Epigenetic con- mosaics (i.e., consisting of haploid male and diploid trol over inheritance is now well established although female tissues), as described for other hymenopterans much of the underlying mechanisms remain to be (Whiting et al. 1934; Grosch 1988; Halstead 1988), uncovered (Constancia et al. 1998; Lyko and Paro or intersexes (i.e., with incompletely differentiated gen- 1999; Vu and Hoffman 2000). Genomic imprints usually der). The fact that our gynandromorphs descend from undergo cyclic application and erasure within single uninseminated females rules out any mechanism in- generations, but Morgan et al. (1999) showed that the volving fertilization, such as polyspermy or fertilization epigenetically inherited agouti locus in mice can retain of binucleate eggs (Stern 1968; Petters and Grosch its imprint for several generations through incomplete 1976). Our results suggest that most gynandromorphs erasure of the modification in the female germline. Al- are diploid. We found only two gynandromorphs with though purely speculative at this moment, it is not in- oyster eyes, which is a recessive mutation, indicating that conceivable that a similar process is responsible for the (at least) their eye tissue was haploid. Moreover, our results found in this study. Such a scenario would im- breeding results show that all fertile uniparental females plicate that one may be able to artificially select for the are diploid (not haploid). Haploidy of females has never trait ‘‘occasional parthenogenetic female production.’’ been described in the Hymenoptera although Whiting This is what we are currently attempting. et al. (1934) reported a type of sex intergrade, consisting Implications for sex determination: Our recovery of of two types of genetically male tissue that interact to diploid daughters and gynandromorphs from unfertil- produce feminization. The higher frequency of femini- ized triploid females indicates that the sex-determining zation in anterior vs. posterior body parts in our gynan- mechanism in Nasonia is most likely more complicated dromorphs suggests that some sort of gradient exists in than the existing models predict. Different models female induction. We are currently investigating ploidy have been reviewed by Cook (1993a) and Dobson and levels of the gynandromorphs to reveal the mechanism Tanouye (1998). According to the FSD model (Whiting of their origin. 1960), sex is determined by fertilization, not by ploidy 988 L. W. Beukeboom and A. Kamping level; i.e., fertilized eggs develop into females and un- production of diploid females and gynandromorphs fertilized eggs into males. Our results now show that through their virgin triploid daughters. Moreover, the female development can also occur without fertilization data show that female and gynandromorph production in N. vitripennis. Together with female development under are largely independent events. These observations in- thelytokous reproduction, which is known from many dicate that sex-determining factors are polymorphic in Hymenoptera, these data discard the FSD model. This the polyploid strain and suggest that at least two seg- model also had previously been rejected on the basis of regating genes are involved, e.g., a sex-determining gene studies with the paternal sex ratio chromosome, which and a gene that modifies its expression. Recombination showed that fertilized eggs can develop into males in triploid females appears to be completely random (Werren et al. 1987; Nur et al. 1988; Beukeboom and (L. Beukeboom, unpublished results). The absence of Werren 1993; Dobson and Tanouye 1998). females and gynandromorphs in the offspring of virgin ´ The GISD model (Poirie et al. 1992; Beukeboom diploid F2 daughters in turn indicates that the ploidy 1995) proposes that paternally and maternally derived level of the mother also plays a role in the production of genes function differently in sex determination and that daughters from unfertilized eggs. at least one paternally inherited set of chromosomes The mode of reproduction of N. vitripennis is called is required for female development. This model was arrhenotoky, which is the common form among hap- prompted by breeding data of the polyploid strain used lodiploid Hymenoptera. Some species are thelytokous in this study. Diploid eggs containing two maternally and produce only daughters from haploid eggs that have derived chromosome sets were found to develop into undergone a diploidy restoration process. Although we males, whereas diploid eggs with one maternal and one have now shown uniparental production of females in paternal set (derived from fertilization of haploid eggs N. vitripennis, the mechanism by which these females of triploid females) resulted in females. This led Dobson arise is different from thelytokous species. They develop and Tanouye (1998) to consider GISD as the only model from unfertilized diploid eggs that result from a reduc- consistent with all available data. However, we now have tion division without any subsequent ploidy increase. shown that diploid eggs without patrigenes can also This is still the consequence of arrhenotokous repro- develop into females that cannot be accounted for easily duction, where it is typically the males that arise by by GISD. We therefore conclude that sex determination automictic parthenogenesis. Polyploid Hymenoptera in Nasonia is more complicated than any of the existing with this mode of reproduction are not known from models predict. nature. The mechanism is most reminiscent of pre- Stouthamer and Kazmer (1994) proposed that trip- meiotic doubling thelytoky, whereby the diploid chro- loid females carry a nonfunctional variant (mutation) mosome complement is doubled before meiosis and of a locus involved in sex determination; i.e., triploid the meiotic products are diploid (Suomalainen et al. females are functionally diploid and diploid males are 1987). This has been reported for a few hymenop- functionally haploid. Assuming Mendelian segregation terans (Doncaster 1916; Dodds 1939; Peacock and of this mutation in the polyploid strain, two-thirds of Sanderson 1939; Ledoux 1954). diploid eggs are expected to carry one mutated and one In conclusion, for the first time we showed unipa- intact copy of the sex locus and develop into diploid rental development of females in N. vitripennis. The males. The other one-third are expected to receive two used polyploid mutant strain proves very useful for intact copies of the sex locus, resulting in female devel- manipulating the number of paternally and maternally opment. Our data do not support these predictions transmitted chromosome sets. Occasional daughter de- because only a very small fraction (,1%) of triploid velopment from eggs without a paternal chromosome daughters of a particular diploid father produces females complement poses a new challenge for the existing or gynandromorphs. Preferential segregation (Conner models of sex determination and additional modifica- 1966) of the mutation to diploid eggs can also be ex- tions seem to be required. Our results will contribute to cluded since the ratio of haploid and diploid individuals the long-term goal of elucidating the genetic regulation in the progeny of these ‘‘exceptional’’triploid females is of haplodiploid sex determination. Further study of similar to that of a random sample of virgin triploid gynandromorphic individuals, such as obtaining data females. on ploidy level and dosage compensation, appears par- Although the proportion of virgin triploid females ticularly promising. that produce daughters is very low, the presence of a We thank Mary Anne Pultz, Richard Stouthamer, Carol Trent, and diploid daughter in the offspring of an ‘‘affected’’ mother John Werren for stimulating discussions on Nasonia sex determina- is not a sporadic event. The frequency of daughters tion; George Saul and John Werren for providing the polyploid mu- among their diploid offspring does not significantly tant; Reyhan O¨ zkan, Bianca Hollebeek, Andy Rumahloine, and Joop deviate from one-third (Table 2). This could be ex- Prijs for technical assistance; and Louis van de Zande, Carol Trent, and Peter Mayhew for their valuable comments on the manuscript. This plained simply by a single dominant mutation that oc- work has been made possible by a fellowship of the Royal Netherlands curred in the germline of these females. However, our Academy of Arts and Sciences and a Pioneer grant of the Dutch data also show a difference between paternal lineages in Scientific Organization to L.W.B. Females From Unfertilized Diploid Eggs in Nasonia 989

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