Experimental heatwaves reduce the effectiveness of ejaculates at occupying female reproductive tracts in a model insect.

Globally, heatwaves have become more common with hazardous consequences on biological processes. Research using a model insect (Tribolium castaneum) found that 5-day experimental heatwave conditions damaged several aspects of male reproductive biology, while females remained unaffected. However, females' reproductive fitness may still be impacted, as insects typically store sperm from multiple males in specialized organs for prolonged periods. Consequently, using males which produce sperm with green fluorescent protein (GFP)-tagged sperm nuclei, we visualized in vivo whether thermal stress affects the ejaculate occupancy across female storage sites under two scenarios; (i) increasing time since insemination and (ii) in the presence of defending competitor sperm. We reconfirmed that sperm from heatwave-exposed males sired fewer offspring with previously mated females and provided new scenarios for in vivo distributions of heat-stress-exposed males' sperm. Sperm from heatwave-exposed males occupied a smaller area and were at lower densities across the females' storage sites. Generally, sperm occupancy decreased with time since insemination, and sperm from the first male to mate dominated the long-term storage site. Reassuringly, although heated males' ejaculate was less successful in occupying female tracts, they were not lost from female storage at a faster rate and were no worse than control males in their offensive ability to enter storage sites occupied by competitor sperm. Future work should consider the potential site-specificity of factors influencing sperm storage where amenable.

Systematic approaches to assessing high-temperature limits to fertility in animals.

Critical thermal limits (CTLs) gauge the physiological impact of temperature on survival or critical biological function, aiding predictions of species range shifts and climatic resilience. Two recent Drosophila species studies, using similar approaches to determine temperatures that induce sterility (thermal fertility limits [TFLs]), reveal that TFLs are often lower than CTLs and that TFLs better predict both current species distributions and extinction probability. Moreover, many studies show fertility is more sensitive at less extreme temperatures than survival (thermal sensitivity of fertility [TSF]). These results present a more pessimistic outlook on the consequences of climate change. However, unlike CTLs, TFL data are limited to Drosophila, and variability in TSF methods poses challenges in predicting species responses to increasing temperature. To address these data and methodological gaps, we propose 3 standardized approaches for assessing thermal impacts on fertility. We focus on adult obligate sexual terrestrial invertebrates but also provide modifications for other animal groups and life-history stages. We first outline a "gold-standard" protocol for determining TFLs, focussing on the effects of short-term heat shocks and simulating more frequent extreme heat events predicted by climate models. As this approach may be difficult to apply to some organisms, we then provide a standardized TSF protocol. Finally, we provide a framework to quantify fertility loss in response to extreme heat events in nature, given the limitations in laboratory approaches. Applying these standardized approaches across many taxa, similar to CTLs, will allow robust tests of the impact of fertility loss on species responses to increasing temperatures.

Pre-fertilization gamete thermal environment influences reproductive success, unmasking opposing sex-specific responses in Atlantic salmon (Salmo salar).

The environment gametes perform in just before fertilization is increasingly recognized to affect offspring fitness, yet the contributions of male and female gametes and their adaptive significance remain largely unexplored. Here, we investigated gametic thermal plasticity and its effects on hatching success and embryo performance in Atlantic salmon (Salmo salar). Eggs and sperm were incubated overnight at 2°C or 8°C, temperatures within the optimal thermal range of this species. Crosses between warm- and cold-incubated gametes were compared using a full-factorial design, with half of each clutch reared in cold temperatures and the other in warm temperatures. This allowed disentangling single-sex interaction effects when pre-fertilization temperature of gametes mismatched embryonic conditions. Pre-fertilization temperature influenced hatch timing and synchrony, and matching sperm and embryo temperatures resulted in earlier hatching. Warm incubation benefited eggs but harmed sperm, reducing the hatching success and, overall, gametic thermal plasticity did not enhance offspring fitness, indicating vulnerability to thermal changes. We highlight the sensitivity of male gametes to higher temperatures, and that gamete acclimation may not effectively buffer against deleterious effects of thermal fluctuations. From an applied angle, we propose the differential storage of male and female gametes as a tool to enhance sustainability within the hatcheries.

Fertility and mortality impacts of thermal stress from experimental heatwaves on different life stages and their recovery in a model insect.

With climate change creating a more volatile atmosphere, heatwaves that create thermal stress for living systems will become stronger and more frequent. Using the flour beetle Tribolium castaneum, we measure the impacts of thermal stress from experimental heatwaves in the laboratory on reproduction and survival across different insect life stages, and the extent and pace of any recovery. We exposed larvae, pupae, juvenile and mature adult male beetles to 5-day periods of heat stress where temperatures were maintained at either 40°C or 42°C, a few degrees above the 35°C optimum for this species' population productivity, and then measured survival and reproduction compared with controls at 30°C. Mortality due to thermal stress was greatest among juvenile life stages. Male reproductive function was specifically damaged by high temperatures, especially if experienced through pupal or immature life stages when complete sterility was shown at reproductive maturity; larval exposure did not damage adult male fertility. High temperatures impaired testis development and the production of viable sperm, with damage being strongest when experienced during pupal or juvenile adult stages. Despite this disruption, males recovered from heat stress and, depending on the stage of exposure, testis size, sperm production and fertility returned to normal 15-28 days after exposure. Our experiments reveal how thermal stress from heatwave conditions could impact on insect survival and reproduction across different life stages, and the potential and timescales of recovery.

