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.