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\begin{document}

\title{Warming increases survival and asexual fitness in a facultatively sexual
freshwater cnidarian with winter diapause}



\author[1]{Jácint Tökölyi}%
\affil[1]{University of Debrecen}%


\vspace{-1em}



  
  \date{May 23, 2024}


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\selectlanguage{english}
\begin{abstract}
Temperature is a key abiotic factor controlling population dynamics. In
facultatively sexual animals inhabiting the temperate zone, temperature
regulates the switch between asexual and sexual modes of reproduction,
initiates growth or dormancy and acts together with photoperiod to
mediate seasonal physiological transitions. Increasing temperature due
to recent global warming is likely to disrupt population dynamics of
facultatively sexual animals because of the strong
temperature-dependence of multiple fitness components. However, the
fitness consequences of warming in these animals are still poorly
understood. This is unfortunate, since facultatively sexual animals --
through their ability for asexual reproduction resulting in quick
population growth and sexual reproduction enabling long-term persistence
-- are key components of freshwater ecosystems. Here, I studied the
fitness effects of warming in Hydra oligactis, a freshwater cnidarian
that reproduces asexually throughout most of the year but switches to
sexual reproduction under decreasing temperatures. I exposed hydra
polyps to simulated short summer heatwaves or long-term elevated winter
temperatures. Since sexual development in this species is dependent on
low temperature, I predicted reduced sexual investment (gonad
production) and elevated asexual fitness (budding) in polyps exposed to
higher temperatures. The results show a complex effect of warming on
sexual fitness: while gonad number decreased in response to warming,
polyps exposed to high winter temperature were capable of multiple
rounds of gamete production. Asexual reproduction and survival rate, on
the other hand, clearly increased in response to higher temperatures,
especially in males. These results predict increased population growth
of H. oligactis in temperate freshwater habitats, which will likely
affect the population dynamics of its' main prey (freshwater
zooplankton), and through that, the whole food web.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Organisms living in the temperate zone are exposed to seasonal
fluctuations in temperature that provide contrasting physiological
environments during summer and winter. Many species inhabiting such
environments have adapted to temperature variation by specializing and
evolving thermal preference to either high or low temperature (Huey and
Kingsolver, 1989) or scheduling their major life events to different
parts of the year (McNamara and Houston, 2008). For instance, among
invertebrates that combine sexual with asexual reproduction in a complex
life cycle (i.e., facultatively sexual organisms, such as aphids,
flatworms, crustaceans, rotifers, sponges or cnidarians), sexual and
asexual reproduction events are often performed in different parts of
the year (Acker and Muscat, 1976; Fuchs et al., 2014; Green, 1966;
Reisa, 1973; Shaffer et al., 2020; Simon et al., 2002). Increasing or
decreasing temperatures in these species can induce a switch from
asexual to sexual reproduction (Acker and Muscat, 1976; Fuchs et al.,
2014; Reisa, 1973; Schr\selectlanguage{ngerman}öder, 2005; Shaffer et al., 2020; Simon et al.,
2002; Vowinckel, 1970), initiate the growth and development of embryos
inside resting eggs (Cáceres and Schwalbach, 2001; Cooley, 1971;
Gilbert, 2017; Gulbrandsen and Johnsen, 1990; Hairston and Kearns, 1995;
Vandekerkhove et al., 2005), and act as a modulator of other cues, such
as photoperiod and crowding (Decaestecker et al., 2009; Gyllström and
Hansson, 2004; Innes, 1997; Reisa, 1973; Schröder, 2005).

Temperature is a key regulator of the dynamics of populations because it
determines locomotor activity and food intake rates (Angilletta et al.,
2002) and acts as a physical factor regulating cellular processes, such
as metabolism or energy budgets (Brown et al., 2004; Huey and
Kingsolver, 2019), ultimately determining growth, reproduction and
survival (Angilletta, 2009). In facultatively sexual organisms both
sexual and asexual modes of reproduction critically depend on
temperature and switches in the mode of reproduction are expected to
have a major influence of population dynamics. Both modes of
reproduction contribute to population persistence and/or growth, albeit
in different ways. Asexual reproduction results in quick population
growth, allowing genotypes to increase in frequency when conditions are
favourable (Scheuerl and Stelzer, 2019). Sexual reproduction, by
contrast, often results in the production of resting eggs that are able
to survive adverse conditions. These resting eggs are able to persist
for years or even decades and replenish populations after stochastic
extinction events (Franch-Gras et al., 2017), provide sources of novel
genetic combinations with higher fitness (McLean et al., 2022), and
enable dispersal of propagules to novel habitats (Panov et al., 2004).

