In a classic example of the invasion of a species by a selfish genetic element, the P-element was horizontally transferred from a distantly related species into Drosophila melanogaster. Despite causing ‘hybrid dysgenesis’, a syndrome of abnormal phenotypes that include sterility, the P-element spread globally in the course of a few decades in D. melanogaster. Until recently, its sister species, including D. simulans, remained P-element free. Here, we find a hybrid dysgenesis-like phenotype in the offspring of crosses between D. simulans strains collected in different years; a survey of 181 strains shows that around 20% of strains induce hybrid dysgenesis. Using genomic and transcriptomic data, we show that this dysgenesis-inducing phenotype is associated with the invasion of the P-element. To characterize this invasion temporally and geographically, we survey 631 D. simulans strains collected on three continents and over 27 years for the presence of the P-element. We find that the D. simulans P-element invasion occurred rapidly and nearly simultaneously in the regions surveyed, with strains containing P-elements being rare in 2006 and common by 2014. Importantly, as evidenced by their resistance to the hybrid dysgenesis phenotype, strains collected from the latter phase of this invasion have adapted to suppress the worst effects of the P-element.
Discovered in the 1950’s by Barbara McClintock [1], transposable elements (TEs) are the ‘ultimate parasite’ [2,3]. They are genetically simple, with autonomous elements consisting of as little as one protein coding gene, but remarkably successful, comprising much of the bulk of large eukaryotic genomes [4,5]. Those that persist do so by inserting copies of themselves into new genomic locations faster than they are eliminated. TEs can also be a source of beneficial mutations; particular TE insertions have been implicated in a number of adaptive phenotypes (reviewed in [6]), and some TE families now form essential components of genomes [7,8]. But most TE insertions are probably deleterious, as insertions can damage genes and may impose other fitness costs on their host [9–11]. In fact, the mutagenic properties of transposons have been harnessed by molecular geneticists to disrupt thousands of Drosophila genes (e. g. , [12]). In the short-term, transposable elements are vertically transmitted, and so can only cause limited damage to their hosts without extinguishing themselves. In the long-term, however, they are also horizontally transmitted [13–15], and, initially, may cause substantial damage to their hosts [16,17]. The best-studied example of a horizontally transferred element is that of the P-element, a DNA transposon. P-element was acquired by D. melanogaster by horizontal transfer from a distant relative, D. willistoni [18], and spread rapidly through the species, as evidenced by its absence from old laboratory stocks collected before the P-element was widespread [19–21]. In its native species, and in current D. melanogaster populations, the P-element is not associated with any gross defects [18,22]. But when males from strains collected after the invasion (‘P strains’) are crossed to females from old laboratory stocks (‘M strains’), the F1 offspring suffer a number of abnormalities, including recombination in males, increased mutation rates, frequent sterility, and abnormally small (dysgenic) gonads [16,19,23–25]. These abnormalities, collectively called ‘hybrid dysgenesis’, appear to be due to DNA damage caused by actively transposing P-element in the germline [17,22,26,27]. In the reciprocal direction, the F1 offspring, though genetically identical, are normal. The reason is that these offspring are protected by piRNAs (PIWI interacting RNAs, a type of small RNA) homologous to the P-element, which are sequestered in the egg cytoplasm of P-type females. As these protective piRNAs target the P-element mRNA for degradation [28], their presence reduces P-element activity and the resulting DNA damage that lead to hybrid dysgenesis. Since its initial discovery, P-element has been one of the most important tools in Drosophila genetics, providing the basis for genetic manipulations and mutagenesis (e. g. , [12,29]). The P-element had been thought to be absent from D. melanogaster’s close relatives [30], but recent work has detected it in D. simulans, apparently horizontally acquired from D. melanogaster [31]. As the D. simulans elements recovered are identical in sequence to a type that segregates at low frequency in D. melanogaster, and do not include the most common D. melanogaster element, this acquisition is likely due to a single transfer event [31]. Here, we report a hybrid dysgenesis-like phenotype in crosses between strains from different D. simulans populations, and show this phenotype occurs in crosses between strains collected before and after a rapid invasion of the P-element.
