# Restrictive Flamenco Alleles Are Maintained in Drosophila melanogaster Population Cages, Despite the Absence of Their Endogenous Gypsy Retroviral Targets.

A. Pelisson, G. Payen-Groschene, C. Terzian, A. Bucheton

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A. Pelisson, G. Payen-Groschene, C. Terzian, A. Bucheton. Restrictive Flamenco Alleles Are Maintained in Drosophila melanogaster Population Cages, Despite the Absence of Their Endogenous Gypsy Retroviral Targets. Molecular Biology and Evolution, 2007, 24 (2), pp.498-504. 10.1093/mol-bev/msl176. hal-00129073

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# Restrictive *flamenco* Alleles Are Maintained in *Drosophila melanogaster*
## Population Cages, Despite the Absence of Their Endogenous gypsy Retroviral Targets

Alain Pélisson,* Geneviève Payen-Groschêne,* Christophe Terzian,†<fnref n="1" /> and Alain Bucheton*

*Institut de Génétique Humaine, CNRS, Montpellier, France; and †UMR5202, CNRS-MNHN-EPHE, Paris, France*

The *flamenco* (*flam*) locus, located at 20A1-3 in the centromeric heterochromatin of the *Drosophila melanogaster* X chromosome, is a major regulator of the *gypsy*/*mdg4* endogenous retrovirus. In restrictive strains, functional *flam* alleles maintain *gypsy* proviruses in a repressed state. By contrast, in permissive strains, proviral amplification results from infection of the female germ line and subsequent insertions into the chromosomes of the progeny. A restrictive/permissive polymorphism prevails in natural and laboratory populations. This polymorphism was assumed to be maintained by the interplay of opposite selective forces; on one hand, the increase of genetic load caused by proviral insertions would favor restrictive *flam* alleles because they make flies resistant to these *gypsy* replicative transpositions and, on the other, a hypothetical resistance cost would select against such alleles in the absence of the retrovirus. However, the population cage data presented in this paper do not fit with this simple resistance cost hypothesis because restrictive alleles were not eliminated in the absence of functional *gypsy* proviruses; on the contrary, using 2 independent *flam* allelic pairs, the restrictive frequency rose to about 90% in every experimental population, whatever the pair of alleles and the allelic proportions in the initial inoculum. These data suggest that the *flam* polymorphism is maintained by some strong balancing selection, which would act either on *flam* itself, independently of the deleterious effect of *gypsy*, or on a hypothetical flanking gene, in linkage disequilibrium with *flam*. Alternatively, restrictive *flam* alleles might also be resistant to some other retroelements that would be still present in the cage populations, causing a positive selection for these alleles. Whatever selective forces that maintain high levels of restrictive alleles independently of *gypsy*, this unknown mechanism can set up an interesting kind of antiviral innate immunity, at the population level.

## Introduction

The coevolution between parasites and their hosts often involves “arms races” that reinforce their mutual interactions. The purpose of the present study was to appreciate some of the evolutionary forces that drive the host–parasite interactions between the genome of *Drosophila melanogaster* and the *gypsy* retrovirus.

*Gypsy*/*mdg4* is the first retrovirus that was recognized, as such, in invertebrates (Kim et al. 1994; Song et al. 1994). Like other retroviruses, *gypsy* is able to integrate its genetic material, as a provirus, in the host genomic DNA. In fact, *gypsy* is an endogenous retrovirus, meaning that the proviruses are integrated into the germ line chromosomes and can thereby be transmitted vertically, through fly generations, along with the proper *Drosophila* genetic material. The endogenous *gypsy* genetic material mainly consists of several dozen old defective proviruses that have accumulated many structural and functional alterations (Lambertsson et al. 1989). Each has generally been inserted at its own genomic location since the ancestor of all present-day flies and is therefore shared by all strains. As this is the case for the other *Drosophila* retroelements, most of these vestiges of a former *gypsy* activity are located in the heterochromatic pericentromeric regions of the genome (Vaury et al. 1989). In addition, the genome of some strains contain functional *gypsy* proviruses that recently integrated in what are now strain-specific euchromatic locations. The strains that are

completely devoid of such potentially active proviruses are called “empty” strains.

