Maternal Control of Haplodiploid Sex Determination in the Wasp Nasonia

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Science  30 Apr 2010:
Vol. 328, Issue 5978, pp. 620-623
DOI: 10.1126/science.1185805


All insects in the order Hymenoptera have haplodiploid sex determination, in which males emerge from haploid unfertilized eggs and females are diploid. Sex determination in the honeybee Apis mellifera is controlled by the complementary sex determination (csd) locus, but the mechanisms controlling sex determination in other Hymenoptera without csd are unknown. We identified the sex-determination system of the parasitic wasp Nasonia, which has no csd locus. Instead, maternal input of Nasonia vitripennis transformer (Nvtra) messenger RNA, in combination with specific zygotic Nvtra transcription, in which Nvtra autoregulates female-specific splicing, is essential for female development. Our data indicate that males develop as a result of maternal imprinting that prevents zygotic transcription of the maternally derived Nvtra allele in unfertilized eggs. Upon fertilization, zygotic Nvtra transcription is initiated, which autoregulates the female-specific transcript, leading to female development.

Mechanisms for sex determination are remarkably variable. In many insect species, a primary signal initiates one of two alternative routes of regulatory gene cascades (1). This cascade leads to sex-specific differential splicing of the gene doublesex (dsx) and the production of either male- or female-specific DSX proteins (211). The splicing factor transformer (TRA) (1215), termed feminizer (FEM) in Apis mellifera (16), mediates the primary sex-determining signal in females by regulating the female-specific splicing of dsx pre-mRNA. In males, no functional TRA/FEM protein is present because of sex-specific splicing of tra/fem pre-mRNA, leading to default male-specific splicing of dsx primary transcripts.

In diploid insects, sex is mostly signaled by components of sex chromosomes (for example, XY and ZW). In Hymenoptera, however, sex is usually regulated by the ploidy of the embryo (17, 18): Males are haploid, developing from unfertilized eggs, whereas diploid females develop from fertilized eggs. In the honeybee A. mellifera, the complementary sex determiner (csd) gene (which exhibits homology to tra/fem) (19, 20) initiates the female sex-determining route when the animal is heterozygous at this locus, whereas homozygosity or hemizygosity leads to maleness. A csd mechanism of sex determination can easily be determined because it results in predictable proportions of homozygous diploids that develop into males (21). Because a number of Hymenoptera, including Nasonia, do not produce diploid males upon inbreeding (22), it was surmised that another mechanism controls haplodiploid sex determination in these species.

We screened the Nasonia genome (22) for motifs matching the Drosophila tra and Apis csd genes, which resulted in the identification of a single gene (16, 22) composed of nine exons and containing two Arg/Ser-domains (SR-domains), of which one is located entirely in exon one and the second spans exons four to seven. In exons seven and eight, a proline-rich (Pro) domain is present. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that female-specific splicing retains only the first part of exon two and yields a single transcript encoding a full-length protein, containing both SR domains and the Pro-rich domain. In male Nasonia, either the complete exon two or different 3′ parts of exon two can be retained by cryptic 3′ splice-site recognition to yield three different transcripts, all of which encode truncated proteins containing only the first SR domain (22). This gene was named Nasonia vitripennis transformer (Nvtra). Nvtra expression was knocked down by injecting double-stranded RNA (dsRNA) against a non–sex-specific part of Nvtra in 1- to 2-day-old female pupae (23) carrying the recessive eye color mutation STDR (stDR/stDR). After emergence, neither phenotypic nor behavioral changes were observed as compared with control uninjected females. Nvtra dsRNA-injected females were capable of mating and ovipositing and were fully fertile. The levels of Nvtra mRNA 5 days after dsRNA injection, when the females were in the late pupal stage, showed a 2.8-fold decrease in Nvtra expression [t(16) = 3.86, P = 0.0007, Fig. 1A] relative to uninjected controls.

Fig. 1

Sex-specific differential splicing of Nvtra and the functional relationship of Nvtra and Nvdsx. (A) Relative levels of Nvtra mRNA after RNAi in control (light gray bar) and Nvtra dsRNA-injected (black bar) females in the late pupal stage. Error bars represent SE. *P < 0.001. (B) RT-PCR analysis of sex-specific splicing of Nvtra (top), Nvdsx (middle), and Ribosomal protein 49 (bottom) mRNA. Lanes 1 to 4, control females; lanes 5 to 10, Nvtra dsRNA-injected females; lanes 11 and 12, haploid male offspring from injected females; lanes 13 and 14, diploid male offspring from injected females; lanes 15 and 16, haploid male offspring from control females. M is a 100-bp molecular size marker. Black arrows indicate male-specific splice forms, gray arrow indicates an unknown splice form, and white arrows indicate female-specific splice forms. A control for amplification from residual genomic DNA is present in the rightmost panel.