Polyandry provides reproductive and genetic benefits in colonising populations.

Polyandry, when females mate with more than one male, is theorised to play an important role in successful colonisation of new habitats. In addition to possible benefits from sexual selection, even mild polyandry could facilitate colonisation by protecting against inbreeding and reducing the costs of mating with incompatible or infertile males. Here, we measure the importance of mild polyandry for population viability and reproductive fitness following experimental founder events into a higher-temperature regime. Using colonisation experiments with the model beetle Tribolium castaneum, in which females can produce offspring for up to 140 days following a single mating, we founded more than 100 replicate populations using single females that had been given the opportunity to mate with either one or two males and then tracked their subsequent population dynamics. Following population viability and fitness across 10 generations, we found that extinction rates were significantly lower in populations founded by females given polyandrous opportunities to mate with two males (9%) compared to populations founded by monogamous females (34%). In addition, populations founded by females that had been provided with opportunities to store sperm from two different males showed double the median productivity following colonisation compared to monogamous-founded populations. Notably, we identified short-term and longer-term benefits to post-colonisation populations from double-mating, with results suggesting that polyandry acts to both protect against mating with incompatible males through the founder event, and reduce inbreeding depression as the colonisation proceeds for 10 generations. Our results therefore show that even mild polyandry provides both reproductive and genetic benefits for colonising populations.

Adaptive thermal plasticity enhances sperm and egg performance in a model insect.

Rising and more variable global temperatures pose a challenge for biodiversity, with reproduction and fertility being especially sensitive to heat. Here, we assessed the potential for thermal adaptation in sperm and egg function using Tribolium flour beetles, a warm-temperate-tropical insect model. Following temperature increases through adult development, we found opposing gamete responses, with males producing shorter sperm and females laying larger eggs. Importantly, this gamete phenotypic plasticity was adaptive: thermal translocation experiments showed that both sperm and eggs produced in warmer conditions had superior reproductive performance in warmer environments, and vice versa for cooler production conditions and reproductive environments. In warmer environments, gamete plasticity enabled males to double their reproductive success, and females could increase offspring production by one-third. Our results reveal exciting potential for sensitive but vital traits within reproduction to handle increasing and more variable thermal regimes in the natural environment.

Experimental heatwaves compromise sperm function and cause transgenerational damage in a model insect.

Climate change is affecting biodiversity, but proximate drivers remain poorly understood. Here, we examine how experimental heatwaves impact on reproduction in an insect system. Male sensitivity to heat is recognised in endotherms, but ectotherms have received limited attention, despite comprising most of biodiversity and being more influenced by temperature variation. Using a flour beetle model system, we find that heatwave conditions (5 to 7 °C above optimum for 5 days) damaged male, but not female, reproduction. Heatwaves reduce male fertility and sperm competitiveness, and successive heatwaves almost sterilise males. Heatwaves reduce sperm production, viability, and migration through the female. Inseminated sperm in female storage are also damaged by heatwaves. Finally, we discover transgenerational impacts, with reduced reproductive potential and lifespan of offspring when fathered by males, or sperm, that had experienced heatwaves. This male reproductive damage under heatwave conditions provides one potential driver behind biodiversity declines and contractions through global warming.

Experimental evolution reveals that sperm competition intensity selects for longer, more costly sperm.

It is the differences between sperm and eggs that fundamentally underpin the differences between the sexes within reproduction. For males, it is theorized that widespread sperm competition leads to selection for investment in sperm numbers, achieved by minimizing sperm size within limited resources for spermatogenesis in the testis. Here, we empirically examine how sperm competition shapes sperm size, after more than 77 generations of experimental selection of replicate lines under either high or low sperm competition intensities in the promiscuous flour beetle Tribolium castaneum. After this experimental evolution, populations had diverged significantly in their sperm competitiveness, with sperm in ejaculates from males evolving under high sperm competition intensities gaining 20% greater paternity than sperm in ejaculates from males that had evolved under low sperm competition intensity. Males did not change their relative investment into sperm production following this experimental evolution, showing no difference in testis sizes between high and low intensity regimes. However, the more competitive males from high sperm competition intensity regimes had evolved significantly longer sperm and, across six independently selected lines, there was a significant association between the degree of divergence in sperm length and average sperm competitiveness. To determine whether such sperm elongation is costly, we used dietary restriction experiments, and revealed that protein-restricted males produced significantly shorter sperm. Our findings therefore demonstrate that sperm competition intensity can exert positive directional selection on sperm size, despite this being a costly reproductive trait.