We assayed for a hybrid dysgenesis-like phenotype in D. simulans, using the absence of developed ovaries as an indicator (Fig 1 and S1 Fig, see also [24,32]). We considered a pair of strains to show hybrid dysgenesis when the F1 females from reciprocal crosses show a significant difference in the number of dysgenic ovaries [with significance defined as p ≤ 0. 05 in a Fisher’s exact test (FET) ]. Differences between F1 females from reciprocal crosses, which have identical nuclear genotypes, are likely due to different maternal contributions to the egg cytoplasm and implicate a cyto-nuclear incompatibility, as in the D. melanogaster hybrid dysgenesis system [19,28]. For our initial survey, we used 22 strains collected in Georgia in 2009 (generously donated by P. Haddrill) and 10 strains collected in Madagascar in 2004 & 2008 (collected by B. Ballard and J. David; S1 Table). We paired strains from the two populations randomly, but ensured that each strain was involved in multiple crosses (at least 5 for the Madagascar strains and at least 2 for the Georgia strains). Among these crosses, 38. 7% (36 of 93) showed unidirectional gonadal dysgenesis in F1 females at 29°C, a temperature which induces hybrid dysgenesis in D. melanogaster, but not at 25°C (S2 Fig). In addition to transposable elements, cyto-nuclear incompatibilities can also be caused by the intracellular bacteria Wolbachia. This is particularly likely here, as Georgia lines harbor a Wolbachia strain [33,34] which appears to induce embryonic lethality in the offspring of crosses to Madagascar males. When we repeated the crosses after curing the strains of their Wolbachia infections, however, we found qualitatively similar results for infected and uninfected versions of the strains (S2 Fig, FET on data from the same cross performed with cured and uncured strains, p > 0. 05 for all). Inspection of the data shows that dysgenic crosses almost always involved one of five Georgia lines as the paternal line (S2 Fig). Control crosses between these “dysgenesis-inducing” (DI) lines showed no significant unidirectional dysgenesis, nor did crosses of these DI lines to 12 of the other Georgia lines (a subset of crosses are shown in S2C and S2D Fig). In contrast, all 10 strains from Madagascar were “dysgenesis-susceptible” (DS) when crossed to DI lines; crosses between these lines also resulted in no hybrid dysgenesis (S2 Fig). The DI and DS classes are analogous to the P-type and M-type of D. melanogaster, respectively. Using the Georgia and Madagascar strains with known DI and DS phenotypes, we unambiguously cytotyped 160 additional strains (cured of Wolbachia) from 23 populations sampled between 2002 and 2014 (Fig 2 and S3 Fig, S1 Table). We reciprocally crossed each strain to at least one known DI and DS strain, and roughly classified tested strains based on the results of the four crosses (see Materials and Methods). In addition to DI and DS types, we also observed a third type of strain, “dysgenesis-resistant” (DR), which produce no dysgenic offspring in either cross. These correspond to the Q types in D. melanogaster, which repress transposition of the P-element, but which contain few or no active elements [25,35]. In fact, the resistant strains probably encompass a range of intermediate cytotypes (i. e. , corresponding to the M’, Q and weak P-types in D. melanogaster [17,25,35–38]). It is likely that these strains contain copies of the dysgenesis-causing element, but in degraded form, at low copy number, or with weak expression. These copies may nevertheless be able to produce piRNAs suppressing the TE [28,39]. Overall, 19. 3% of strains were DI, 26. 5% DR, and 54. 2% DS. Fertility assays performed on a subset of these crosses show that F1 females from dysgenic crosses show higher rates of sterility and produce fewer offspring than their genetically identical sisters from reciprocal crosses (S4 Fig; Mann-Whitney U test, p < 0. 05). We reasoned that, as in D. melanogaster, the DS and DI strains might show temporal and/or geographical differences [20,21,49–51]. In fact, the frequency of the DI and DR dysgenesis types increase over time (binomial GLM; Africa: z = 5. 822, p = 5. 81x 10−9), and previous work shows differences in P-element content between a samples from 2010 and 2012 [31]. We tested a collection of 631 samples (isofemale lines, ethanol-preserved flies, and DNA extracted from wild flies) collected from 54 locations between 1984 and 2014 for the presence or absence of the P-element (S1 Table, Figs 3 and 4). To detect P-element, we used PCR, as before, with multiple sets of primers and repeated independent DNA extractions (see Materials and Methods). As before, the primers were used to amplify products that correspond to the full-length element and to each of the four exons. As for the cytotyped lines, we found strains with full-length copies, strains with only partial copies (in which at least one exon consistently failed to amplify), and strains with no successful amplification of any of the tested P-element fragments. The partial copies were usually missing internal exons, a common by-product of imperfect excision events in D. melanogaster [16,52]. Strains with full-length elements often also contained other, partial copies with similar internal deletions. The fraction of strains containing the P-element increases over time on all continents surveyed. In collections from 2004 to 2009, strains free of the P-element predominate (Fig 3A). After 2009, most strains harbour some form of P-element, and many have full-length elements (Fig 3B). D. sechellia, a close relative of D. simulans with which it is known to hybridize [53,54], remains P-element free in our 2014 samples (kindly provided by D. Matute, Fig 3B). We find a significant association between when a strain was collected and whether it contains any P-element copies, regardless of the geographic region from which it was collected (Fig 4, binomial GLM with year, geographical region, and an interaction term fit to the P-element for each strain; year as z = 5. 218 p = 1. 81x 10−7). By 2013, almost all sampled strains contained P-elements in some form (Fig 3). It is formally possible that this apparent spread is instead due to the loss of P-element in strains propagated for several years in the laboratory. This process could not have occurred, however, in the Portuguese samples, for which genomic DNA was extracted from directly wild flies, and these show a similar pattern (compare Portuguese samples from 2006 and 2013 in Fig 3). The rapidity of the spread makes it difficult to identify a likely origin point for the invasion, as the P-element appears to have invaded all three regions nearly simultaneously (Fig 4), with statistical differences between regions (S3 Table) apparently due to low prevalence of the P-element in a few late samples, rather than pointing to a geographic origin of the invasion.
