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\begin{document}

\title{Plastic responses of survival and fertility following heat stress in
pupal and adult \emph{Drosophila virilis}}



\author[1]{Benjamin Walsh}%
\author[1]{Steven Parratt}%
\author[1]{Natasha Mannion}%
\author[2]{Rhonda Snook}%
\author[3]{Amanda Bretman}%
\author[1]{Tom Price}%
\affil[1]{University of Liverpool}%
\affil[2]{Unversity of Stockholm}%
\affil[3]{University of Leeds}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
The impact of rising global temperatures on survival and reproduction is
putting many species at risk of extinction. In particular, it has
recently been shown that thermal effects on reproduction, especially
limits to male fertility, can underpin species distributions in insects.
However, the physiological factors influencing fertility at high
temperatures are poorly understood. Key factors that affect somatic
thermal tolerance such as hardening, the ability to phenotypically
increase thermal tolerance after a mild heat shock, and the differential
impact of temperature on different life stages, are largely unexplored
for thermal fertility tolerance. Here, we examine the impact of high
temperatures on male fertility in the cosmopolitan fruit fly Drosophila
virilis. We first determined whether temperature stress at either the
pupal or adult life-history stage impacts fertility. We then tested the
capacity for heat-hardening to mitigate heat-induced sterility. We found
that thermal stress reduces fertility in different ways in pupae and
adults. Pupal heat stress delays sexual maturity, whereas males heated
as adults can reproduce initially following heat stress, but lose the
ability to produce offspring. We also found evidence that while
heat-hardening in D. virilis can improve high temperature survival,
there is no significant protective impact of this same hardening
treatment on fertility. These results suggest that males may be unable
to prevent the costs of high temperature stress on fertility through
heat-hardening which limits a species' ability to quickly and
effectively reduce fertility loss in the face of short-term high
temperature events.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Climate change is increasing the frequency of extreme temperature events
(Christidis, Jones \& Stott 2015). A major research priority is to
assess which organisms will be able to maintain fitness and cope with
the changing climate. Initial efforts to explore the impact of rising
temperatures on biodiversity mostly considered how thermal stress
affects survival (Deutsch \emph{et al.} 2008; Kellermann \emph{et
al.}2012; Pinsky \emph{et al.} 2019). While the impact of climate change
on survival is clearly important, it has also been known for around a
century that fertility is vulnerable to high temperatures in some
species (Young \& Plough 1926; Cowles 1945). Heat-induced sterility
occurs across diverse taxa including crops (Matsui \emph{et al.} 1997)
and livestock (Karaca \emph{et al.} 2002), so species where fertility is
lost at temperatures far below the lethal limit may represent both a
major economic and conservation concern (Walsh \emph{et al.} 2019) with
potentially worrying implications for humanity's resilience against
climate change. Fertility loss is generally sex-specific, with males
often more sensitive to fertility loss than females (Sales \emph{et
al.}2018; Iossa 2019; Walsh \emph{et al.} 2020; Zwoinska \emph{et
al.}2020). Recent work has found that the highest
temperatures\emph{Drosophila} species are found at worldwide is strongly
correlated to laboratory measurements of their lethal temperature, or
the temperature at which males lose fertility, whichever is the lower
(Parratt \emph{et al.} 2021; van Heerwaarden \& Sgr\selectlanguage{ngerman}ò 2021). This
suggests that species distributions may often be restricted by their
upper thermal limits to fertility in nature. However, we still know
relatively little about the physiological factors that affect fertility
loss at high temperatures.

In holometabolous insects, it is widely known that survival at high
temperatures can be affected by the life-stage at which thermal stress
occurs (Zhang \emph{et al.} 2015; Moghadam \emph{et al.} 2019). Studies
on heat-induced sterility in males typically use either a single
long-term stress across age-groups (Rohmer \emph{et al.} 2004;
Porcelli\emph{et al.} 2016), or an acute stress to individuals from a
single age-group (Jørgensen, Sørensen \& Bundgaard 2006; Sales \emph{et
al.}2018; Walsh \emph{et al.} 2020; Jørgensen \emph{et al.} 2021).
However, it has recently been shown in the flour beetle \emph{Tribolium
castaneum} that the extent of male fertility loss depends on the
life-stage exposed to thermal stress (Sales, Vasudeva \& Gage 2021).
Here, pupal and immature adults show the highest sterility after thermal
stress as compared with larval and mature adults. This study reveals a
critical period in the life-cycle of \emph{T. castaneum} where fertility
is particularly vulnerable to heat-stress of immature individuals. In
order to uncover any general patterns in thermal sensitivity of
fertility across life-stages, research should examine this across
species.

