Robustness of recombination frequency estimation assay
A total of 31,229 flies were scored for this experiment. These flies were roughly evenly distributed among treatments, with between 16 % and 23 % of the total flies corresponding to each treatment. Within the control treatment, the average crossover frequency observed among replicates is 21.4 cM, which corresponds well to the published genetic map distance of 20.4 cM [29]. Given that our assay for estimating recombination frequency depends on visible markers, we first confirmed that, as previously suggested [29], there were no fitness effects associated with these mutations. To assess this, we test for a deviation from the expected 1:1 ratio of phenotype classes. Specifically, if there are no fitness effects associated with the e and ro mutations, we expect to see equal numbers of wild-type (++) and double-mutant progeny (e ro), and equal numbers of both recombinant phenotype classes (+ro and e+). We summed the progeny counts across all replicates within a given treatment and used a G-test for goodness of fit to test for viability defects associated with the marked chromosomes. The data are presented in Additional file 1: Table S1. For each treatment, we saw no significant deviation from the expected 1:1 ratio for either the ratio of two nonrecombinant phenotypes (++:e ro) or the ratio of the two recombinant phenotypes (e+:+ro) (Bonferroni-corrected P > 0.1, all tests). This suggests that any viability defects associated with the marked chromosome are too small in magnitude to markedly affect recombination frequency as estimated by our backcrossing assay. Coupled with the observation that crossover frequency observed in our control treatment aligns well with the expected map distance between these two markers, these data suggest that this recombination assay is both robust and accurate.
Effects of heat-shock on recombination frequency
The observation that meiotic recombination frequency in Drosophila females increases in response to acute heat shock was first made fifty years ago [21]. Though this has been confirmed more recently [27], the timing of the recombinational response to heat shock appears quite variable. While it was initially shown that recombination frequency increases in response to heat shock, that increase did not manifest until 5 days after treatment [21]. In contrast, later work showed clearly that recombination frequency increases in the three-day window following treatment [27]. This difference is critical for understanding the mechanism underlying the observed increase in recombination frequency; while the 5 day delay between treatment and an increase in recombination is indicative of a meiotic origin of the increased crossing over [20, 21], an immediate effect of heat-shock points to an alternative mechanism such as transmission distortion as has been argued elsewhere [30]. This stems from the knowledge that D. melanogaster crossovers are initiated and resolved in developing oocytes 4-5 days before eggs are fertilized and laid [31, 32]. Thus, an increase in recombination frequency due to transmission distortion may manifest very soon after an environmental perturbation, while an increase in recombination frequency due to increased crossing-over during meiotic prophase is not expected to manifest for 4-5 days.
Another striking observation that emerges from the literature is the marked variation in the magnitude of the recombinational response to heat shock relative to controls, ranging from a 2-3 fold increase [21] to a 10-fold increase [27]. While some of this variation is likely due to the size of the interval used for estimating recombination frequency, which necessarily imposes limits on the maximum possible increase in recombination frequency one could observe, it may also be due in part to experimental differences in administering heat shock with respect to exposure temperature, exposure duration, and the age of the exposed females. To shed additional light on the timing of the recombinational response to heat shock and to test the hypothesis that the magnitude of the increase in recombination frequency in response to heat shock is dependent on the duration of treatment, we measured recombination frequency in females exposed to 35° for 0, 6, 12, 18, and 24 h. We only surveyed eggs laid in the first 5 days following exposure for recombination frequency estimation; any perturbation in recombination frequency observed in this period is likely not meiotic in origin but rather primarily due to transmission distortion.
Results from our linear regression model indicate that both duration of exposure (‘time’) and ‘batch’ (see Materials and Methods) both significantly affect recombination frequency. The effect of batch (P = 0.0026 for recombination rate and 7.8 × 10−4 for number of offspring) is likely due to small variations in fly media and humidity conditions between the two batches. Our data confirm that recombination frequency increases in response to acute temperature stress. We further find that the magnitude of the increase in recombination frequency in heat-shocked females relative to control females significantly increases with increasing duration of heat-shock (Fig. 2). Interestingly, while there is a linear component to this relationship (P = 3.6 × 10−8), recombination frequency appears to increase quadratically across treatments and indeed, the time2 term in our linear model (see Materials and Methods) also significantly contributes to the variation in recombination frequency observed in this experiment (P = 0.021). Non-parametric analysis confirms a significant effect of exposure time on recombination frequency (P = 4.4 × 10−6, Kruskal-Wallis test).
Though previous work consistently indicated that heat shock is associated with an increase in recombination frequency, the extent to which such an effect could be observed in the first few days following treatment was inconsistent among studies [20, 21, 27]. That we observe a perturbation in recombination frequency so quickly after heat-shock has been administered strongly suggests that this perturbation is mediated at least in part by transmission distortion. A rapid change in recombination frequency in response to environmental conditions including heat shock, cold shock, and mating stress in Drosophila has been documented previously [11, 27], and this likely implicates transmission distortion as an underlying mechanism rather than an increase in crossing-over during meiosis as explained above. We hypothesize that the transmission distortion is mediated by an asymmetry in meiosis II, and future work will be aimed at testing this hypothesis.