In control females, only the female-specific Nvtra splice form was present. However, Nvtra dsRNA-injected females had a decreased amount of female-specific splice form and produced all three male-specific Nvtra splice forms (Fig. 1B). Apparently, repression of Nvtra also disrupted female-specific splicing of Nvtra pre-mRNA itself. For control females, N. vitripennis doublesex (Nvdsx) female-specific splicing along with very low quantities of a male-specific Nvdsx splice form (11) were observed. In Nvtra dsRNA-injected females, the expression of the predominant female splice form of Nvdsx decreased, whereas expression of the male-specific splice form increased (Fig. 1B). This indicates that, in Nasonia, an active NvTRA is necessary for female-specific splicing of Nvdsx mRNA. The presence of both male- and female-specific splice forms of Nvtra and Nvdsx was observed to be correlated with the degree of femaleness in haploid Nasonia gynandromorphs (11, 22, 24), indicating that these genes function in sex-specific phenotype establishment. The fact that a similar Nvtra and Nvdsx transcript composition in dsRNA-injected females nevertheless leads to complete morphological and functional females indicates either that the essential period of this Nvtra/Nvdsx-mediated phenotypic establishment is before the pupal stage or that the lower level of female-specific Nvtra is still sufficient to elicit female development.

To monitor the relative levels of Nvtra and Nvdsx during early and late embryonic development, we sampled embryos over time and determined the ratio of Nvtra and Nvdsx transcripts. In 0- to 1-hour-old embryos, an eightfold excess of Nvtra over Nvdsx was observed [t(18) = 3.62, P = 0.0020, table S1]. Because no appreciable zygotic gene expression occurs at this early stage (25), this relatively high level of Nvtra mRNA must be provided to the egg during oogenesis as a maternal factor and should be the female-specific splice variant only. RT-PCR confirmed this expectation, by showing only female-specific transcripts of Nvtra in 0- to 5-hour-old embryos from fertilized and unfertilized eggs (Fig. 2A).

Fig. 2

Maternal input in early embryos. (A) Maternal input of female-specific Nvtra mRNA in early embryos from unfertilized (top) and fertilized (bottom) eggs shown 1, 3, and 5 hours after egg laying. Open arrows indicate female-specific Nvtra splice forms of 228 bp. M is a 100-bp size marker. (B) Relative Nvtra mRNA levels in equally mixed embryos from mated and unmated control (gray bar) or Nvtra dsRNA-injected (black bar) females. Error bars represent SE. *P < 0.001.

As expected, virgin Nvtra dsRNA-injected STDR females produced only stDR males (fig. S1). When injected STDR females were mated to wild-type (st+) males, they still produced only male offspring of which 44% had the stDR red-eye phenotype (representing unfertilized eggs) and 56% had wild-type eyes and must therefore be diploid (stDR/st+) (Table 1). Both haploid and diploid adult males had only the male-specific splice forms of both Nvtra and Nvdsx (Fig. 1B). Because neither intersex nor female offspring were observed, Nvtra dsRNA-injected females exhibit a complete sex reversal in their offspring. Flow cytometry confirmed the diploidy of the stDR/st+ males (fig. S2). We mated a subset of these diploid stDR/st+ males to STDR females. The female offspring of this cross all had wild-type eyes. Because male gametogenesis does not involve reduction division, we assume that these males had transmitted their complete diploid genome to generate triploid stDR/stDR/st+ daughters, as reported earlier for diploid males from a triploid strain (26).

Table 1

Nvtra dsRNA-injected females and their offspring numbers. Number of Nvtra dsRNA-injected females [P: parental females (RNAi)] that produced offspring [P: parental females (fertile)] as virgin or as mated to AsymC males and the offspring they produced (F1: haploid males; F1: diploid females; and F1: diploid males).