One way organisms can cope with thermal stress is to plastically invest
resources into thermal protection after receiving a signal that the risk
of extreme high temperatures has increased. For example, exposure to a
short-term moderately stressful sub-lethal heat can cause organisms to
make physiological changes that allow them to better survive extreme
temperatures (Loeschcke \& Hoffmann 2007; Moghadam \emph{et al.} 2019).
This response is called heat hardening, and is widespread in animals and
plants (Bilyk, Evans \& DeVries 2012; Neuner \& Buchner 2012;
Moghadam\emph{et al.} 2019). The positive impacts of hardening in
ectotherms are generally thought to occur through the upregulation of
heat-shock proteins such as HSP70 (Sørensen, Dahlgaard \& Loeschcke
2001). When the individual thereafter experiences extreme temperatures,
the increased concentration of heat-shock proteins reduces the thermal
damage. Hardening has been shown to mitigate the deleterious effects of
high temperatures on a multitude of traits, including survival
(Heerwaarden, Kellermann \& Sgrò 2016; Moghadam \emph{et al.} 2019) and
the ability to locate resources such as food or mating sites (Loeschcke
\& Hoffmann 2007). In the fruit fly \emph{Drosophila}
\emph{melanogaster} , individual survival is improved at high
temperatures through hardening, however the amount of protection
provided changes depending on the life-stage measured (Moghadam \emph{et
al.} 2019). In this case, pupae show strong protection through
heat-hardening, whereas adults' hardening capacity is minimal. Clearly,
a full understanding of heat-hardening itself is difficult without
examining multiple life-stages.

While the capacity of individuals to improve survival through
heat-hardening is widespread, it remains unclear whether individuals can
utilise hardening to mitigate heat-induced sterility. Some studies
suggest that there is a trade-off between hardening and reproduction
(Krebs \& Loeschcke 1994), but other examples found hardening improves
mating behaviour (Sambucetti \& Norry 2015) and, in a few species
heat-hardened individuals show greater offspring production after
thermal stress (Sarup \emph{et al.} 2004; Jørgensen, Sørensen \&
Bundgaard 2006). Heat-induced sterility occurs at sub-lethal
temperatures in many organisms (Walsh \emph{et al.} 2019), including
\textasciitilde{}44\% of a panel of 43 \emph{Drosophila} species
(Parratt \emph{et al.} 2021). So it is likely that, in the marginal
populations of particularly vulnerable species, a male's fitness could
be greatly improved by maintaining fertility at sub-lethal stress
temperatures. If males can plastically harden to prevent fertility loss
at extreme temperatures, then populations may have the capacity to
better cope with sub-lethal but stressful heat events.

Here, we explore the impact of high temperatures on male fertility in
the cosmopolitan fruit fly \emph{Drosophila virilis} , an extremely
widespread model species. Critically, it has previously been
demonstrated that male \emph{D. virilis} can be sterilised by thermal
stress well below their lethal temperature limit (80\% of adult males
sterile after four hours at 35°C, 80\% of adult males dead after four
hours at 38°C) (Walsh \emph{et al.} 2020; Parratt \emph{et al.} 2021).
This sterilisation of males at survivable temperatures makes \emph{D.
virilis} an ideal species to look for heat hardening of fertility. We
test the impact of temperature stress on fertility across life-history
stages, heating individuals as either pupae or adults. Further, we
demonstrate the capacity for heat-hardening to improve survival at
extreme temperatures and subsequently test if this hardening response
can also mitigate heat-induced sterility. Importantly, we measure how
fertility changes over an individual's age, to better understand the
long-term fitness implications of thermal stress and hardening at
different life-stages.