Recent climate change, however, is affecting temperatures worldwide and
rapidly rising temperatures have negative effects on organisms that
evolved under a different thermal regime (McCarty, 2001; Walther et al.,
2002). Documented effects of global warming include changes in phenology
(i.e. timing of major life history events such as migration in birds or
flowering in plants; (Jenni and Kéry, 2003; Molnár V et al., 2012)),
shifts in geographical range size (Thomas, 2010) and a widely detected
decline in animal body size (Sheridan and Bickford, 2011). Facultatively
sexual organisms, due to their adaptation to seasonally varying
temperature regimes, should be strongly impacted by warming
temperatures, although the complexity of facultatively sexual life
cycles makes prediction of the expected consequences of global warming
difficult. In aphids, for instance, documented population responses to
warming can be either positive, neutral or negative (Blanchard et al.,
2019; van Baaren et al., 2010). Since aphids are reproducing
parthenogenetically during summer and switch to sexual reproduction
during summer, rising temperatures are expected to yield short-term
increases in population sizes (until temperature becomes stressful) and
the disappearance of sexually produced offspring (Blanchard et al.,
2019). Warming temperatures have been shown in mesocosm experiments to
boost spring population growth of zooplankton, with a higher effect in
parthenogenetically reproducing cladocerans than non-parthenogenetic
copepods (Ekvall and Hansson, 2012). Due to the altered population
growth and differential thermal sensitivity, zooplankton communities
experience changes in dominance patterns (Ekvall and Hansson, 2012), but
also experience trophic mismatches that can result in population
declines (Winder and Schindler, 2004). However, despite these examples,
the number of studies examining the expected consequence of temperature
warming on facultatively sexual organisms is still very low. This is
unfortunate, since facultatively sexual organisms -- through their
ability to achieve quick population growth through asexually
reproduction and long-term persistance and dispersal through the
production of dormant stages -- are key components of the ecosystems
they inhabit, both as prey and as predators. Therefore, changes in their
population dynamics due to climate warming is likely to have wide-range
consequences at the level of the whole ecosystem.

The freshwater cnidarian \emph{Hydra oligactis} Pallas 1766 is a small,
sessile predator inhabiting the temperate zone. It is a cold-adapted
species that prefers low temperatures, and even short-term exposures to
temperatures above 30 ºC are lethal (Bosch et al., 1988). They reproduce
asexually throughout much of the year through budding, however, they
rapidly switch to a sexual mode of reproduction upon exposure to cold
temperatures (Reisa, 1973). Males produce testes, and females produce
ovaries, and upon fertilization the egg develops a into a resting embryo
surrounded by a thick shell that is extremely resistant to adverse
conditions (Reisa, 1973). Hence, the production of resting eggs appears
to be an adaptation to survive freezing water through dormancy, and
indeed, sexually reproducing individuals are found in nature during
autumn, before the freezing of water surface occurs (Miklós et al.,
2021; Sebestyén et al., 2018). Following sexual reproduction, hydra
polyps experience a senescence-like process and increased mortality
risk, although some of them survive and revert to asexual reproduction
(Tökölyi et al., 2017; Yoshida et al., 2006), depending on their age,
size, and genotype (Miklós et al., 2022; Ngo et al., 2021; Sebestyén et
al., 2020). Because of the presence of these asexual
individuals,\emph{H. oligactis} can reach huge population densities
during late winter and early summer ((Bryden, 1952); J.T. personal
observation) which could influence the population dynamics of their prey
(mainly cladocerans and copepods) in a manner that amplifies with
climate change. However, whether warming temperatures affect sexual or
asexual fitness, and thereby population dynamics in hydra is still
unclear.

Here, I set out to address this question by exposing \emph{H.
oligactis}polyps belonging to six strains (three male and three female)
to elevated temperature. Since climate warming is predicted to be
unequal across seasons (Meehl et al., 2007), I considered two scenarios:
an increased frequency of summer heatwaves and an increased average
winter temperature, both of these predicted based on current climate
projections for the temperate and boreal zone (Meehl et al., 2007). To
this end, hydra polyps were exposed either to a short (1-week long)
period of high summer temperature (23 ºC, compared to 18 ºC for
controls), which was followed by a winter treatment consisting either of
low (8 ºC) or high winter temperature (12 ºC) maintained for five
months. Throughout the experiment sexual and asexual fitness components
were recorded. Because of the sensitivity of \emph{H. oligactis} to high
temperature I predicted reduced performance (both sexual and asexual) of
polyps exposed to short heatwaves. Furthermore, because of the
dependence of sexual reproduction on cold temperatures in \emph{H.
oligactis} I predicted a reduced sexual fitness in groups exposed to
warm winter temperatures. Conversely, a higher asexual fitness and
survival might be predicted due to the negative relationship between
sexual reproduction and other fitness components (Ngo et al., 2021;
Roff, 1993; Stearns, 1989).

