Discussion
We here investigated the recovery dynamics of a temperate D. melanogaster strain with a special focus on effects on the male reproductive tissues. Overall, sublethal temperatures severely affected a male’s ability to reproduce as found in other ectotherms (Conrad et al., 2017; Nguyen et al., 2013; Parratt et al., 2020; Rodrigues et al., 2022, 2021; Sales et al., 2018; Vasudeva et al., 2019; Walsh et al., 2019; Zheng et al., 2017; Zwoinska et al., 2020). In accordance with previous findings (Chakir et al., 2002; Petavy et al., 2001), the males used here became temporarily sterile when exposed to temperatures above 29°C during development. Moreover, we found negative effects on most of our measured reproductive traits that can be explained by males transferring a sub-optimal ejaculate and despite the ability to recover, we found temperature stress to still lead to severe fitness reductions with recovery dynamics depending on the developmental temperature experienced.
The observed reduction in output could not be explained by reduced male mating rates, i.e. due to males becoming unattractive as found in male red mason bees (Osmia bicornis, (Conrad et al., 2017)), as we found little effect of a moderate heat-challenge of four degrees over the optimal temperature on mating behaviour. We rather suspected male ability to produce or transfer sperm to be affected. Developmental temperature can result in aberrant sperm in D. melanogaster(Rohmer et al., 2004) potentially explaining our reduced egg-to-adult survival. Even if males can produce sperm, they might transfer less, reducing their overall fertility (Kraaijeveld and Chapman, 2004; Seo et al., 1990; Taylor et al., 2001). Females of the parasitoid waspAnisopteromalus calandrae stored 100 time less sperm when mated with a male exposed to a heatwave and even though males were transferred to the optimum temperature after a heat shock, they were not able to produce mature sperm (Nguyen et al., 2013). Constant exposure to heat stress during development resulted in reduced testes and sperm size in the bruchid beetle Callosobruchus maculatus (Vasudeva et al., 2014). However, while C. maculatus males had a lower sperm viability, no reduction in fertility in the absence of sperm competition was observed. Hence, while sperm number is important, other factors might be at play as well.
In addition to single mating productivity, we also tested male sperm competitive ability after developmental heat-exposure. Competitive ability is key to male reproductive success (Simmons, 2001) and was sensitive to thermal conditions in T. castaneum (Sales et al., 2018). We similarly document a severe negative impact of heat on male sperm defense ability, even after we allowed males to recover for 5 days. Thus, overall we also observe the previously described sensitivity of male reproductive function to elevated, but sub-lethal temperatures (Chakir et al., 2002; David et al., 2005; Sales et al., 2018; Walsh et al., 2019) in both competitive and non-competitive contexts. Reduced ability to fertilise eggs and win in sperm competition could be due to reduced sperm transfer (Kraaijeveld and Chapman, 2004; Seo et al., 1990; Taylor et al., 2001) and/ or reduced sperm storage by females (Nguyen et al., 2013). Both traits are important determinants in D .melanogaster sperm competition outcomes (Lüpold et al., 2013; Manier et al., 2010). Sperm storage and sperm competitive ability are mediated by receipt of SFPs (Avila et al., 2011). Thus, we continued by looking at the SVs and the AGs for possible heat damage and we will discuss those two in turn.