Our data thus support the idea that Drosophila females can plastically alter their recombination fraction in rapid response to environmental conditions. The extent to which this is generalizable remains unknown. Future work will be aimed at determining the role of genetic background in the recombinational response to heat shock, as well as whether other acute or chronic stressors can perturb recombination frequency via transmission distortion. It will be particularly interesting from an evolutionary perspective to assess whether this transmission-distortion mediated increase in recombination frequency is observed in other taxa, as it would represent a powerful mechanism by which organisms could generate genetic diversity within their offspring in response to environmental conditions on an ecologically-relevant timescale.
Also unexpected was the non-linear relationship between the recombination frequency and the duration of exposure to heat shock conditions. That the relationship appears to be quadratic in nature may point to some feedback mechanism at the organismal level that serves to mediate the recombinational response to acute or chronic stress. However, it is important to note that in spite of the quadratic dependence of recombination frequency on exposure time, the increase in recombination frequency between the treatment with the longest exposure (24 h) and the control remains modest, at 25 %, in comparison to previous work which showed up to a 1000 % increase in recombination frequency [27]. Though this is certainly due in part to our choice of interval which, at 20 cM, only enables detection of increases in recombination frequency up to ~150 %, other genetic and environmental factors are likely to contribute to this as well. Previous work on the frequency of homologous recombination in somatic tissue of thermally-stressed plants illustrates clearly that the magnitude and direction of the change in homologous recombination frequency depends on temperature, the age at which the plants were exposed, and the duration of exposure [33]. As alluded to earlier, recombination frequency in Drosophila is remarkably plastic, varying in response to a number of factors including age, temperature, nutritional status, and genetic background [7, 9, 17, 19, 24, 27, 30, 34]. It therefore seems likely that some of the differences among studies are due to differences in environmental conditions.
Further work is required to assess the extent to which factors such as genetic background and maternal age mediate the magnitude of the recombinational response to stress. An interesting and open question is whether, for instance, phenotypic plasticity in recombination frequency varies as a function of maternal age. In addition, exploring the relationship between recombination frequency and the duration of exposures to other types of stressors will be of interest, and will illustrate the extent to which the quadratic dependence of recombination frequency on exposure time observed in the current study is generalizable. If this appears to be a canonical response, this may ultimately help in identifying signaling pathways underlying the organismal response to heat shock in particular or stress in general that lead to perturbations in recombination frequency.
Effects of heat-shock on offspring production
The evolution of plastic recombination remains puzzling. As described earlier, one model for the evolution of plastic recombination is the fitness-associated recombination model, which was developed as a haploid model [25] and subsequently studied in the diploid case [26]. The foundation for this model is a negative correlation between organismal fitness and recombination frequency; such a correlation allows for all of the benefits of recombination (potentially bringing together favorable alleles to create higher-fitness haplotypes) without incurring the cost of recombination (destroying favorable combinations of alleles in high-fitness haplotypes). This correlation can manifest in several ways, including stress-associated recombination. Our results thus provide an opportunity to test whether the negative correlation between fitness and recombination frequency underlying the fitness-associated recombination model is supported in the case of acute thermal stress.
The general relationship between fitness and recombination in Drosophila appears far from straightforward. Early work found a strong, negative correlation between fitness and recombination frequency [35] based on natural, population-level genetic variation in both of these traits. More recent work exploiting a combination of wild-type and marked laboratory lines revealed no significant correlation between offspring production and recombination frequency [10]. Recombination and fitness have also been found to be negatively correlated in heat-shocked flies, but not in control, mating-stressed, or cold-stressed flies [27]. Our results show that fitness (as measured by progeny production) significantly decreases with increasing duration of exposure to heat shock conditions (P = 2.1 × 10−4; Fig. 3). Non-parametric analysis confirms this finding (P = 0.0007, Kruskal-Wallis test). The average progeny count for control flies was 487 progeny/bottle, with average progeny counts of 450, 418, 386, and 341 for the 6, 12, 18, and 24 h heat shock treatment flies, respectively (Fig. 4). This corresponds to a ~30 % decrease in progeny production between the control and 24-h treated flies. The increase in recombination frequency with increased exposure (Fig. 2) coupled with the decrease in progeny production with increased exposure (Fig. 3) yields the requisite negative correlation between fitness and recombination frequency required in the fitness-associated recombination model. However, it is important to note that while fitness and recombination are negatively correlated, this relationship does not appear causal. Rather, exposure time appears to be independently driving both the increase in recombination frequency and the decrease in offspring production. This is evidenced by the observation that within any given treatment, there is no significant correlation between recombination frequency and offspring production (Fig. 4). If recombination frequency was functionally linked to offspring production or vice versa, then we would expect that within an exposure treatment, changes in recombination frequency would be associated with changes in fitness, which is not what is observed. Thus, the overall correlation observed between fitness and recombination appears driven by the third variable, exposure time.
Although our findings support the correlation between fitness and recombination frequency that is central to the fitness-associated-recombination model [25], it is important to note that this does not necessarily suggest that this model is likely to be applicable for natural populations of Drosophila. Theoretical work illustrates that the fitness-associated recombination is less likely to evolve in diploids relative to the haploid case. Specifically, the evolution of fitness-associated recombination in diploids requires 1) a mechanism by which a recombination modifier is provided information about the haplotype on which it resides, 2) cis-trans epistasis, or 3) maternal effects on fitness [26]. While there is certainly evidence in support of maternal effects on fitness in Drosophila (e.g. [36]), it is yet unknown whether stress-associated reductions in fitness such as those observed in the current study also yield maternal fitness effects.