Abstract
The predicted temperature increase caused by climate change is a threat
to biodiversity. Male reproduction is particularly sensitive to elevated
temperatures resulting in sterility. Here we investigate temperature
induced changes in reproductive tissues and the fertility reduction in
male Drosophila melanogaster. We challenged males during
development and either allowed them to recover or not in early
adulthood, while measuring several determinants of male reproductive
success. We found significant differences in recovery rate, organ sizes,
sperm production and other key reproductive traits among males from our
different temperature treatments. Spermatogenesis and hence sperm
maturation was impaired before reaching the upper thermal sterility
threshold. While some effects were reversible, this did not compensate
the earlier damage imposed. Surprisingly, developmental heat stress was
damaging to accessory gland growth and female post mating responses
mediated by seminal fluid proteins were impaired regardless of the
possibility of recovery. We suggest that sub-lethal thermal sterility
and the subsequent fertility reduction is caused by a combination of
malfunctioning reproductive traits: inefficient functionality of the
accessory gland and alteration of spermatogenesis.
Temperature is a critical abiotic factor for many organisms and can turn
into an environmental stressor, particularly for ectotherms. Elevated
temperatures are known to affect an ectotherms physiology, behavior and,
on a broader scale their overall performance (Huey and Stevenson, 1979).
When critical thermal limits are exceeded, both viability and
reproductive potential are harmed. Within the last years, most research
assessing fitness loss under increasing temperatures have incorporated
mainly measures that evaluate an organism physiological failure
(viability thresholds), like death or heat coma (Walsh et al., 2019).
However, often the fertility range is narrower than the viability range,
such that sub-lethal temperatures already pose an important fitness
loss, impeding organisms to reproduce, threatening population stability
and persistence (Kellermann et al., 2012; van Heerwaarden and Sgrò,
2021; Walsh et al., 2019). Given the strong ecological implications that
sterility and fertility loss exert on organisms, study the capacity of
species and the mechanisms to respond ecologically and evolutionarily to
the challenges of increasing temperatures has become a research
priority, especially within the climate change context (Parmesan 2006).
Hence, understanding the reproductive and resulting fitness consequences
of exposure to elevated temperatures is key.
Sterility is one consequence when reaching thermal fertility limits at
both the upper and lower ends. Most of the literature refers to males,
as these have repeatedly been found to be more temperature sensitive
than females (e.g. Sales et al., 2018; Zwoinska et al., 2020). However,
there are also examples of female sterility due to high temperatures
(e.g. in the Nile tilapia, Oreochromis niloticus (Byerly et al.,
2005)). Based on the premise that spermatogenesis is more
thermosensitive than oogenesis, the study of male fertility thresholds
is of special interest, not only to understand the damage imposed on
male fertility, but also to gauge the consequences for reproductive
capacity and thus, fitness (Parratt et al., 2021). Even though this
phenomenon has been documented for a range of taxa like insects (e.g.
several Drosophila species such as D. melanogaster and D.
simulans (Chakir et al., 2002)), fishes (e.g. Nile tilapia, O.
niloticus (Byerly et al., 2005) and channel catfish, Ictalurus
punctatus (Strüssmann et al., 1998)), reptiles (e.g. yucca night
lizard, Xantusia vigilis (Cowles and Burleson, 1945)) and some
vertebrates (e.g. Arbor Acres roosters, Gallus gallus (McDaniel
et al., 1996), zebra finch, Taeniopygia guttata (Hurley et al.,
2018) and rams, Ovis aries (Hafez, 1964)), the mechanisms
underlying sterility at extreme temperatures are still unknown. The fact
though that some species can recover fertility after being transferred
to milder temperatures (e.g. D. melanogaster males (Chakir et
al., 2002)) indicates that the destruction of germ cells might not
explain the observed temperature induced infertility.
Previous research on D. melanogaster (Rohmer et al., 2004) has
shown that elevated temperatures disrupt spermatogenesis causing
cytological abnormalities. As a result, males have shorter cysts, show
abnormalities in the shape and position of sperm nuclei, an impairment
of spermatid elongation and an increase in spermatid death rate.D. simulans instead had shorter cysts at higher temperatures
(David et al., 2005) indicating phenotypic plasticity, with temperature
dependent plasticity in sperm length being adaptive in Tribolium
castaneum (Vasudeva et al., 2019). Despite these first studies into
spermatogenesis dynamics under thermal stress, little is yet known about
recovery dynamics on sperm production. Furthermore, effects on the
second reproductive tissue, the male accessory glands (AGs), have not
been considered. The importance of the AGs for male reproductive success
has been widely studied (Chen, 1984; Gillott, 2003; Wolfner, 1997).
Seminal fluid proteins (SFPs), secreted mainly by the AGs, are
transferred together with sperm to the female during mating causing
changes in female postmating responses (e.g. behavior and physiology
(Chapman, 2001; Chen et al., 1988)). In addition, SFPs affect male sperm
competitive ability, and modulate sperm storage dynamics inside the
female’s sperm storage organs (Avila et al., 2011) together ensuring
fertility. Moreover, SFPs might have protective functions as in
honeybees, Apis mellifera (den Boer et al., 2009) and leaf-cutter
ants, Atta colombica (den Boer et al., 2007), where SFPs increase
sperm viability. Hence, whether temperature damages either or both
tissues, needs to be considered to understand the mechanisms of
temperature induced sterility.
With the predicted temperature increase (at least 1.5 - 2°C for
2081-2100 (Collins et al., 2013)) and the occurrence of longer and more
severe heat waves (Meehl and Tebaldi, 2004) due to global climate
change, we are convinced that studying the responses of reproductive
traits to stressful thermal conditions is of special interest in order
to determine species persistence under possible new environmental
conditions (Hoffmann, 2010; Huey and Kingsolver, 1993; Huey and
Stevenson, 1979; Kellermann et al., 2012; Sinclair et al., 2016; van
Heerwaarden and Sgrò, 2021; Walsh et al., 2019).
In this context, we assessed fitness loss and the ability to recover,
focusing on the mechanistic basis of heat-induced sterility, of males
exposed to sub-lethal developmental temperatures. Life stages undergoing
fundamental changes might be particularly sensitive to environmental
stressors (Lowe et al., 2021), as e.g. the pupal stage in the oriental
fruit moth, Grapholita molesta (Zheng et al., 2017) orDrosophila larvae (Hoffmann et al., 2003). In addition, the lack
of mobility of many species at both early and late developmental stages
adds a challenge to elude thermal stress. Hence, we here exposed larvae
to heat-stress and considered the resulting consequences on male
fertility in early adulthood. To determine the causes of male
temperature-induced sterility, we tested whether spermatogenesis is
disrupted impairing mature sperm formation and secondly, measured
whether a delay in AG maturation (Ruhmann et al., 2016) contributes to
reduced reproductive success. With this extensive analysis of male
reproductive traits, we suggest that impaired functionality of both
reproductive tissues is causing temperature induced male sterility.
Materials and methods