\textbf{Materials \& methods}

\textbf{Strains and culture conditions}

For the experiment presented here six \emph{H. oligactis} strains were
used (three male strains: C2/7, M83/4 and T3/2 and three female strains:
X11/14, M26/9/10 and T3/1). All strains were established from a single
individual that was collected from freshwater bodies in Eastern Hungary
between 2016 and 2020 and asexually propagated in the lab under
simulated summer conditions (Gergely \& Tökölyi \emph{in press} ). These
conditions include 18 ºC temperature and 12/12 hours light/dark cycle.
Polyps were kept individually in 6-well tissue culture plates containing
artificial hydra medium (1mM Tris, 1mM NaCl, 1mM CaCl\textsubscript{2},
0.1mM KCl, 0.1mM MgSO\textsubscript{4}; pH: 7.6) and they were fed and
cleaned four times per week.

\textbf{Experimental design}

All data recorded for this experiment comes from polyps that were kept
individually. The experiment consisted of two treatments: a summer
treatment and a winter treatment. The summer treatment lasted for one
week and aimed at simulating a heat wave. The winter treatment lasted
for five months and aimed at simulating elevated winter temperatures.
For both treatments, the control values were chosen such that both
summer and winter conditions are comparable to our previous studies
investigating \emph{H. oligactis} life history (Ngo et al. 2021;
Sebestyén et al. 2020; Tökölyi et al. 2017). Hence, the control
temperature for summer was set as 18 ºC (cold summer, CS) and the
control temperature for winter was set to 8 ºC (cold winter, CW). To
simulate summer heatwaves, I exposed polyps to 23 ºC for one week (warm
summer, WS). Water temperatures as high as 25 ºC regularly occur in
natural populations at the end of the summer (J. T. personal
observation), however, in the laboratory larger or longer heat
treatments (i.e., above 23 ºC or longer than one week) result in excess
mortality. For the winter treatment, I exposed experimental polyps to 12
ºC temperature for five months (warm winter, WW). A four degree increase
in winter temperature is in accordance with IPCC predictions for Europe
by the end of the century (Meehl et al., 2007). The summer and winter
treatments were applied in a factorial design, such that there were four
experimental groups: cold summer -- cold winter (CS-CW); cold summer --
warm winter (CS-WW); warm summer -- cold winter (WS-CW) and warm summer
-- warm winter (WS-WW).

During the one-week summer treatment (either 18 or 23 ºC) polyps were
fed four times per week. After the summer treatment, polyps were
photographed on a 1-mm grid paper sheet to measure their size, since
size at cooling is an important predictor of sexual reproduction and
post-reproductive survival (Ngo et al., 2021). Next, they were moved to
either 8 or 12 ºC, depending on their treatment and an 8/16 hours
light/dark cycle. Lowering temperature induces sexual reproduction in
this species and I recorded the presence of male or female gonads four
times per week as a proxy for sexual fitness. In addition, the number of
detached buds was recorded twice per week as a proxy for asexual
fitness. The winter treatment lasted for five months during which polyps
were fed and cleaned twice per week. A five month winter treatment is
necessary and sufficient time period to quantify sexual reproduction and
post-reproductive survival in most polyps in these strains. At the end
of the winter treatment, polyps were scored as survived if they had an
intact body with tentacles and had the ability to feed, and they were
scored dead if they disappeared during the experiment or consisted only
of necrotic tissue. All experimental groups were started with 30-36
polyps per strain, with a total starting sample size of N = 843,
although some individuals were excluded because they were accidentally
lost during the experiment, or they proved to be sex-changed individuals
(see Results). The experiment was initiated in batches over a period of
about two months, depending on the availability of experimental animals.
Each batch consisted of animals randomly divided into four treatment
groups and any batch effects were statistically taken into account (see
below).