An increase in *gypsy* copy number requires not only the functionality of the proviruses but also the permissive character of the host. The latter feature is genetically determined by an as-yet-uncloned X-linked heterochromatic locus (Prud'homme et al. 1995; Robert et al. 2001) called *flamenco* (*flam*). The restrictive function of *flam* represses *gypsy* expression in the somatic follicle cells of the ovary (Pélisson et al. 1994). As a result, proviral integration in the germ line chromosomes of the progeny is prevented (Chalvet et al. 1999). Deficiencies of the *flam* region behave like natural permissive alleles. Both are recessive because their combination with a single dose of a restrictive *flam* allele is sufficient to prevent *gypsy* replicative transposition (Prud'homme et al. 1995). Because these large deficiencies uncover vital flanking genes, they cannot be used to tell what the phenotype of the complete loss of *flam* function would look like. Whatever role this function plays in the biology of *D. melanogaster*, it does not seem to be affected in any of the natural *flam* alleles because, in our laboratory conditions, they do not show any visible phenotype other than the permissive character.

The *flam* genotype can be inferred from the expression level of ovarian β-galactosidase encoded by a *gypsy-lacZ* reporter transgene (Pélisson et al. 1994). This assay shows a semidominance effect because homozygosity of a restrictive allele can repress more efficiently than a heterozygote with a permissive allele (Pélisson et al. 1997).

According to previous genomic analyses (Robert et al. 2001), the *flam* locus spans at least 130 kb of a repeat-rich heterochromatic region that includes a sequencing gap of unknown size. Although the molecular characterization of *flam* is still in progress, we have some hints about the mechanism of *gypsy* repression in which *flam* is involved (Sarot et al. 2004). Available evidence supports a model whereby some defective *gypsy* proviruses can trigger the

<footnote n="1">¹ Present address: UMR 754, INRA-ENVL-UCBL-EPHE, Lyon, France.</footnote>

<footnote>Key words: *Drosophila melanogaster*, *flamenco*, anti-*gypsy* resistance cost, natural and experimental populations, balancing selection, linkage disequilibrium.</footnote>

<footnote>E-mail: alain.pélisson@igh.cnrs.fr.</footnote>

<footnote>Mol. Biol. Evol. 24(2):498–504. 2007
doi:10.1093/molbev/msl176
Advance Access publication November 21, 2006</footnote>
production of small interfering RNAs that would target the homologous transcripts for repression.

The results of a thorough genetic study of *flam* indicate that this locus displays a surprisingly high level of polymorphism both in the laboratory and in the nature (Pelisson et al. 1997). This is especially intriguing when one considers that it maps in the non-recombination-prone 20A1-3 pericentromeric region. Such regions are usually characterized by reduced levels of polymorphism as a result of genetic hitchhiking associated with the fixation of advantageous mutants (Begun and Aquadro 1992). This paradox could be explained by assuming that the *flam* polymorphism results from opposite selective forces. As suggested previously (Pelisson et al. 1997), selection could be driven, on one hand, by the positive impact of the enhanced fitness of the *flam* restrictive individuals in the presence of *gypsy* and, on the other hand, by a negative impact of the restrictive alleles on host fitness in the absence of *gypsy*, that is, a cost of resistance. However, the population cage study presented here does not support the resistance cost hypothesis because the *flam* polymorphism could be maintained in the absence of any functional *gypsy* provirus.