\textbf{Materials and Methods}

In overview, we test if heat-shocks experienced during pupal and adult
life-history stages result in male sterility. We also test if a brief
period of heat-hardening can ameliorate these effects. In a series of
experiments, adult and pupal male \emph{D. virilis} were exposed to a 1
hour heat hardening treatment followed immediately by a 4 hour heat
stress, and assayed for survival and fertility over 1-2 weeks to reveal
temporal patterns in fertility loss and restoration. We chose a 4 hour
stress because midday rises to high temperature are relatively common
(Geleti\selectlanguage{polish}č, \selectlanguage{english}Lehnert \& Jurek 2020), and we think it is ecologically
reasonable that a fly in nature might be exposed to these conditions for
a few hours. Moreover, it is an experimentally tractable time period,
and previous work has demonstrated this method can create male sterility
in many \emph{Drosophila} species, including \emph{D. virilis}
(Walsh\emph{et al.} 2020; Parratt \emph{et al.} 2021).

\textbf{Animal stock maintenance}

Stocks of \emph{Drosophila virilis} (Cambridge Fly Facility StrainvS-4,
isolated in 1991), were kept in a temperature-controlled room at 23\selectlanguage{ngerman}°C,
12:12 L:D and ambient humidity. Although a long term laboratory stock,
this stock was included in a recent analysis of upper thermal limits
from 36 \emph{Drosophila} species that found no significant association
between time in culture and any upper thermal limit (Parratt \emph{et
al.} 2021), suggesting it is a reasonable model for the species. Stocks
were maintained at moderate density (50 -- 100 flies per 300ml bottle
culture) on `Propionic' medium (10g agar, 20g yeast extract, 70g
cornmeal, 10g soya flour, 80g malt extract, 22g molasses, 14ml 10\%
nipagin, 6ml propionic acid, 1000ml H\textsubscript{2}O). Ovipositing
adults were tipped to new food every week to prevent overlapping
generations and were replaced with fresh sexually mature adult flies
every 4-6 weeks.

\textbf{Pupal heat-stress}

\textbf{Survival}

Pupae were collected from stock vials within 24 hours of pupation,
allocated to vials of 20 pupal flies. Three vials were allocated to each
treatment (giving 60 flies total per treatment, \textasciitilde{}30
males, as sex cannot be determined in young pupae). These vials were
randomly assigned to 3D-printed floating racks into pre-heated water
baths (Grant TXF200) for 1 hour at either a control non-hardening
temperature at 23°C (`no hardening') or a range of hardening
temperatures (`hardening': 34, 35 \& 36°C). These are non-lethal pupal
temperatures that also do not significantly sterilise males
(Walsh\emph{et al.} 2020). After this hardening treatment, they were
immediately moved into different pre-heated water-baths for 4-hours at
either 23°C (`benign') or at a range of sub-lethal to lethal
temperatures between 37 - 41°C (`stress'). Immediately following
treatment, vials were returned to benign conditions (23°C) and emerging
individuals were collected and sexed. This allowed us to assess survival
of pupae at extreme temperatures, and gave us an idea of whether
survival may be sex specific. However, as we were unable to determine
the sex of the pupae prior to stress, we could not explicitly test for
sex differences in survival thermal tolerance.

\textbf{Fertility}

Pupae were allocated to 3D-printed floating racks in pre-heated
water-baths set to 23°C (`no hardening') or 36°C (`hardening') for 1h as
above. Immediately following hardening, they were transferred into
pre-heated water baths at 23°C (`benign') or 38°C (`stress'), chosen as
the highest temperature not resulting in significant mortality from a
prior study (Walsh \emph{et al.} 2020). After four hours at their
treatment temperature, vials were subsequently removed from the
water-baths and returned to benign temperatures (23°C). Emerging males
were collected and immediately moved into individual vials with 4
sexually mature virgin female partners each. These groups were moved
into new vials every 2 days for 7 times, giving a total of 8 vials
across 16 days where fertility was recorded. Estimates
of\emph{Drosophila} survival rates in nature suggest 16 days represents
a substantial portion of their expected adult lifespan (Powell 1997).
Males were deemed as qualitatively fertile at any given time-point if
there was evidence of larvae present in the vial (either via direct
observation of larvae or observing larval tracks in the food).

