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 et al. 2008; Kellermann et al.2012; Pinsky 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 et al. 1997) and livestock (Karaca 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 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 et al.2018; Iossa 2019; Walsh et al. 2020; Zwoinska et al.2020). Recent work has found that the highest temperaturesDrosophila 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 et al. 2021; van Heerwaarden & Sgrò 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 et al. 2015; Moghadam et al. 2019). Studies on heat-induced sterility in males typically use either a single long-term stress across age-groups (Rohmer et al. 2004; Porcelliet al. 2016), or an acute stress to individuals from a single age-group (Jørgensen, Sørensen & Bundgaard 2006; Sales et al.2018; Walsh et al. 2020; Jørgensen et al. 2021). However, it has recently been shown in the flour beetle 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 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 et al. 2019). This response is called heat hardening, and is widespread in animals and plants (Bilyk, Evans & DeVries 2012; Neuner & Buchner 2012; Moghadamet 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 et al. 2019) and the ability to locate resources such as food or mating sites (Loeschcke & Hoffmann 2007). In the fruit fly Drosophila melanogaster , individual survival is improved at high temperatures through hardening, however the amount of protection provided changes depending on the life-stage measured (Moghadam 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 et al. 2004; Jørgensen, Sørensen & Bundgaard 2006). Heat-induced sterility occurs at sub-lethal temperatures in many organisms (Walsh et al. 2019), including ~44% of a panel of 43 Drosophila species (Parratt 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 Drosophila virilis , an extremely widespread model species. Critically, it has previously been demonstrated that male 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 et al. 2020; Parratt et al. 2021). This sterilisation of males at survivable temperatures makes 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.