## Materials and Methods

### Laboratory *D. melanogaster* Strains

Flies were maintained on standard fly food (Gans et al. 1975) at 26 °C. All strains used in this study were “empty” strains, devoid of euchromatic *gypsy* proviruses (data not shown). The absence of active *gypsy* in these stocks was checked in Southern blots (data not shown) by the lack of the *Hind*III 1.6-kb band that has been reported to be diagnostic of functional *gypsy* copies (Chalvet et al. 1998). Finally, the absence of any *gypsy*-induced mutability in the permissive strains was also consistent with the lack of active provirus.

A70, an attached-X tester strain, is homozygous for the *#12a gypsy-lacZ* reporter transgene (Sarot et al. 2004). The X-linked *Df(1)l11* deficiency carried by this strain uncovers the *flam* locus as well as vital flanking genes (Prud’homme et al. 1995). The recessive lethality of males is rescued by the Y-linked $y^+$*Ymal*<sup>106</sup> duplication.

OR(R), OR(P), SS(R), and SS(P) are the generic names of the 16 independent X chromosomal clones (4 replicates for each genotype) that were isolated from the previously described white-eyed wOR(P) and wSS(P) permissive empty stocks (Pelisson et al. 1997; Chalvet et al. 1999) as follows. Individual wOR(P) and wSS(P) males were successively mated with an attached-X stock and a *FM3* balanced stock, both devoid of active *gypsy* proviruses. Each *w/w* homozygous chromosomal clone was established by crossing, respectively, the *w/Y* male with the *w/FM3* female progenies and by getting rid of the *FM3* balancer at the next generation. Females of each clone were crossed with A70 males to test its ability to repress the expression of the *gypsy-lacZ* reporter (see below), and 8 clones of each strain, 4 restrictive (R) and 4 permissive (P), were chosen for the present study. When genotyped with the *ZAM-LacZ* assay described below, all these clones were shown to be able to repress the ZAM retroelement.

<figure><img src="image_2.png" /><figcaption>FIG. 1.—Mating scheme used to combine the *gypsy-lacZ* reporter (*#12a*) with each of the 16 X chromosomes cloned (w). The *#12a* transgene was introduced into 4 permissive and 4 restrictive X chromosomal clones extracted from each of the wOR and wSS polymorphic strains. Two successive backcrosses using females of the same chromosomal clone were followed by 2 successive sibmatings. In the w background, flies with the *miniwhite*-containing *#12a* transgene have colored eyes, 2 doses of the transgene being discriminated from a single dose by the corresponding darker eye color phenotype.</figcaption></figure>

As illustrated in figure 1, introduction of the *gypsy-lacZ* transgene into each of the 16 X chromosomal clones was achieved by crossing transgenic males to females of each clone, backcrossing the male progeny and then sibmatating to make the transgene homozygous in each of these 16 genetic backgrounds.

### Natural Populations

In October 2000, at least 50 individuals were captured in each of 2 different cellars of the Montpellier area, Corconnex and Vendargues. DIS13-1 to DIS13-11 are 11 isofemale lines trapped by Dr Daniel Lachaise in Uganda in 2004.

### Population Cages

Population cages were inoculated with samples of 200 fertilized females containing various proportions of either of the 2 allelic pairs. In each case, the 4 replicate clones were used to buffer the genetic background. So, for instance, the wSS population was inoculated with 10% of permissive alleles contained in the sample of 200 female founders corresponding to 5 and 45 females of each of the 4 SS(P); #12a and SS(R); #12a clones, respectively.

Cages were kindly provided by the Rouyer's laboratory. They consist of plastic boxes (30 × 20 × 10 cm), the bottom of which is fitted with 12 cups. Three times a week, they were supplied with 2 cups each containing about 15 g of standard fly food. Each cup therefore stayed 2 weeks in the cage, which is far enough for 1 generation to develop at
26 °C. Although limited, this regular supply of food could sustain continuous populations of several thousands of tiny starving individuals.

Determination of the allelic frequencies was performed as follows: 200–300 eggs were collected during several hours on a cup of fresh food and were allowed to develop outside of the cage, without food restriction. About 100 newly hatched females were fed for 2 days on fresh yeast, and the *lacZ* staining of one of their ovaries was performed as described below.