\textbf{Data analysis}

The effects of treatments were analyzed for the following dependent
variables: (1) body size after summer treatment; (2) sexual development
time (days elapsed after the beginning of the winter treatment until the
appearance of first gonads); (3) sexual fitness (total number of eggs
produced by females and the maximum number of testes seen on a male);
(4) asexual fitness (total number of buds produced by an individual from
the start to the end of the experiment, i.e. during both the summer and
winter treatments) and (5) survival rate. I used Generalized Linear
Mixed Models implemented in the glmmTMB package (v1.1.4) in R version
4.2.2 (Brooks et al., 2017; R Core Team, 2020). Gaussian errors were
assumed for body size and binomial errors for survival rate. For sexual
development time, sexual and asexual fitness (count data), four
different error structures were considered: Gaussian, Poisson, negative
binomial with linear parametrization (\emph{nbinom1} ) and negative
binomial with quadratic parametrization (\emph{nbinom2} ) (Brooks et
al., 2017). The model best fitting to the data was selected with Akaike
Information Criteria corrected for small sample size (AICc). After
selecting the right distribution, I fitted a model with experimental
treatment as a fixed effect and strain ID and batch ID as random
effects. This model was compared to a random effects null model via
Likelihood Ratio Tests (LRTs). \emph{Post-hoc} treatments-versus-control
comparisons with Dunnett's correction were obtained using
the\emph{emmeans} R package (Lenth, 2022). For the model analyzing body
size after summer treatment only one predictor was included (summer
treatment) and I used LRT to evaluate the effect of treatment on size.
Finally, I generated model diagnostics plot using the DHARMa R package
(v0.4.5; (Hartig, 2022)) to check that model residuals did not show
outstanding deviations from model assumptions. Since the model
diagnostics for sexual development time showed substantial deviations
from the expectation for both males and females, I also analyzed these
variables with non-parametric Kruskal-Wallis tests, followed by pairwise
Wilcoxon tests with Holm's correction.

\textbf{Results}

Of the total N = 843 individuals 12 were accidentally lost during
routine handling and nine individuals were sex-changed. All data were
removed for these individuals, yielding a sample size of N = 822, with N
= 206 individuals in the CS-CW group, N = 200 individuals in the WS-CW
group, N=210 individuals in the CS-WW group and N = 206 individuals in
the WS-WW group.

\textbf{Body size after summer treatment}

A one-week heat treatment reduced body size in male polyps by
approximately 17\% and female polyps by approximately 24\%, with the
difference being statistically highly significant in both sexes
(Gaussian GLMM, LRT males: chi-square = 117.59, p \textless{} 0.001;
females: chi-square = 42.984, p \textless{} 0.001; Fig. 1.).

\textbf{Sexual development time}

In both male and female strains, sexual development time was
significantly influenced by treatment (males: LRT, Gaussian GLMM,
\selectlanguage{greek}λ\selectlanguage{english}\textsuperscript{2} = 257.19, p \textless{} 0.001; females: LRT,
Negative binomial GLMM with quadratic parametrization,
\selectlanguage{greek}λ\selectlanguage{english}\textsuperscript{2} = 133.34, p \textless{} 0.001). Both male and
female polyps exposed to one week heat treatment showed delayed sexual
development, while those exposed to elevated winter temperatures showed
slightly, but significantly faster sexual development (Table 1., Fig.
2., 3.). Polyps in the WS-CW group delayed their reproduction most (Fig.
2A., 3A.), implying that a large sudden drop in temperature has negative
effects on sexual development. The results were corroborated by the
Kruskal-Wallis test and the pairwise Wilcoxon test as
\emph{post-hoc}analysis, with the exception of males in the CS-WW group,
where advancement of sexual development time was only marginally
significant (p = 0.081).

\textbf{Sexual fitness}

The number of gonads produced was also significantly influenced by
treatment (males: LRT, Gaussian GLMM, \selectlanguage{greek}λ\selectlanguage{english}\textsuperscript{2} = 56.43, p
\textless{} 0.001; females: LRT, Negative binomial GLMM with linear
parametrization, \selectlanguage{greek}λ\selectlanguage{english}\textsuperscript{2} = 34.66, p \textless{} 0.001).
Male polyps exposed to a simulated summer heatwave had reduced number of
testes (Table 1.; Fig. 2B.). The negative effects of a warm summer were
accentuated if they were followed by cold winter: a sudden large drop in
temperature was associated with markedly reduced male fitness (Table 1.;
Fig. 2B.). In females, on the other hand, only the WS-CW group had
significantly reduced number of eggs compared to the CS-CW group (Table
1., Fig. 3B.).

Surprisingly, polyps exposed to a warm winter treatment underwent a
second round of gametogenesis after the first one. This second
gametogenesis event happened in individuals that recovered from their
first gametogenesis event, around 3-4 months after cooling (Fig. 2C.,
3C.).