\textbf{Adult heat-stress}

\textbf{Survival}

Virgin males and females (all 7 days old) were separated and allocated
to vials of 10 flies per vial of their respective sex. These vials were
randomly allocated to 3D-printed floating racks in pre-heated
water-baths for one hour at a hardening temperature at 23°C (`no
hardening') or 33°C (`hardening', determined as the highest temperature
in which no sterility is observed (Parratt \emph{et al.} 2021)). After
this hardening treatment, vials were immediately moved into different
pre-heated water-baths for four hours at either 23°C (`benign') or 38°C
(`stress', determined as lowest lethal temperature from (Parratt\emph{et
al.} 2021)). Immediately following treatment, vials were returned to
benign conditions (23°C) and left for 24 hours to ensure that any flies
that were immobilised by heat but not killed could recover. After 24
hours, the number of surviving males and females from each treatment was
assessed.

\textbf{Fertility}

Virgin males were allocated to vials (10 per treatment) and treated in
pre-heated water-baths at 23°C (`non-hardening') or 33°C (`hardening')
for 1h as above. Immediately following heat-hardening, flies were
transferred into pre-heated water baths at 34°C for a further 4 hours
(`stress', chosen as the lowest whole-degree Celsius temperature at
which \emph{D. virilis} are sterilised (Parratt \emph{et al.} 2021)).
Vials were subsequently removed from the water-baths and males were
placed in new individual vials with 4 virgin female partners each.
Previous experiments have shown that, when stressed as adults,
male\emph{D. virilis} initially retain fertility for several days and
then become sterilised (Walsh \emph{et al.} 2019). Hence, unlike our
assay with pupal-stress flies, we did not passage males to new vials
every 2 days immediately. Instead, we gave males an initial 7-day period
in a single vial with 4 females. We then gave each male 4 new virgin
females and passaged each group every 2 days for 4 times.

\textbf{Statistical analyses}

Measuring fertility which is a long-term adult trait when individuals
are heated during different life-stages introduces significant temporal
biases. We decided to measure fertility from the earliest possible
time-point post-stress, and continue to measure over time. This allowed
us to capture any visible loss/regain of fertility. Flies do not breed
as pupae, so fertility cannot be measured immediately following
heat-stress during this stage. Therefore, in order to understand how
these responses change depending on life-stage, we measured fertility
over a substantial period of time after stress for both pupae and
adults. Due to the inherent differences this introduced, we analysed
pupal and adult heat-stress separately, so comparisons of responses
between stages can only be qualitative.

Data were analysed using variations on linear models. We assessed model
fit by plotting patterns in residuals against fits and against
predictors. All statistical analyses were completed in R (version
3.5.0), using the packages: binom (Dorai-Raj 2014), car (Fox 2011),
``ggplot2'' (Wickham 2016) and ``survival'' (Therneau 2015). We did
model selection using Wald Chi-squared likelihood ratio-tests, removing
non-significant interactions. We retained all main effects and reported
statistics of these from type II likelihood ratio tests using the
`Anova' function from the `car' package (Fox 2011).

\textbf{1a) Pupal survival after heat-stress}

We chose 36°C as our single experimental `hardening' temperature since
it is the highest temperature that does not reduce fertility when males
experience it for 4h (Walsh \emph{et al.} 2020; Parratt \emph{et
al.}2021). We analysed pupal survival after heat stress using a logistic
regression with survival as a Bernoulli response variable. Stress
temperature, hardening treatment (non-hardened or hardened at 36°C), and
their interaction were fitted as explanatory variables. To determine
whether the hardening temperature altered its protective effect, we
analysed pupal survival of all flies hardened at 34, 35, and 36°C prior
to heat stress at the key stress temperature of 40°C where protection is
observed. We performed a logistic regression with survival as a
Bernoulli response variable. We used hardening temperature as the
explanatory variable. Note that the 34 and 35°C hardening temperatures
were not measured at 37 and 38°C temperature stress at this preliminary
stage, as these temperatures are non-lethal after a 4h stress
(Walsh\emph{et al.} 2020).

\textbf{1b) Adult survival after heat-stress}

As every fly stressed at control temperatures (23°C) survived, we
analysed adult survival at the chosen stress temperature (38°C) only,
using a logistic regression with survival as a Bernoulli response
variable and sex (male or female), hardening treatment (non-hardened or
hardened), and their interaction as explanatory variables.

\textbf{2a) Pupal fertility over time}

We analysed the effect of heat stress on fertility over time with
inverse Cox proportional hazard survival analyses (using the
``survival'' package (Therneau 2015)). This allowed us to model the time
in days post-eclosion until focal individuals become fertile. We fit the
time point at which fertility (scored as the presence of larvae) was
observed as our response variable with heat treatment (benign or
stress), hardening treatment (non-hardened or hardened) and their
interaction as independent variables.