## Genotyping

*The *flam*<sup>P</sup>/flam<sup>R</sup> Polymorphism.* A *flam* allele is characterized as restrictive or permissive depending on whether or not it can repress the ovarian expression of the *gypsy-lacZ* reporter transgene. The β-galactosidase staining assay was performed essentially as previously described (Sarot et al. 2004). Briefly, ovaries were fixed for 5 min at room temperature in 2% formaldehyde and 0.2% glutaraldehyde, thoroughly rinsed, and then stained for 3 h at 23 °C with 3 mg/ml X-Gal. The genotype of females originating from cage populations could be directly determined because they already contained the *gypsy-lacZ* reporter. The 3 different female genotypes (*flam*<sup>P</sup>/flam<sup>P</sup>, *flam*<sup>P</sup>/flam<sup>R</sup>, and *flam*<sup>R</sup>/flam<sup>R</sup>) were distinguished owing to the semidominance effect. Indeed, the whole follicular epithelium of stage 10 homozygous permissive egg chambers was already homogeneously stained after 30 min, whereas no significant staining was visible in homozygous restrictive ovaries, even after the 180-min incubation time (fig. 2A). In heterozygous ovaries, β-galactosidase activity was only detected in the centripetal follicle cells, which cover the anterior part of the oocyte and in a few patches of cells randomly distributed throughout the epithelium. The same criteria were used to operationally define 3 levels of *gypsy-lacZ* expression in a tentative classification of the X chromosomes extracted from natural populations as permissive, “strongly” restrictive, and “weakly” restrictive, respectively. In this case, each tested X chromosome was combined with the *Df(1)l11 flam* deficiency in the presence of the #12a *gypsy-lacZ* reporter. Such hemizygous ovaries were dissected in the progeny of mass crosses involving at least 10 tested females and 10 males of the A70 tester stock.

*The XmnI Restriction Polymorphism.* Polymerase chain reaction (PCR) amplification of a DNA fragment in the *flam* region was performed with 1 µl of genomic DNA isolated as follows. Individual fly heads were crudely squashed with a pipet tip in 50 µl of squashing buffer (10 mM Tris pH 8.2, 1 mM ethylenediaminetetraacetic acid, 25 mM NaCl, and 0.2 mg/ml Proteinase K) and incubated for 30 min at 29 °C, then for 2 min at 85 °C. The following pair of primers was used at an annealing temperature of 56 °C: 5'-CCTCAGTTAAAAATATTGGACCACTT-3' and 5'-TAGACATTTCACACTGGCCATAGTAA-3'. The PCR products were purified using the Qiaquick PCR spin columns (Qiagen, Courtaboeuf, France) and were then either sequenced or digested with *XmnI*, according to the manufacturer's recommendations (Promega, Charbonnières, France). The digestion products were run on a 1.5% agarose gel in 1× tris-borate-EDTA.