\textbf{Asexual fitness}

In males, asexual fitness was significantly affected by treatment (LRT,
Negative binomial GLMM with linear parametrization,
\selectlanguage{greek}λ\selectlanguage{english}\textsuperscript{2}= 26.96, p \textless{} 0.001). The number of buds
produced increased if polyps were exposed to a simulated summer heat
wave and if they were cultured under elevated winter temperatures, or
they received both treatments compared to a cold summer-cold winter
scenario (Table 1, Fig. 4A.). In females, by contrast, treatment had no
effect on asexual fitness (LRT, Negative binomial GLMM with quadratic
parametrization, \selectlanguage{greek}λ\selectlanguage{english}\textsuperscript{2} = 6.11, p = 0.106).

\textbf{Survival rate}

Male survival rate was significantly affect by treatment (LRT, Binomial
GLMM, \selectlanguage{greek}λ\selectlanguage{english}\textsuperscript{2} = 44.36, p \textless{} 0.001). Male polyps
exposed to simulated summer heatwaves, elevated winter temperatures, or
both treatments had significantly higher survival than polyps in the
control group (Table 1, Fig. 4C.). The highest survival rate was seen in
male polyps exposed to the CS-WW treatment, where about 80\% of the
polyps survived compared to \textasciitilde{}50\% in the control CS-CW
group. Female survival was only marginally significantly affected by
treatment (LRT, Binomial GLMM, \selectlanguage{greek}λ\selectlanguage{english}\textsuperscript{2} = 7.36, p = 0.061),
with the WS-WW group being significantly more likely to survive compared
to the control group (\textasciitilde{}45\% of female polyps survived
compared to 30\% in the control group; Table 1., Fig. 4C.).

\textbf{Discussion}

Organisms are heavily affected by warming temperatures and show short-
or long-term biological responses that include advancing phenologies,
shifting geographical distribution ranges, and altered physiology (Huey
and Kingsolver, 2019; McCarty, 2001; Thomas, 2010; Walther et al.,
2002). Warming also affects biological fitness, such that organisms have
altered reproduction and/or survival, with important consequences for
population dynamics and ecosystem stability (Anderson, 2016; Deutsch et
al., 2008; Kingsolver et al., 2013; Sinclair et al., 2016). While the
fitness consequences of warming are widely considered to be negative,
detailed investigations show a much more varied picture, with both
negative and positive effects observed depending e.g. on the thermal
tolerance and phenotypic plasticity of the species (Chamaill\selectlanguage{ngerman}é-Jammes et
al., 2006; Deutsch et al., 2008; Pelini et al., 2012; Weitere et al.,
2009).

Here, I studied the fitness consequences of warming across the life
cycle of a facultatively sexual freshwater cnidarian \emph{H.
oligactis,} an important predator of zooplankton in freshwater
ecosystems. This species reproduces asexually during summer, switches to
a sexual mode of reproduction after the temperature drops during autumn
and experiences a post-reproductive senescence with increased mortality
risk (Sebestyén et al., 2020, 2018). However, some individuals do not
initiate sexual reproduction at all, or survive and continue reproducing
asexually during the winter. Due to the heat-intolerance of this species
(Bosch et al., 1988) and the dependence of sexual reproduction on cold
temperature in \emph{H. oligactis} , I hypothesized that warming might
have negative fitness effects and an overall shift from sexual to
asexual reproduction. The results paint a more complex picture of
fitness effects of warming in \emph{Hydra} .

Exposure of hydra polyps to simulated summer heat waves had immediate
negative effects on the body size of hydra polyps. The reduction of body
size with warming in animals is a widespread phenomenon (Gardner et al.,
2011; Sheridan and Bickford, 2011), and is thought to be due to altered
metabolic requirements and nutrient-dependence (Audzijonyte et al.,
2022; Lee et al., 2015), oxygen limitation (Verberk et al., 2021;
Walczy\selectlanguage{polish}ń\selectlanguage{english}ska et al., 2015) or temperature-dependent size-fecundity or
size-mortality relationships (Arendt, 2015; Audzijonyte et al., 2022).
The size declines observed here were the result of phenotypic
flexibility and occurred very quickly (observed after a week of exposure
to simulated heat waves), suggesting a likely involvement of heat stress
in the shrinkage of hydra polyps. Although such an immediate reduction
in body size due to heat stress might not occur in most animals with a
relatively fixed adult body size (e.g. insects), several other groups
are able to plastically change adult size in response to environmental
conditions (see e.g. Thommen et al., 2019 for an example of body size
fluctuations in response to food availability in planarians), and in
these animals warming, and the associated heatwaves could have an
immediate effect on body size, just like in hydra. Furthermore, the
changes in body size in hydra are significant because body size at
cooling is a strong predictor of subsequent performance in this species:
small polyps have delayed sexual maturation, reduced fecundity, but a
higher post-reproductive survival (Ngo et al., 2021). In accordance, I
found that polyps in the ``Warm Summer'' groups, exposed to simulated
summer heat wave, required more time to produce the first gonads, had
reduced sexual fitness and at least in males, they had a higher
post-reproductive survival rate and higher asexual fitness.