Here, we identified a hybrid dysgenesis-like system in D. simulans. Using next generation sequencing information and PCR, we find that the P-element, which has recently invaded D. simulans [31], is associated with the hybrid dysgenesis phenotype, and spread rapidly across Europe, Africa, and North America. In fact, compared to D. melanogaster, the spread of the P-element in D. simulans is surprisingly fast: In D. melanogaster, the first P-type fly was collected from the wild in 1954, and most wild strains were infected by 1974 [20]. We find the first evidence of the P-element in D. simulans samples from 2006, with P-elements appearing in nearly all lines sampled since 2013, seven years later (apart from one sample from Ethiopia; Fig 3, Fig 4, S1 Table). There are a number of reasons the P-element invasion may have occurred more rapidly in D. simulans than in D. melanogaster. For one, D. simulans appears to enjoy lower rates of P-element induced gonadal dysgenesis than D. melanogaster; high rates of sterility are expected to hamper the spread of the P-element by reducing the fitness of its carriers. Direct comparisons between the species are problematic, as the severity of hybrid dysgenesis increases with copy number, which differs between the species ([19,24,26,31]; S2, S4 and S6 Figs). But in comparisons between artificially constructed D. melanogaster and D. simulans strains with comparable copy numbers, P-element does appear to be less active in D. simulans [32,45,55]. Another, simpler explanation for the difference in the speed of invasion is higher migration rates between D. simulans populations than for D. melanogaster as they experienced the P-element invasion. Such a scenario seems reasonable: Unlike D. melanogaster, D. simulans cannot over-winter in temperate regions, and instead reinvades the northern limits of its range annually. Sequence data correspondingly suggests a high rate of homogenization between D. simulans populations at nuclear loci [56–61]. Furthermore, for both species, migration might be largely human-aided, and the global movement of fruit has increased dramatically in the years since the spread of the P-element in D. melanogaster. That is, global imports and exports of fleshy fruits (measured in tons) grew about 4% per year from 1961–2013 (the years tracked by the Food and Agriculture Organization of the United Nations Statistics Division). As a result, the average tonnage of fruit imports was 5-fold higher in the years measured during the spread of the P-element in D. simulans compared to D. melanogaster (for imports, 1961–1974: 12. 5 vs. 2006–2014: 61. 5 million tons/year; exports tracked imports closely). While these statistics capture only the movement of fruit between countries, the increase in movement seems likely to have boosted the global spread of the P-element in later years. The P-element in D. simulans adds to a handful of documented cases of the spread of a horizontally transmitted element through a Drosophila species [33,62–65]. Several of the other cases include intracellular bacteria, which are capable of behaving selfishly, but in these cases also appear to be conferring a benefit to their hosts [64–67]. In contrast, the P-element appears to be spreading mainly via a selfish mechanism; though the occasional insertion may be beneficial [22,68]. In response, both D. simulans and D. melanogaster have quickly evolved to suppress the dysgenesis phenotype by the end of the invasion (present study, [20,38]), suggesting strong selection to ameliorate the the P-element’s worst effects, and underscoring the evolutionary challenge posed by selfish elements.