<figure><img src="image_3.png" /></figure>

FIG. 2.—Validation of the β-galactosidase genotyping method by the XmnI restriction site criterion. (A) Examples of each of the 3 different levels of *gypsy-lacZ* expression (as well as the 3 corresponding inferred genotypes), which could be typically distinguished by β-galactosidase assaying the ovaries of females extracted from the experimental populations. In fact, the samples of ovaries shown here were dissected either from a permissive (left) or a restrictive (right) wOR; #12a X chromosomal clone or from the heterozygous F1 progeny (middle) obtained by crossing them with each other. The dense blue β-galactosidase staining of *flam*<sup>P</sup>/flam<sup>P</sup> follicle cells appears black on this grayscale picture. In *flam*<sup>R</sup>/flam<sup>R</sup> ovaries, only the centripetal follicle cells of stages 10–12 show this strong level of staining. (B) Illustration of the corresponding XmnI restriction site polymorphism. Heads of 3 individuals whose ovaries are shown in (A) were used to perform genomic DNA amplification in the *flam* region. The 1.27-kb PCR fragment produced by the homozygous permissive female (*flam*<sup>P</sup>/flam<sup>P</sup>) was missing the XmnI diagnostic restriction site. By contrast, the *flam*<sup>R</sup>/flam<sup>R</sup> female genomic PCR product was cut into 2 fragments (0.67 and 0.6 kb) characteristic of XmnI-containing X chromosomes. As expected, the heterozygous female produced the 3 possible bands in stoichiometric ratios (1.27 = 0.67 + 0.6). (C) The rare XmnI-containing restrictive X chromosomes inoculated into one of the most permissive populations (seeded with 95% of homozygous permissive founders devoid of this XmnI restriction site) had almost invaded the population at the 27th generation. Shown are the XmnI-digested amplification products of 8 pools of 5 genomic DNAs extracted from the heads of females that had been individually sorted into the major *flam*<sup>R</sup>/flam<sup>R</sup> class, according to their ovarian *lacZ* staining. The absence of uncut DNA indicates that each of these 40 females was also homozygous for the XmnI haplotype.

*The ZAM-LacZ Assay (Desset et al. 2003).* The ability of the 16 independent X chromosomal clones to repress the ZAM retroelement was tested using a similar assay as the aforementioned anti-*gypsy* resistance test. The reporter transgene consists of the ZAM promoter fused to the *Escherichia coli* *lacZ* gene. Tester females, containing this ZAM-*lacZ* transgene and unable to repress ZAM, were crossed to males under test and the level of presence/
<table><caption>**Table 1**
Assay of the *gypsy*-Repressing Ability of *flam* Alleles
Sampled from the DIS, Vendargues, and Corconnes
Natural Populations</caption><thead><tr><th>gypsy-lacZ Ovarian Expression Genotypical Class<sup>a</sup></th><th>Null Restrictive (strong)</th><th>Moderate Restrictive (weak)</th><th>Strong Permissive</th></tr></thead><tbody><tr><td>DIS13-1</td><td>5</td><td>0</td><td>10</td></tr><tr><td>DIS13-2</td><td>4</td><td>1</td><td>6</td></tr><tr><td>DIS13-3</td><td>12</td><td>4</td><td>0</td></tr><tr><td>DIS13-4</td><td>14</td><td>0</td><td>0</td></tr><tr><td>DIS13-5</td><td>7</td><td>6</td><td>1</td></tr><tr><td>DIS13-6</td><td>2</td><td>3</td><td>15</td></tr><tr><td>DIS13-7</td><td>10</td><td>3</td><td>0</td></tr><tr><td>DIS13-8</td><td>2</td><td>8</td><td>4</td></tr><tr><td>DIS13-9</td><td>12</td><td>2</td><td>0</td></tr><tr><td>DIS13-10</td><td>12</td><td>1</td><td>0</td></tr><tr><td>DIS13-11</td><td>8</td><td>12</td><td>1</td></tr><tr><td>Total<sup>b</sup></td><td>88 (54%)</td><td>40 (24%)</td><td>37 (22%)</td></tr><tr><td>Vendargues<sup>c</sup></td><td>60 (47%)</td><td>0</td><td>67 (53%)</td></tr><tr><td>Corconnes<sup>c</sup></td><td>39 (46%)</td><td>20 (24%)</td><td>25 (30%)</td></tr></tbody></table>

absence of β-galactosidase activity was monitored in the follicle cells of the heterozygous daughters.

## Results

### Further Evidence for a High Level of *flam* Polymorphism in Natural Populations

Table 1 shows that, when combined with a large deficiency of the *flam* locus (*Df(1)l<sub>11</sub>*, only half of the X chromosomes from the Uganda isofemale lines allowed complete expression of the *gypsy-lacZ* reporter transgene in the follicle cells of the ovary. About the same proportions were observed with 2 other natural populations, Vendargues and Corconnes, that were sampled and tested immediately after their capture en masse in the south of France.