The effects of the summer heat wave, however, were modulated by the
winter treatment in a complex way. In \emph{H. oligactis} , temperatures
below 12 \selectlanguage{ngerman}ºC degrees are required for gamete differentiation to occur
(Littlefield, 1991; Littlefield et al., 1991), and researchers
previously used temperatures from 4 to 10 ºC to induce sex in this
species (Boutry et al., 2022; Kaliszewicz, 2015; Tomczyk et al., 2020).
Higher simulated winter temperatures advanced sexual maturation in both
males and females, which is consistent with a hypothesis that higher
temperatures are more permissive for cell proliferation and the
differentiation of gametes and reproductive tissue (Álvarez and Nicieza,
2002). These results suggest that, while all temperatures below or equal
to 12 ºC promote sexual development in \emph{H. oligactis} , the exact
temperature can affect fine details of sexual development. The strongest
effects on sexual development and fecundity were, however, observed in
the WS-CW group, where simulated summer heatwaves were followed by a
sudden drop in temperature. Animals in this group needed the most time
to produce the first gonads and, at least in males, they had the lowest
number of reproductive organs. This suggests that sudden drops in
temperature are stressful for Hydra, and this stress could contribute to
sex induction in this species. While temperature fluctuations as large
as this (from 23 to 8 ºC within one day) are unlikely to occur under
natural conditions in a freshwater habitat, both heat waves and cold
spells, i.e. temperature variability are predicted to be more common in
the future (Meehl et al., 2007) and these are likely to have negative
physiological consequences in Hydra. Remarkably, I detected an
unexpected consequence of simulated winter warming: polyps cultured
under 12 ºC underwent a second round of gonadogenesis and continued to
show signs of sexual reproduction five months after cooling, while all
surviving polyps cultured on 8 ºC were asexual. Hence, while warming
might have immediate negative effects on sexual reproduction in general
in this species (especially if it demonstrates in increased temperature
fluctuations), these negative effects could be counterbalanced on the
longer term by the positive effects of warm winters on the number of
reproductive cycles.

Compared to sexual reproduction, the effects of elevated temperatures on
asexual fitness were much more clearcut (although sex-dependent). Polyps
exposed to simulated heatwaves produced a higher number of asexual buds
in male strains, and males exposed to elevated winter temperatures
produced a higher number of asexual buds. The increased asexual fitness
might be explained on one hand by a shift from sexual to asexual
reproduction (possibly mediated by the reduced size of polyps exposed to
simulated heat waves) and by the higher survival rate of polyps exposed
to higher temperatures. Since asexual reproduction allows very quick
population growth, these results suggest that increased temperatures
will result in higher hydra population sizes in late winter due to
climate warming. Hence, temperate freshwater bodies might experience
``hydra blooms'' similar to the warming-induced jellyfish blooms
observed in marine environments (Goldstein and Steiner, 2020; Holst,
2012; Purcell et al., 2007), with potential up- and down-stream
consequences on the whole aquatic food web. Unfortunately, little is
known about hydra population dynamics under natural settings. Based on
the phenology of resting egg production in \emph{H. oligactis,} which
peaks before the onset of the winter, one could assume that population
size collapses during winter either due to freezing, low food
availability, or increased mortality due to reduced somatic maintenance
of sexually reproducing polyps (Sebestyén et al., 2018). The limited
number of field observations, however, seem to contradict this
assumption, since all observations point to the fact that \emph{H.
oligactis} thrives during the winter and reaches very large population
densities (Bryden, 1952; Ribi et al., 1985; Welch and Loomis, 1924). The
experiment presented here suggests that Hydra population sizes could
become even higher in the future due to climate warming. Future studies
should aim to test these predictions in a more natural setting, e.g., in
mesocosms simulating the complexity of an aquatic food web to gain more
insight into this problem.