We observed males to recover fertility to some extent within six days, indicating that spermatogenesis was not completely damaged. Spermatogenesis in D. melanogaster lasts 10 days from the initial stem cell division (reviewed in (Fabian and Brill, 2012)) and starts in the early larval stages (Le Bras and Van Doren, 2006) with the reproductive system fully active during the pupal stage (Bodenstein, 1950) when most of sperm individualisation and maturation occurs (Fabian and Brill, 2012). In our assay, males started producing offspring by day 4 of the recovery process with the exception of males that had developed at 31°C, who needed much longer to recover fertility. As sperm individualisation is temperature sensitive (Ben-David et al., 2015), high developmental temperatures might disrupt proper sperm maturation. As the last step, the 64 interconnected spermatids individualize and finally the mature sperm coils into the base of the testis (Fabian and Brill, 2012; Steinhauer, 2015) and already at 29°C Ben-David and colleagues (Ben-David et al., 2015) observed the formation of fewer and more abnormal individualization complexes. Our proxy for availability of mature sperm - SV size and sperm presence in the SV- corroborated these findings with a major impact of elevated developmental temperatures and the opportunity to recover on the presence of mature sperm in the SVs, but also highlighted a delay in mature sperm formation. Although we found that sperm presence in the SV of males allowed to recover improved over time, the sperm quantity was lower than in control males. Apart from having fewer sperm, this sperm also seems more sensitive as sperm viability decreased faster in males exposed to 29°C during development within 30 mins after collection with the possibility to lead to reduced fertilisation success in the long-run. While this data shows that recovery of spermatogenesis is possible to some extent, the effects of temperature are not completely compensated during recovery. A reduction in the number of sperm ejaculated was also found in T. castaneummales when facing a heat shock of 5°C above the optimum temperature (Sales et al., 2018). This reduction might be the result of a significant increase in sperm cell death of exposed males (Sales et al., 2018) as we found with time. We additionally show, when elevated temperatures persist and recovery is not allowed, sperm maturation and/ or movement into the SV is not possible, as no sperm was found in the SVs of six day old males grown and kept at 31°C. A result similarly found in D. suzukii males raised at 30°C (Kirk Green et al., 2019) and in line with the idea that spermiogenesis is affected as found previously (Ben-David et al., 2015) halting the maturation of sperm. However, there is the potential for strong variation across genotypes in their ability to produce mature sperm as indicated by the variation in fertility at sub-lethal temperatures across isogenic lines of the Drosophila Genetic Reference Panel (Rodrigues et al., 2021).
In addition to the SVs, we also investigated the response to elevated developmental temperature on accessory gland maturation as it is the main production site of SFPs, which are important determinants of male reproductive success (Avila et al., 2010; Chapman et al., 2003). The interplay between male SFPs, sperm and the female reproductive tract is integral to ensure all stages of the reproductive cascade can proceed and culminate in the fertilization of a passing ova (Avila et al., 2010). Additionally, male SFPs can protect sperm and enhance sperm viability (den Boer et al., 2009, 2008; Holman, 2009; King et al., 2011). The growth of the accessory gland is key during sexual maturation (Ruhmann et al., 2016) and accompanied by an increase in functionality (Leiblich et al., 2012; Prince et al., 2019). We here observed a negative impact of heat-stress during development with a clear reduction in AG growth during the early stages. Surprisingly, recovery had little effect and did not aid AG maturation, which could result in reduced AG functionality, affecting SFP properties and/or composition. This hypothesis is tentatively supported by our phenotypic data, as temperature challenged males were not able to prevent female remating, induce increased oviposition (a trait mediated by ejaculatory sex peptide (Chapman et al., 2003; Liu and Kubli, 2003) and ovulin (Rubinstein and Wolfner, 2013)) and defend their ejaculate against subsequent rivals, regardless of the possibility for recovery. These traits are determined by seminal fluid proteins like the sex peptide (Avila et al., 2010; Chapman et al., 2003; Fricke et al., 2009; Liu and Kubli, 2003) and our results point towards the possibility that heat-challenged males could not transfer functional or adequate amounts of sex peptide and potentially other SFPs. We worked under the premise that larger AG size is indicative of SFP accumulation, which is an adequate proxy for at least the first three days after eclosion (Koppik et al., 2018).
Under normal circumstances, rapid growth of the AG can be observed in the first ten days after eclosion (Box et al., 2019; Ruhmann et al., 2016) and continues at a lower rate during male adulthood (Box et al., 2019). In general, the change in AG size occurs due to changes in both its cell types – the secondary and the main cells (Box et al., 2019; Leiblich et al., 2012). Main cells increase in size due to endocycling throughout male life (Box et al., 2019). In secondary cells the vacuole like compartments (VLCs) increase in size and change nature and location (Prince et al., 2019). VLCs are vital to secondary cell functionality (Corrigan et al., 2014; Gligorov et al., 2013; Prince et al., 2019) by secreting their content into the gland lumen and communicating with neighbouring main cells (Hopkins et al., 2019). Secondary cell and proper VLC maturation is important for overall AG functionality and thus the question arises, whether development at elevated temperatures disrupts proper formation of secondary cells and/ or interferes with main cell growth. As, we did not observe an improvement for males allowed to recover compared with males kept at the growth temperature, this might indicate that processes involved in AG growth cannot be rescued and developmental temperatures might produce an irreversible damage, which could explain the inability of heat-damaged males to recover and reach the fitness of control males. However, the AG can regenerate from damage (Box et al., 2019) and possibly our chosen time span was too short to see this effect after heat-damage warranting further investigations.