Discussion
We found functionally different impacts of thermal stress at different life-history stages on fertility in Drosophila virilis . Pupal heat stress delays the age of reproductive maturity (ARM), whereas adult heat stress sterilises most males. Many stressed adult males are fertile immediately post-heat stress but lose fertility over a week and remain permanently sterile for the duration measured. Heat-induced sterility inDrosophila melanogaster has been associated with disruptions to spermatid elongation during spermatogenesis (Rohmer et al. 2004). Therefore, it is possible that mature sperm stored in the seminal vesicles of adult males are relatively unharmed and can be used by stressed males, whereas immature sperm are destroyed and the capacity to produce sperm is disrupted. However, it is unclear why pupae appear to recover fertility over the course of the experiment, whereas adults remain sterile. Benign adult males saw a drop-off in fertility over the last two time-points. Therefore, it is possible that the combination of heat-induced sterility and natural ageing prevent heated adult males from recovering fertility over the experiment. Exploring how fertility is affected by high temperature at the pupal and adult stages by looking at sperm production over an individual’s lifetime may be necessary to disentangle these differences.
We found pupae were more thermally robust than adults. At 38°C, non-hardened adult D. virilis cannot survive, whereas pupae show high survival, and their ARM is delayed but eventually recovers. This contrasts with some previous studies that find pupae to be a particularly sensitive life-stage to thermal stress. For example, a recent study examining flour beetles found that pupae and immature males are the most vulnerable life-stages to both fertility loss and survival at high temperatures (Sales, Vasudeva & Gage 2021). Similarly, inD. melanogaster , non-hardened pupae have only slightly lower upper lethal limits than adults (Moghadam et al. 2019). With no obvious pattern in how life-stage interacts with heat-induced death and sterility across species groups, it is clear that studies on thermal limits should consider examining all life stages that are likely to be exposed to high temperatures in the wild.
As expected, we found D. virilis can improve high temperature survival through prior hardening at sub-lethal stress temperatures. This response occurs in both life-history stages measured. The effect is sex-specific in adults such that heat-hardened males show higher survival over heat-hardened females at lethal temperatures. A meta-analysis on sex differences in acclimation capacity, including fourDrosophila species, found no significant differences in overall acclimation capacity between males and females (Pottier et al.2021). However, the authors found that where differences between sexes exist, females appear to have higher acclimation capacity than males. It has previously been shown that D. virilis female fertility is robust to high pupal temperatures when compared with male fertility (Walsh et al. 2020). It follows that females would be able to utilise the improved survival at high temperatures by reproducing. This makes the finding that heat-hardened males actually show higher survival than females surprising, as it is difficult to see the fitness benefit gained by permanently sterilised males surviving high temperatures.
In contrast to survival, we found no significant protective impact of this same hardening treatment on fertility at sterilising temperatures. This is true for both pupae and adults, suggesting that, although prior heat-hardening improves survival at lethal temperatures, it does not protect male fertility. Whereas previous studies found a positive impact of heat-hardening on reproduction (Jørgensen, Sørensen & Bundgaard 2006), here we find no measurable benefit of heat-hardening on fertility. Given the clear physiological plastic response we demonstrate for survival, it is highly surprising that fertility is not also protected. This suggests it may be difficult for thermal limits to fertility to be improved during short-term high-temperature events.
We tested relatively short periods of hardening and stress, but longer-term acclimation to high temperatures can influence reproduction. In the flour beetle Tribolium castaneum , adult male development at stressful temperatures results in males producing sperm with shorter tails (Vasudeva et al. 2019). This is shown to be an adaptive morphological shift, with shorter sperm doubling performance when males are reproducing at high temperatures. Similarly, a recent study inD. melanogaster found that a three-day acclimation period prior to mating increases mating success by around 70% at stressful temperatures (Stazione, Norry & Sambucetti 2019). It is known that the timing of heat-shock and heat-hardening/acclimation can drive differences in the response to temperature stress (Weldon, Terblanche & Chown 2011; Zhang, Storey & Dong 2021). Possibly, there is a delay for any physiological response to ‘kick-in’ before components of fertility can be protected. Many experiments demonstrating thermal plasticity of reproductive traits utilise multiple-day stress treatments (Stazione, Norry & Sambucetti 2019; Vasudeva et al. 2019), or delays between ‘hardening’ and thermal stress (Jørgensen, Sørensen & Bundgaard 2006). However, natural populations caught during the peak midday sun of a heatwave may not realistically have the opportunity to ‘ramp-up’ their physiological response. Clearly plasticity in reproductive traits is possible, however its general capacity to allow organisms to cope with climate change is still unclear (Sgrò, Terblanche & Hoffmann 2016). If a similar lack of strong or robust short-term heat-hardening for fertility is found across taxa, then organisms may be more vulnerable to climate change than previously thought.
Superficially, it seems that improving survival of males via heat-hardening may be less beneficial to fitness than previously thought, given that males may be alive but permanently sterilised. Parratt et al. (2021) found that males from 19 of 43 Drosophila species could survive apparently permanently sterilising temperatures, suggesting there must be a biological explanation. The adaptive benefit of heat-hardening is particularly confusing if it protects survival without allowing individuals any opportunities to reproduce. However, a key finding here is that both life-stages measured still have a limited capacity to reproduce after heat-shock. Males heated as pupae are eventually sexually mature, and heated adult males can reproduce within a few days, before long-term sterility manifests. Therefore, the improved survival at extreme temperatures may provide more males with these limited opportunities to use up surviving mature sperm, without protecting reproductive traits directly. It is also possible that if males sterilised as adults were kept long term, they may restore some fertility over time. Alternatively, male hardening could simply be a neutral by-product of selection on females for survival at high temperatures, as females are far better able to maintain fertility at near-lethal temperatures (Walsh et al. 2020).
To gain a more complete understanding of how natural populations will be affected by heat-waves, measuring the difference of survival and fertility between life-stages will be important. Our findings also suggest that research needs to consider that heat-hardening may not be a sufficient plastic rescue mechanism, although heat hardening effects on fertility in more taxa need to be tested. Importantly, studies showing the positive effects of heat-hardening should consider whether surviving individuals are fully fertile. This will allow researchers to more fully understand the adaptive benefits of these responses.