\textbf{2b) Adult fertility over time}

We examined whether there was an immediate effect of heat stress on
fertility, and whether hardening affects this response. We used a
logistic regression with day 1 fertility as a Bernoulli response
variable and stress (benign or stressed), hardening treatment
(non-hardened or hardened), and their interaction as explanatory
variables.

Adult fertility over time was analysed using two separate approaches due
to the observed delayed sterility and how the experimental design was
constructed around it. This allowed us to pull apart different
hypotheses and test them. We first tested whether heat-stress reduced
fertility from day 7 onwards compared to benign temperature controls,
due to delays in adult sterility. To do this we used a mixed effect
logistic regression on non-hardened flies, with fertility as a Bernoulli
response variable and stress, time, and their interaction as explanatory
variables. Fly ID was used as a random effect to account for
non-independence in the data.

We then tested whether hardening can improve fertility over time in
stressed males. We used a mixed effect logistic regression on stressed
flies, with fertility as a Bernoulli response variable and hardening,
time, and their interaction as explanatory variables. Fly ID was used as
a random effect to account for repeated measures in the data.

\textbf{Results}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1:} Proportion of surviving individuals after a 4-hour
heat stress. Focal individuals were subjected to a pre-stress
`hardening' treatment for 1-hour immediately prior to temperature
stress. \textbf{a)} \emph{D. virilis} individuals of unknown sex were
heated during the pupal stage and subjected to a range of stressful
temperatures. A range of hardening temperatures were also used to
examine the hardening response. Note that the 34 and 35\selectlanguage{ngerman}°C hardening
temperatures were not measured at 37 and 38°C temperature
stress.\textbf{b)} Male and female \emph{D. virilis} were heated during
the adult stage 7 days post-emergence, and subjected to two stress
temperatures (23°C: benign, 38°C, stress). Error bars represent 95\%
confidence intervals.

\textbf{1a) Survival after pupal heat-stress}

When focusing on a single hardening temperature (36°C) compared with
non-hardened controls, we found that pupal survival probability was
significantly affected by the interaction between hardening and
heat-stress temperature (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(5)} =
33.74, p\textless{}0.001; Figure 1a). Specifically, pupae heat-hardened
at 36\selectlanguage{ngerman}°C showed significantly improved survival at higher stress
temperatures over non-hardened pupae. Between the 3 hardening
temperatures of 34, 35 and 36°C, we found no effect of hardening
temperature (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(2)} = 2.040, p=0.361;
Figure 1a) on individual survival at the pupal stress temperature of
40\selectlanguage{ngerman}°C.

\textbf{1b) Survival after adult heat-stress}

There was no interaction between hardening and sex for adult survival at
38°C (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 0.000, p=0.999; Figure
1b). However, we found a main effect of hardening on survival
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 41.321, p\textless{} 0.001;
Figure 1b)\textbf{.} Survival is significantly higher if adults have
experienced a 1h hardening treatment at 33\selectlanguage{ngerman}°C, as compared to
non-hardened controls. We also found a main effect of sex, with lower
survival in females than males (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)}=
16.891, p\textless{}0.001; Figure 1b).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2:} Cumulative fertility of male \emph{D. virilis} over
time after a 4h heat-stress. Focal individuals were subjected to a
pre-stress hardening treatment for 1h immediately prior to temperature
stress. The age at heat-stress is represented using an arrow, and the
life-stage of the individual is represented using grey (pupal) and white
(adult) background. \textbf{a)} Individuals were heated during the pupal
stage at either benign (23\selectlanguage{ngerman}°C) or stressful (38°C) temperatures.
Individuals were exposed to a 1h hardening treatment at 23°C
(`non-hardening') or 36°C (`hardening') prior to heat-stress. Focal
males were given a single group of virgin females at the first day
post-eclosion. \textbf{b)} Individuals were heated during the adult
stage at either benign (23°C) or stressful (35°C) temperatures.
Individuals were exposed to a 1h hardening treatment at 23°C
(`non-hardening') or 33°C (`hardening') prior to heat-stress. Focal
males were given access to 2 groups of virgin females: one from days 1
to 6 post-heat, and another fresh set of virgin females from day 7
post-heat, to account for delayed sterility of males. Error bars
represent 95\% confidence intervals.