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To assess whether Nvtra dsRNA-injected mothers provided lower amounts of Nvtra to the eggs, we measured the relative levels of Nvtra in the offspring of Nvtra dsRNA-injected and uninjected females. We found that very early embryos (0 to 3 hours old), in which zygotic gene expression has not yet started (25), resulting from both virgin and mated Nvtra dsRNA-injected females, had decreased levels of Nvtra mRNA to about 20% of that of early embryos from control noninjected females [t(35) = –3.92, P = 0.0002, Fig. 2B].

Our results suggest that a threshold level of maternally provided female-specific Nvtra mRNA is essential for female development of the fertilized egg, because knockdown of Nvtra in mothers leads to the production of diploid male offspring. They also indicate that female-specific Nvtra splicing depends on an autoregulatory loop. First, knockdown of Nvtra in the mother leads to the disruption of the female-specific splicing of both Nvtra and Nvdsx in these mothers. Second, the diploid male offspring from Nvtra dsRNA-injected mothers had only male-specific spliced Nvtra transcripts, indicating the dependence of a functional NvTRA protein for female-specific splicing. Third, the high sensitivity of the diploid embryos from the injected mothers to the lowered levels of female-specific Nvtra resulting in a full sex reversal indicates that sufficient NvTRA is needed for female-specific splicing. Fourth, eight putative TRA/TRA2 binding motifs (U/G)GAAGAU(U/A) in the tra/fem-regulated dsx and fruitless (fru) genes of N. vitripennis and A. mellifera (27) are located in the male-specific exon 2m1 (22) and in the intronic region between exons two and three of the Nvtra gene. Based upon similar arguments, tra autoregulatory loops have been proposed for the dipterans Ceratitis capitata, Bactrocera oleae, Lucilia cuprina and A. mellifera (14, 20, 28, 29). We conclude that Nvtra is part of the Nasonia sex-determining cascade and is responsible for the sex-specific splicing of Nvdsx. In addition, sufficient levels of female-specific Nvtra transcripts are necessary to maintain the female-specific splicing pattern of Nvtra itself.

In diploid houseflies (Musca domestica), which lack haplodiploidy, the dominant male-determining M factor represses the sex-determining F factor, resulting in male development (30). In the absence of M, F, which is an ortholog of the Ceratitis tra gene (13), is activated, leading to female development. In M. domestica, the M factor can be located on the Y chromosome and/or on one of the autosomes. In other Diptera, such as Ceratitis and Lucilia, the M factor leads to male development by blocking the transcription or translation of female tra or by interfering with tra splicing (13, 29). Only males can provide the M factor for the next generation. Therefore, M factors are incompatible with haplodiploid sex determination, where only unfertilized eggs develop into males. This implies that in Nasonia, a different mechanism is responsible for the development of males in the presence of maternally provided Nvtra mRNA or protein. Thus, we conclude that maternal Nvtra mRNA is most likely provided to all eggs as a means to start the female-specific autoregulatory loop.

Because fertilization per se had been ruled out as a sex-determining factor in Nasonia before (31), and because unfertilized eggs will develop as males, we asked whether the presence of a paternal genome together with a maternal genome explains why only fertilized eggs develop as females. Quantitative PCR (qPCR) showed that in 1- to 3-hour-old embryos from both fertilized and unfertilized eggs, the maternally provided Nvtra mRNA input gradually decayed (Fig. 3A). In embryos from unfertilized eggs, a low level of Nvtra mRNA was maintained throughout the 23 hours of embryonic development (Fig. 3A). In sharp contrast, a 15-times-higher expression of Nvtra in embryos from fertilized eggs was observed 7 hours after egg laying [t(8) = 4.18, P = 0.0031, Fig. 3A], which cannot be explained by the presence of two versus one Nvtra alleles in these embryos and calls for a regulatory explanation. After this peak expression, a significantly higher level (F15,63 = 5.25, P < 0.0001) of Nvtra mRNA was maintained as compared with embryos from unfertilized eggs (Fig. 3A). We used a Russian strain of N. vitripennis that harbors a deletion of 18 base pairs (bp) in the first exon of the Nvtra gene, which apparently does not affect the function of the gene, to monitor the paternal genome for the onset of zygotic Nvtra production. RT-PCR of Nvtra transcripts in these samples showed that in offspring from fertilized (diploid) eggs, zygotic Nvtra mRNA is transcribed from the paternal genome 5 hours after egg laying (Fig. 3B) and confirmed our assumption that, in early (0 to 3 hours) Nasonia embryos, no zygotic Nvtra transcription takes place. A reciprocal cross yielded identical results. Unfortunately, because of the repetitive nature of the indel and its flanking region, we were unable to design primers to perform qPCR to quantify the relative contributions of the paternal and maternal Nvtra alleles, respectively.