In parallel with the higher asexual fitness of polyps exposed to
simulated warming, I also found a positive effect of summer heatwaves
and warmer winters on post-reproductive survival. Indeed, the higher
asexual fitness of polyps exposed to higher temperature might be at
least partly the consequence of their increased survival rate, although
this effect was clearly observed only in males, while in females it was
much more subtle. Hence, temperature appears in a growing list of
intrinsic and extrinsic factors that affect post-reproductive survival
in this species (age, size, genotype; Miklós et al., 2022; Ngo et al.,
2021; Sebestyén et al., 2020). Exposure to high temperature could have
resulted in higher survival for at least two different reasons. First,
simulated summer heatwaves caused a reduction in body size, which is
known to directly influence survival rate in this species (Ngo et al.,
2021), most likely through a shift of resources from reproduction to
survival. However, even male polyps that were not exposed to simulated
summer heatwaves experienced improved survival rate if they were
cultured at 12 ºC simulated winter temperatures, which points at a
second, independent mechanism. The higher temperature could have caused
improved survival by altering the metabolic cost of tissue maintenance
in these polyps (Gillooly et al., 2001). Alternatively, it could also be
the consequence of a shift from reproduction to survival functions in
polyps experiencing warmer winters, although no clear evidence of that
was observed, since polyps in the ``Warm Winter'' groups did not show
evidence of reduced testes number compared to the ``Cold Winter''
groups. Further studies should explore the effect of temperature on
post-reproductive survival in more detail.

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Table 1. Sexual development time, sexual fitness (number of gonads),
asexual fitness (number of buds) and survival rate of male and
female\emph{H. oligactis} polyps exposed to simulated summer heatwave
(WS-CW), elevated winter temperature (CS-WW), or both (WS-WW), compared
to polyps exposed to a cold summer -- cold winter scenario (CS-CW). The
table shows estimated marginal means contrasts from Generalized Linear
Mixed Models (GLMMs) that included treatment as a fixed effect, and
strain ID and batch ID as random effects (see Methods for more detail).
The type of model is indicated above the contrasts: Gaussian
(``\emph{gaussian} ''), Negative Binomial with linear parametrization
(``\emph{nbinom1} ''), Negative Binomial with quadratic parametrization
(``\emph{nbinom2} '') or Binomial (``\emph{binomial} ''). P-values are
after Dunnett's correction for multiple comparisons. Significant
differences are highlighted in bold.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{Males} & \textbf{Males} & \textbf{Males} & \textbf{Females} &
\textbf{Females} & \textbf{Females}\tabularnewline
\midrule
\endhead
Contrast & Estimate & SE & P-value & Estimate & SE &
P-value\tabularnewline
\textbf{Sexual development time} & \emph{gaussian} & \emph{gaussian} &
\emph{gaussian} & \emph{nbinom2} & \emph{nbinom2} &
\emph{nbinom2}\tabularnewline
(WS-CW) - (CS-CW) & \textbf{6.53} & \textbf{0.44} &
\textbf{\textless{}0.001} & \textbf{0.26} & \textbf{0.03} &
\textbf{\textless{}0.001}\tabularnewline
(CS-WW) - (CS-CW) & \textbf{-1.04} & \textbf{0.43} & \textbf{0.047} &
\textbf{-0.08} & \textbf{0.03} & \textbf{0.016}\tabularnewline
(WS-WW) - (CS-CW) & \textbf{3.01} & \textbf{0.44} &
\textbf{\textless{}0.001} & \textbf{0.13} & \textbf{0.03} &
\textbf{\textless{}0.001}\tabularnewline
\textbf{Sexual fitness} & \emph{gaussian} & \emph{gaussian} &
\emph{gaussian} & \emph{nbinom1} & \emph{nbinom1} &
\emph{nbinom1}\tabularnewline
(WS-CW) - (CS-CW) & \textbf{-2.89} & \textbf{0.43} &
\textbf{\textless{}0.001} & \textbf{-0.61} & \textbf{0.12} &
\textbf{\textless{}0.001}\tabularnewline
(CS-WW) - (CS-CW) & -0.03 & 0.43 & 0.998 & -0.04 & 0.10 &
0.910\tabularnewline
(WS-WW) - (CS-CW) & \textbf{-1.32} & \textbf{0.43} & \textbf{0.007} &
-0.21 & 0.10 & 0.082\tabularnewline
\textbf{Asexual fitness} & \emph{nbinom1} & \emph{nbinom1} &
\emph{nbinom1} & \emph{nbinom2} & \emph{nbinom2} &
\emph{nbinom2}\tabularnewline
(WS-CW) - (CS-CW) & \textbf{0.30} & \textbf{0.07} &
\textbf{\textless{}0.001} & 0.19 & 0.10 & 0.114\tabularnewline
(CS-WW) - (CS-CW) & \textbf{0.24} & \textbf{0.07} & \textbf{0.001} &
0.13 & 0.09 & 0.387\tabularnewline
(WS-WW) - (CS-CW) & \textbf{0.28} & \textbf{0.07} &
\textbf{\textless{}0.001} & 0.21 & 0.09 & 0.067\tabularnewline
\textbf{Survival} & \emph{binomial} & \emph{binomial} & \emph{binomial}
& \emph{binomial} & \emph{binomial} & \emph{binomial}\tabularnewline
(WS-CW) - (CS-CW) & \textbf{1.75} & \textbf{0.45} &
\textbf{\textless{}0.001} & 0.49 & 0.37 & 0.408\tabularnewline
(CS-WW) - (CS-CW) & \textbf{2.65} & \textbf{0.48} &
\textbf{\textless{}0.001} & 0.35 & 0.37 & 0.648\tabularnewline
(WS-WW) - (CS-CW) & \textbf{2.40} & \textbf{0.48} &
\textbf{\textless{}0.001} & \textbf{0.97} & \textbf{0.37} &
\textbf{0.025}\tabularnewline
\bottomrule
\end{longtable}