We used D. simulans isofemale lines collected across multiple locations over 15 years (see S1 Table for details). Flies were reared on molasses–yeast–agar Drosophila medium at 18°C unless otherwise stated. The species identity of all lines was confirmed either by visual inspection of male genitalia or, when this was not possible, by amplification of PCR products that yield species-specific product lengths (using primer sequences for Argonaute2, FBgn0087035, kindly provided by D. Obbard: Mel_Sim_F 5′-CCCTAAACCGCAAGGATGGAG-3′, D_sim_303_R 5′-GTCCACTTGTGCGCCACATT-3′, D_mel_394_R 5′-CCTTGCCTTGGGATATTATTAGGTT-3′). Unless otherwise stated, all lines were cured of Wolbachia infections before crosses were performed, in order to obtain sufficient offspring from crosses that normally show high levels of cytoplasmic incompatibility. (If known, the infection status of lines prior to curing is given in S1 Table.) To cure lines, we raised larvae on food supplemented with 0. 05mg/ml tetracycline-hydrochloride for two generations. After curing, flies were tested for Wolbachia using PCR, with primer sequences wsp81F (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and wsp691R (5′-AAAAATTAAACGCTACTCCA-3′), and a cycling program of 94°C for 5' followed by 30 cycles of 94°C 30" , 55°C for 60" , 72°C for 2' , and ending with one cycle of 72°C for 10' , as in [69]. To find TEs expressed in the Florida lines, we used RNA-seq data form a pooled sample of lines from this population (accession number PRJEB7936 [31]). Paired end Illumina reads were mapped to a D. simulans reference genome produced from a Madagascar strain [44] and 179 Drosophila TE sequences (http: //flybase. org/; [71,72]) using GSNAP ([73]; parameters: -n = 1, -N = 1). We examined whole genome sequence from the Florida strains using paired-end Illumina reads (SRA PRJEB7936 [31], and PRJNA308281) collected from 12 barcoded individual F1 offspring of the M252 reference strain females (sequence data under SRX504933) crossed to males from Florida (2010) isofemale lines, and the paired end reads from the M252 reference strain [44]. We mapped these data to the D. simulans M252 reference genome using BWA-SW with the default parameters [74,75] and following the mapping protocol described in [76]. We counted the number of insertions per individual sequenced in two ways: First, we used PoPoolationTE [74], with the requirement that each insertion be supported by at least 3 reads. Second, due to the fragmented nature of the D. simulans genome sequence, some insertion sites may not be recovered by PoPoolationTE, which relies on mapping reads to a reference. We therefore also estimated TE copy number per family by comparing average coverage of the TE sequence to average coverage of chromosome arm 2L. Primers for TEs were designed based on the EMBL sequences (S4 Table, http: //flybase. org/; [71,72]). The following PCR program was used: 94°C for 5' followed by 30 cycles of 94°C 30" , 55°C for 60" , 72°C for 1–3' (depending on the expected length of the PCR product, 1 minute per kilobase); 72°C for 10' . PCR products were run on a 1% agarose gel to test for the presence or absence of a TE. To survey for the P-element, primers were designed for each exon of P-element (S4 Table). The presence of full P-elements was assessed using the forward primer for exon 0 and the reverse primer for exon 3, and using a primer annealing to both ends of the P-element (in the TIR region; sequence kindly provided by L. Teysset). To ensure that PCR failures are due to the absence of P-element, DNA quality was checked with a positive control PCR for an essential gene, and D. melanogaster Harwich P was used as a positive control in all PCRs. Sanger sequencing (performed by LGC Genomics, http: //www. lgcgroup. com/) of a subset of PCR products was used to confirm that the amplicons correspond to P-element sequence (Genbank accession numbers KU719478-KU719502); the sequence of additional sequencing primers is available upon request. We required a minimum of two independent DNA extractions to consistent results for each line. To perform RT-PCR, RNA was isolated from Florida, Georgia, Harvard, Maryland and Madagascar lines using Peqlab TriFast RNA isolation protocol, and extractions checked on a 1% agarose gel following treatment with DNase. We then performed RT-PCR using QIAGEN OneStep RT-PCR Kit, using the following program to detect full-length P-element RNA (using the forward primer for exon 0 and the reverse primer for exon 3): 45°C for 30' for reverse transcription, then 95C for 15' , followed by 30 cycles of 94C for 15" , 55C for 60" and 68C for 6' ; 68C for 10' to amplify cDNA. Using the same program, reverse transcription was also performed separately for both forward and reverse of the P-element (using either the forward primer for exon 0 or reverse for exon 3) to generate unidirectional cDNA for PCR. All statistical analyses were performed using R [77]; https: //www. r-project. org/). To estimate global trade in fruits, we downloaded data for total worldwide imports and exports of fruits and vegetables for the years 1961 to 2013 (measured in tons) from the FAOstat website (http: //faostat3. fao. org/download/T/TP/E). From these, data for nuts, vegetables, and processed foods were excluded, resulting in data for 29 categories of fleshy fruit and berries. Using the nls function in R, we fit exponential growth models to these data of the form y = a exp (bx), where y is the exports in tons, x is the number of years since1961, and a and b are the estimated intercept and growth rate, respectively.
Some genes perform necessary organismal functions, others hijack the cellular machinery to replicate themselves, potentially harming the host in the process. These ‘selfish genes’ can spread through genomes and species; as a result, eukaryotic genomes are typically saddled with large amounts of parasitic DNA. Here, we chronicle the surprisingly rapid global spread of a selfish transposable element through a close relative of the genetic model, Drosophila melanogaster. We see that, as it spreads, the transposable element is associated with damaging effects, including sterility, but that the flies quickly adapt to the negative consequences of the transposable element.
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Hybrid Dysgenesis in Drosophila simulans Associated with a Rapid Invasion of the P-Element