The fact that 2 of the 3 populations exhibited what looks like different restrictivity levels is reminiscent of the whole range of variations due to the *flam* allelic series, previously described in the laboratory (Pelisson et al. 1997). However, because these intrapopulation variations were not further analyzed genetically, it is not known whether they actually reflect the presence of different types of *flam* restrictive alleles in the wild or if they result from the effect of modifiers on the restrictivity phenotype. Note, however, that such putative modifiers should be dominant unless they are either located in the small portion of the X chromosome uncovered by the *Df(1)l<sub>11</sub>* deficiency or already present in the tester X chromosome.

At the time of the test, 4 months after their capture, 5 out of the 11 DIS lines were still containing each of these

3 phenotypic categories, suggesting that this polymorphism, whatever its genetic determinism, could be maintained in these lines, at least during this short period of time.

### Isolation of 2 Pairs of *flam* Alleles from 2 Polymorphic Laboratory Strains

We then chose to study 2 simpler cases of *flam* polymorphism that each involved a single pair of alleles. As reported previously (Pelisson et al. 1997; Chalvet et al. 1999), wOR(P) and wSS(P) are 2 of the permissive strains that do not display any *gypsy*-induced mutability because they are devoid of functional *gypsy* copies. Surprisingly, some restrictive flies once happened to be serendipitously detected in a subline of each of these stocks. The frequencies of these intruders were high enough to enable their isolation, as described in Materials and Methods, in 4 restrictive X chromosomal clones that were called OR(R) and SS(R) to distinguish them from their 4 permissive siblings called OR(P) and SS(P), respectively.

A 1.27-kb fragment, located in the *flam* region, upstream of the DIP1 gene, was PCR amplified and sequenced in each of the 8 OR chromosomal clones (see Materials and Methods). All sequences were found to be identical except for a single T/C nucleotide change at position 677 (corresponding to position 21,441,509 in the genome assembly, release 4.2.1), which created a *XmnI* restriction polymorphism and enabled to discriminate the 4 *XmnI*-containing OR(R) clones from the 4 OR(P) permissive clones (data not shown). The fact that this single nucleotide polymorphism was linked to the restrictive/permissive character in the small sample of 4 OR(R) and 4 OR(P) clones suggested that the restrictive allele did not arise inside the OR(P) stock as a revertant, but more likely resulted from the casual contamination of this stock. Whatever the case, the putative contamination event has not introduced any wild-type *white* allele or any functional provirus that would otherwise have invaded the stock and caused its mutability. Southern blotting data (not shown) also confirmed the absence of effective contamination by functional proviruses containing the diagnostic *Hind*III site (Chalvet et al. 1998). The same putative contaminant chromosome cannot be at the origin of the SS(P)/SS(R) polymorphism because SS(R) chromosomes are missing the *XmnI* restriction site (data not shown).

A locus controlling the activity of the *ZAM* and *Idefix* retroelements was mapped at the base of the X chromosome (Desset et al. 2003). However, this locus was not reported to be very polymorphic, and indeed, as described in Materials and Methods, we could check that the 16 independent X chromosomal clones all contained the “stable” allele that is able to repress *ZAM* expression in the ovary (data not shown). So, our population cage study could be performed in the absence of any interference with this additional host-parasite interaction.