\textbf{2a) Fertility after pupal heat-stress}

There was no interaction between pupal hardening and stress temperatures
on the age of reproductive maturity (ARM) (Cox proportional hazard test
interaction term: HR= 0.3831,
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)}=1.096, p=0.295; Figure 2a).
However, high pupal stress temperatures increase the time after eclosion
until males can produce offspring (Cox proportional hazard test
interaction term: HR= -0.8862, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)}
=23.27, p\textless{}0.001; Figure 2a). This extends the ARM, with many
males eventually becoming fertile. Pupal hardening does not
significantly reduce ARM at the stress temperature of 38\selectlanguage{ngerman}°C (Cox
proportional hazard test interaction term: HR= 0.1034,
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} =0.338, p=0.561; Figure 2a).

\textbf{2b) Fertility after adult heat-stress}

Adult males were given an initial group of virgin females to mate with,
and there was no interaction between stress temperature and hardening
treatment on immediate fertility of adult males
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 0.244, p=0.621; Figure 2b,
days 13-19). We also found no effect of heat-stress on immediate
fertility (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 2.286, p=0.130;
Figure 4, days 13-19), and no main effect of hardening on fertility at
this initial time point (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} =
0.590, p= 0.443; Figure 2b, days 13-19).

From 7 days post heat-stress onwards in non-hardened flies, there was no
interaction between heat-stress and time
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} 3.333, p=0.068; Figure 2b, days
19-27). However, we found that heat stress significantly reduced
fertility through a main effect of stress
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 28.444, p\textless{} 0.001;
Figure 2b, days 19-27). Stressed males had lower fertility than controls
after 7 days post heat-stress. We found no significant effect of time on
fertility after day 7 (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 2.413,
p= 0.120; Figure 2b, days 19-27) meaning fertility remained low post 7
days.

There was no interaction between hardening and time on fertility at the
stress temperature of 34\selectlanguage{ngerman}°C when measured after day 7
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 2.1824, p= 0.140; Figure 2b,
days 19-27). Hardening also did not affect fertility of heat-stressed
adults (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 0.1319, p= 0.717;
Figure 2b, days 19-27) meaning hardening does not change the sterility
pattern induced by thermal stress, even though there was a main effect
of time on fertility (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}\textsubscript{(1)} = 4.265,
p= 0.039; Figure 2b, days 19-27), where fertility increased slightly as
the experiment progressed.

\textbf{Discussion}

We found functionally different impacts of thermal stress at different
life-history stages on fertility in \emph{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
in\emph{Drosophila melanogaster} has been associated with disruptions to
spermatid elongation during spermatogenesis (Rohmer \emph{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\selectlanguage{ngerman}°C,
non-hardened adult \emph{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,
in\emph{D. melanogaster} , non-hardened pupae have only slightly lower
upper lethal limits than adults (Moghadam \emph{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 \emph{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
four\emph{Drosophila} species, found no significant differences in
overall acclimation capacity between males and females (Pottier \emph{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 \emph{D. virilis} female
fertility is robust to high pupal temperatures when compared with male
fertility (Walsh \emph{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 \emph{Tribolium castaneum} , adult male development
at stressful temperatures results in males producing sperm with shorter
tails (Vasudeva \emph{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
in\emph{D. 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 \emph{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 \emph{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 \emph{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.

\textbf{Acknowledgements}

The authors thank Jolanta Tanianis-Hughes for assistance with
experiments. \textbf{Author contributions:} Conception: BW, SP \& TP.
Methodology \& data collection: BW, NM \& SP. Data curation: BW.
Analysis: BW \& SP. Original Draft: BW \& TP. Review \& Editing: All
Authors. \textbf{Funding} : This work was funded by a Natural
Environment Research Council (NERC) ``Adapting to the Challenges of a
Changing Environment'' (ACCE) Doctoral Training Partnership studentship
to B.S.W., and NERC grant NE/ P002692/1 to T.A.R.P., AB and
RS.\textbf{Data and materials availability:} The data and analysis code
are available at:
https://datadryad.org/stash/share/bw3gtN7IvgxhlgknEXDK\_oODNOqOiRiZ5ZV5YJ4gjLg

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