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