Figure legends

Fig. 1. Body size of \emph{H. oligactis} polyps from three male and
three female strains and cultured for one week on 18 \selectlanguage{ngerman}ºC (cool summer,
CS) or exposed to a simulated one-week heatwave of 23 ºC (warm summer,
WS). A simulated summer heatwave significantly reduces both male and
female body size (see Results).

Fig. 2. Sexual reproduction in male \emph{H. oligactis} polyps belonging
to three distinct strains exposed to four distinct temperature regimes:
CS-CW (18 ºC summer -- 8 ºC winter; control group), WS-CW (23 ºC summer
-- 8 ºC winter), CS-WW (8 ºC summer -- 12 ºC winter) or WS-WW (23 ºC
summer -- 12 ºC winter). The summer treatment lasted for one week, while
the winter treatment lasted 21 weeks. The graph shows the time elapsed
until the appearance of the first gonads after cooling (A), the maximum
number of testes (B) and temporal changes in the frequency of sexual
individuals (C). Photograph of a male individual with mature testes is
shown on panel D for illustration.

Fig. 3. Sexual reproduction in female \emph{H. oligactis} polyps
belonging to three distinct strains exposed to four distinct temperature
regimes: CS-CW (18 ºC summer -- 8 ºC winter; control group), WS-CW (23
ºC summer -- 8 ºC winter), CS-WW (8 ºC summer -- 12 ºC winter) or WS-WW
(23 ºC summer -- 12 ºC winter). The summer treatment lasted for one
week, while the winter treatment lasted 21 weeks. The graph shows the
time elapsed until the appearance of the first gonads after cooling (A),
the total number of eggs (B) and temporal changes in the frequency of
sexual individuals (C). Photograph of a female individual with mature
eggs is shown on panel D for illustration.

Fig. 4. Asexual reproduction and survival in \emph{H. oligactis} polyps
belonging to three male and three female strains, exposed to four
distinct temperature regimes: CS-CW (18 ºC summer -- 8 ºC winter;
control group), WS-CW (23 ºC summer -- 8 ºC winter), CS-WW (8 ºC summer
-- 12 ºC winter) or WS-WW (23 ºC summer -- 12 ºC winter). The summer
treatment lasted for one week, while the winter treatment lasted 21
weeks. The graph shows the total number of buds produced during the 22
weeks of the experiment (A) and proportion of individuals surviving (C).
The number of surviving animals from each strain and treatment group is
shown on the bars. Photographs of an asexual individual (B) and a
senescent individual (D) are shown for illustration.

\textbf{Data accessibility statement:}

Raw data is available on Figshare
(\emph{https://doi.org/10.6084/m9.figshare.21750188.v1}).

R code to analyze the data is available on Figshare
(\emph{https://doi.org/10.6084/m9.figshare.21750170.v1}).

\textbf{Competing interests statement:}

The author declares no competing interests.

\textbf{Author contributions:}

\textbf{Jácint Tökölyi} : Conceptualization (lead); data curation
(lead); formal analysis (lead); funding acquisition (lead); methodology
(lead); project administration (lead); visualization (lead); writing
(lead).

\textbf{Acknowledgements}

The author wishes to thank Réka Gergely, Erzsébet Ágnes Nehéz, Mariann
Emese Zöld and Hanna Révész for lab assistance.\selectlanguage{english}
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