A Functional Dosage Compensation Complex Required for Male Killing in Drosophila

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Science  04 Mar 2005:
Vol. 307, Issue 5714, pp. 1461-1463
DOI: 10.1126/science.1107182


Bacteria that selectively kill males (“male-killers”) were first characterized more than 50 years ago in Drosophila and have proved to be common in insects. However, the mechanism by which sex specificity of virulence is achieved has remained unknown. We tested the ability of Spiroplasma poulsonii to kill Drosophila melanogaster males carrying mutations in genes that encode the dosage compensation complex. The bacterium failed to kill males lacking any of the five protein components of the complex.

Certain isofemale lines of Drosophila only give rise to daughters following the death of male embryos (1). Male death is due to the presence of intracellular bacteria that pass from a female to her progeny and that selectively kill males during embryogenesis (2). These male-killing bacteria are found in a wide range of other insect species, and many different bacteria have evolved male-killing phenotypes (3). In some host species, male-killers drive the host population sex ratio to levels as high as 100 females per male (4) and alter the pattern of mate competition (5). However, the underlying processes that produce male-limited mortality are unclear (6). Here we examine the interaction between the male-killing bacterium Spiroplasma poulsonii and the sex determination pathway of D. melanogaster (7).

The primary signal of sex in Drosophila is the X-to-autosome ratio. This signal is permanently established in expression of Sex-lethal (Sxl) in females and its absence in males (8, 9). This, in turn, effects three processes: germline sexual identity, somatic sexual differentiation, and dosage compensation, the process by which the gene expression titer on the X chromosome is equalized between two sexes despite their difference in X chromosome number. Mutations in the gene tra that convert XX individuals to male somatic sex do not induce female death (7). Our observations indicate germline formation and migration happen correctly in male embryos and that dying male embryos do not express Sxl. We, therefore, examined the requirement of the Spiroplasma for genes within the system of dosage compensation.

In Drosophila, the single X of males is hypertranscribed. This process of hypertranscription requires the formation of the dosage compensation complex (DCC) and its binding to (and modification of) the X chromosome (10). SXL in female Drosophila inhibits the production of MSL-2 protein, which is thus only present in male Drosophila. MSL-2 forms a complex with four other proteins, MSL-1, MSL-3, MLE, and MOF, which collectively form the DCC. MSL-1, MSL-3, MLE, and MOF are constitutively present in both males and females and are also supplied maternally. The complete DCC binds, with JIL-1, to the male X chromosome at various entry points, and, with the products of two noncoding RNAs, RoX1 and RoX2, it affects the modification of the single X chromosome and its hypertranscription.

We examined the effect of mutations within the host DCC on the ability of the male-killer to function (11). The survival of male progeny beyond embryogenesis to L2/L3 (and in one case adult) was scored in the presence of different loss-of-function mutations within the dosage compensation system (normal male-killing occurs during embryogenesis) (2), in the presence and absence of infection. Because many genes within this group additionally show strong maternal effects (12), the effect of mutations was in each case tested by using both mothers that were heterozygous for the loss-of-function mutation and mothers homozygous for it.

Uninfected males homozygous for loss-of-function mutations within the dosage compensation system generally survive to the third larval instar. We tested survival to the third larval instar for loss-of-function alleles of msl-1 (alleles msl-11 and msl-1b), msl-2 (msl-2g227 and msl-2g134), msl-3 (msl-3132), mle (mle9, mle1), and mof (mof1), and survival to adult for mle1/mle6 transheterozygotes. In the case of all alleles of msl-1, msl-3, mle, and mof, a similar pattern is observed: Males homozygous or hemizygous for the loss-of-function mutation have appreciable survival in the presence of infection when their mother is also homozygous for the loss-of-function mutation (Figs. 1 and 2; tables S1–S4). In contrast, heterozygous male embryos that were siblings of the above (that will have a wild-type DCC) were always killed, as were all male embryos in the case where the mother was heterozygous (where maternal supply of these proteins enables dosage compensation to be initiated, although not maintained) (12). In the case of mle1/mle6 transheterozygotes, male survival to adult was observed (table S1). For the case of mof, male-killing was restored to full efficiency when 18H1, a transgenic copy of mof (13), was added to the mof1 loss-of-function background. Within the above crosses, three observations make us sure the Spiroplasma was fully operational. First, crosses involving heterozygous mothers, where male-killing was complete, were conducted concurrently with those using homozygous mothers, and the females in these crosses were siblings from the same vials. Second, in each cross and vial where homozygous males survived, heterozygous males (with wild-type function) still died. Finally, F1 females derived from these crosses, when mated to wild-type males, produced a full, female-biased sex ratio.

Fig. 1.

Percentage survival through to day 5 of F1 males of differing genotype with respect to loss-of-function mutations in the dosage compensation pathway in the presence (red) and absence (blue) of infection. Left-hand side graphs represent male survival rates in crosses where the female parents were homozygous for the loss-of-function mutations in question and the male parent heterozygous for it. Right-hand side graphs represent crosses where both male and female parents were heterozygous for the loss-of-function mutations. Percentage survival of males is taken relative to females of the same genotype within the cross; mean of at least eight independent crosses taken for each cross type.

Fig. 2.

Percentage survival to day 5 of male progeny in crosses between females of different genotype with respect to the mutation mof1 (given above the bar) and wild-type males. 18H1 is a transgenic copy of mof + inserted on chromosome 2.

In the case of msl-2, where there is no maternal supply of MSL-2, survival of homozygous sons was observed for both homozygous and heterozygous mothers for the case of the mutation msl-2g134 (Fig. 1). For the case of msl-2 g227, no male progeny were observed from infected females (table S5). This mutation does not rescue males in our assay, probably because the two mutations have different effects on msl-2 expression. The msl-2g134 allele prevents formation of MSL-2 protein, whereas msl-2 g227 potentially encodes the RING-finger element of a truncated MSL-2 protein (14).

Thus, absence or reduced function of any of the proteins of the DCC can reduce the efficiency of male killing, and a functional DCC is required for male killing by S. poulsonii. The fact that the genes mediating this process in Drosophila have been well studied can be exploited to yield further insights into the mechanism of male killing.

Supporting Online Material

Materials and Methods

Tables S1 to S5

References and Notes

References and Notes

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