### The Permissive/Restrictive Polymorphisms of the wOR and wSS Strains Are Maintained in Experimental Populations Devoid of Active *gypsy* Proviruses

With the aim of an easy *flam* phenotyping, we wanted the *#12a* autosomal *gypsy-lacZ* reporter transgene

<footnote><sup>a</sup> Flies of natural populations were tested as previously described (Sarot et al. 2004), by crossing sets of 10 females with males of the A70 tester stock and assaying the β-galactosidase activity of the *gypsy-lacZ* reporter in individual ovaries of the hemizygous progeny. Each tested X chromosome, which had been thus combined with the *Df(1)l<sub>11</sub>* deficiency of the A70 stock, in the presence of the *#12a* *gypsy-lacZ* reporter was operationally classified as permissive or restrictive (strong or weak) according to the level of β-galactosidase expression observed in the corresponding hemizygous ovary (see Materials and Methods).</footnote>

<footnote><sup>b</sup> Pooled results of the tests performed with the 11 DIS isofemale lines collected in Uganda.</footnote>

<footnote><sup>c</sup> Pooled results of 6 and 3 tests, which were performed with females of both french populations, Vendargues and Corconnes, respectively.</footnote>
<figure><img src="image_4.png" /><figcaption>FIG. 3.—Evolution of the frequencies of 2 *flam* allelic pairs in experimental populations inoculated at various initial proportions. The data from the only population cage that was seeded with flies containing the wSS pair of *flam* alleles (10% of homozygous permissive and 90% of homozygous restrictive founders) can be distinguished from the others by the filled circle symbol. All populations were devoid of functional *gypsy* proviruses. One experiment (inoculated with 90% of wOR permissive alleles) had to be stopped prematurely at the 44th generation, before the equilibrium was reached, because of a contamination of the cage by flies devoid of the *miniwhite* transgene. The evolution of all the other populations slowed down after they had reached the frequency of about 10% permissive alleles.</figcaption></figure>

(Sarot et al. 2004) to be present in every fly of the experimental populations. That is why this transgene was first introduced into each of the 16 X chromosomal clones used to seed the populations, as illustrated in figure 1 and described in Materials and Methods. These *gypsy-lacZ*-containing stocks were used to seed 6 independent experimental populations (see Materials and Methods).

As shown in figure 2A, we could take advantage of the semidominance effect to phenotypically discriminate between the 3 different genotypes. In most cases, any ovary could thus be unambiguously assigned to 1 of the 3 genotypic classes. The robustness of this phenotyping could be further validated by the 2 following criteria. First, the frequencies of the 3 genotypes were always in the Hardy-Weinberg proportions (data not shown). Second, at the 27th generation of a population seeded with 95% OR(P); #12a founders, females were simultaneously tested both for the level of *lacZ* expression in their ovary and for the *XmnI* restriction site polymorphism described above. While most ovaries disclosed the absence of staining typical of the homozygous restrictive genotype, the corresponding genomic DNAs were all found to be homozygous for the *XmnI* restriction site (fig. 2C).

This haplotype, which had been introduced into the population by only 5% of restrictive founders, had therefore already invaded 80% of the cage by generation 27 (fig. 3). This observation ruled out the possibility of an unusually high rate of mutation that would have transformed most of the permissive alleles into restrictive alleles. Instead, the decrease of the permissive allelic frequency in all 4 permissive populations (fig. 3) was more likely to result from selection. In fact, a balancing selection might have operated because every population eventually tended toward the same frequency of about 10% permissive alleles, whatever the initial frequency (fig. 3). However, because this frequency is as low as that of the lowest inoculation frequency, we could not observe a rise of the permissive frequency up

to the plateau. So, we cannot discriminate between a stable equilibrium and a continuous decrease of permissive frequency. Although only studied in a single cage, the wSS *flam* polymorphism seemed to behave similarly and to be therefore driven by the same unknown selective forces as the wOR one.