Fig. 3

Expression and splicing of Nvtra during embryonic development. (A) Relative Nvtra mRNA levels in embryos from unfertilized (gray bars) and fertilized (black bars) eggs at different developmental times, indicated as hours after egg laying. Error bars represent SE. (B) Nvtra mRNA originating as maternal input (light gray arrows) or transcribed from the paternal genome (dark gray arrows) in embryos from unfertilized (top) and fertilized (bottom) eggs. Open dotted arrow indicates amplification resulting from residual genomic DNA. M is a 100-bp size marker. (C) Temporal pattern of sex-specific splicing of Nvtra mRNA in embryos from unfertilized (top) and fertilized (bottom) eggs. Black arrows indicate male-specific splice forms. White arrows indicate female-specific splice form.

Because 1- to 5-hour-old embryos from fertilized and unfertilized eggs contained only female-specific Nvtra mRNA (Figs. 2A and 3C), we hypothesize that the absence of sufficient zygotic Nvtra expression to initiate the autoregulatory loop results in default male-specific splicing (Fig. 3C). However, in embryos from fertilized eggs, the female-specific splicing of Nvtra is maintained (Fig. 3C) because of the availability of zygotic Nvtra mRNA. The low levels of the male-specific splice forms observed in these pooled embryo samples most likely result from the unfertilized eggs laid by the mated STDR females (a typical brood contains 20% males). One explanation could be that only the paternal allele of Nvtra is transcribed in the early embryo, thus allowing the loop of autoregulatory splicing to take place. Alternatively, a trans factor necessary for the timely onset of zygotic Nvtra transcription may be silenced in the maternal genome set of the embryo.

Our data show that maternal provision of Nvtra to all embryos, followed by sufficient early zygotic Nvtra expression, which occurs only in fertilized eggs, is necessary for female development in Nasonia. RNA interference (RNAi) treatment decreased the maternal provision of Nvtra to the eggs, which alone would be sufficient for the production of diploid males. It is possible that the resulting small interfering RNAs (siRNAs) were also transmitted to the eggs, resulting in a decrease in zygotic Nvtra transcript expression in addition to a decrease in maternal Nvtra input. Either way, the simplest explanation for the mechanism behind Nasonia sex determination appears to be maternal input of Nvtra mRNA combined with a form of maternal imprinting (31).

Several insects have maternal input of tra mRNA followed by an autoregulatory loop for the continuous production of female-specific tra (13, 20, 29). However, in Nasonia, male development does not result from disruption of the Nvtra autoregulatory loop by paternal repression (for example, an M factor) or a nonfunctioning CSD, but is most likely caused by maternal silencing of the tra gene. The presence of a paternal genome leads to zygotic expression of Nvtra, but maternally provided Nvtra mRNA is required to initiate female-specific splicing. Hence, in Nasonia, females regulate the sex of the offspring by providing a feminizing effect by maternal input of Nvtra, while at the same time preventing zygotic expression of Nvtra in haploid offspring. Pane et al. (13) suggested that the sensitivity of the tra autoregulation is evolutionarily important for the recruitment of upstream regulators. Indeed, in A. mellifera, csd originated as a duplication of fem (=tra) (16). The gregarious lifestyle of Nasonia implies potential high levels of inbreeding, so the evolution of a csd sex-determining mode is under constraint. Instead, a maternal imprinting event seems to be an upstream regulator, rendering the system dependent on zygotic expression. This is analogous to the observed evolutionary modulation of the maternal provision versus zygotic transcription of patterning determinants by Rosenberg et al. (32). The interplay of maternal and zygotic provision of sensitive sex-determination regulatory factors may facilitate the recurrent appearance of thelytokous reproduction in haplodiploid insects.

Supporting Online Material

Materials and Methods

Figs. S1 and S2

Tables S1 and S2


References and Notes

  1. The authors thank the Werren lab (University of Rochester, United States), D. Bopp, M. H. Linskens, A. Rensink, and A. Kamping for technical assistance and discussions and four anonymous reviewers for helpful suggestions. This work was partly funded by Pioneer grant ALW 833.02.003 from the Netherlands Organization for Scientific Research (NWO).
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