## Discussion

*Gypsy* is controlled by a *Drosophila* gene called *flam*, the restrictive alleles of which, unlike permissive alleles, maintain this retrovirus in a repressed state. Permissive strains containing functional *gypsy* proviruses are unhealthy probably because they undergo frequent mutations that result from efficient *gypsy* replicative transposition. Stabilization of such mutable strains sometimes happens because of the fixation of restrictive alleles that stop the increase of genetic load (Pelisson et al. 1997). The persistence of permissive alleles, both in the laboratory and in the wild (Pelisson et al. 1997), might be explained by a low number of euchromatic, possibly functional, *gypsy* proviruses (Modolell et al. 1983; Bayev et al. 1984; Biemont et al. 1994) and a putative selective cost of the restrictive alleles in the absence of these parasites. To address these issues we set up cage populations and followed the evolution of a permissive–restrictive allelic pair that had been extracted from the wOR laboratory strain. In spite of the absence of functional *gypsy* proviruses in these populations, the frequency of the restrictive phenotype rose to a surprisingly high level, clearly demonstrating the absence of a cost of resistance.

The characteristics of this evolution were the following. 1) The dynamics of the restrictive/permissive phenotypes studied here truly resulted from selection, not mutation. The molecular nature of this allelic pair could not be assayed because the DNA sequence of *flam* is not yet available. However, we could take advantage of the fact that a polymorphic *XmnI* restriction site was specifically
linked to the 5% of restrictive alleles inoculated into one of the experimental populations. Twenty-seven generations later, when most chromosomes were phenotypically characterized as restrictive, this marker was still linked to each of them, providing strong evidence for an almost complete invasion of the population by the restrictive haplotype. 2) Indeed, whatever the composition of the initial inoculate (5, 10, 50, or 90% of restrictive alleles), every population tended to stabilize at the same restrictive frequency of 90%. However, we could not definitely discriminate between a real plateau due to some unknown mechanism of balancing selection, on one hand, and a continuous decrease of permissive allelic frequency due to directional selection, on the other hand. 3) The restrictive-permissive polymorphism originating from the unrelated SS strain also seemed to be stabilized at the same 90% restrictive level, suggesting that the same selective forces were operating on the restrictive and permissive chromosomes of both strains.

Even on the hypothesis of a true balancing selection, the effect of an additional selection, due to the presence of active gypsy proviruses in the nature, is expected to tend to the final elimination of permissive alleles from natural populations. However, as shown previously (Pelisson et al. 1997) and in the present study (table 1), such populations still exhibit high frequencies of permissive *flam* alleles. This might mean either that functional copies are too rare in the nature to have any significant impact on the *flam* polymorphism or that they are held in check by additional (copy-dependent, cosuppressionlike?) mechanisms able to take over the repression in permissive females.

Alternatively, because we could not perform the experiments for an infinite number of generations, we may have missed the ultimate elimination of the permissive alleles from the cages. Such a selection against homozygous permissive females could be explained, despite the absence of functional *gypsy*, if they were also sensitive to the deleterious presence of another hypothetical transposable element. The presence of this unsuspected cause of selection in the cages would have antagonized and eventually masked the selection cost of restrictive alleles. We are currently investigating the possibility that *flam* would confer resistance to other retroelements than *gypsy*.

Finally, it would be interesting to check that the probability of infecting an experimental population by *gypsy* virions, as previously described for a permissive genotype (Kim et al. 1994; Song et al. 1994), will be reduced by the presence of restrictive alleles in this population. Should this prediction be correct, then the unknown mechanism that maintains high levels of restrictive alleles independently of the presence of *gypsy* might be able to set up a sort of innate immunity, at the population level, against the horizontal transfer of an otherwise vertically transmitted retrovirus.

## Acknowledgments

We are very grateful to François Rouyer and André Klarsfeld for generously sharing the population cage system and to Daniel Lachaise, our late colleague, who kindly gave us the DIS lines. We thank Nicolas Gilbert for critically reading the manuscript. Significant improvements

of the manuscript are also due to insightful comments by unknown reviewers. This work was supported by grants from the Centre National pour la Recherche Scientifique, the Association pour la Recherche sur le Cancer, and the E.C.'s Research Training Network Silencing in Different Organisms.

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Koichiro Tamura, Associate Editor

Accepted November 15, 2006