Increasing developmental temperatures are well-known to impact fertility, yet their effects on pre-copulatory behaviors, despite having clear fitness consequences, are often overlooked. In many species, male nuptial gift presentation during courtship plays an important role in sex-specific mate choice, fitness and subsequent co-evolutionary dynamics. However, developmental temperature effects on nuptial gift behaviors and their implications for population fitness remain unknown. Heat-induced changes to male behavior may signal fertility, diving female discrimination, particularly in monandrous systems where exclusively pairing with an infertile male threatens population growth. Additionally, as nuptial gift production is costly, the differential allocation hypothesis suggests males should adjust gift investment based on female fitness. Here, we investigated how elevated developmental temperature affects nuptial gift behavior, mating likelihood and reproductive output in the monandrous species Drosophila subobscura. Individuals developed at either a control or stressful temperature, and fully factorial no-choice mating tests were used to identify sex-specific effects of heat stress. Heat-stressed males were largely infertile, less likely to mate, present a gift, or have a gift accepted, suggesting nuptial gifts may signal male fertility and influence female mate choice. Heat-stressed females were also less likely to mate or receive a gift, and were presented with fewer gifts from heat-stressed males. As heat-stressed females required more gifts to match the reproductive output of controls, selection may drive male mate choice through strategic resource allocation. These findings highlight how climate change may significantly impact sex-specific mate choice, with important implications for selection on pre-copulatory courtship traits and population dynamics.

The ecology of mating interactions determines a species’ mating system, yet whether environmental change can alter the mating system of a species remains unclear. Elevated temperatures can cause male sterility, prompting females to remate for fertility assurance. In monandrous systems, heat-induced male infertility poses a significant extinction risk, as females may mate exclusively with infertile males. A key question is whether male sterility could drive polyandry in a typically monandrous system. Here we address this by examining genetic variance underlying both male fertility resilience to heat stress and facultative polyandry, and assessing the fitness consequences of each mating system. We used isofemales lines of Drosophila subobscura, a monandrous species, exposing males to developmental heat stress. Male heat stress generated sterility and females mated to these males typically remated. While significant genetic variation in male fertility sensitivity and female remating emerged at moderate to high temperatures, we found little genetic variation in plasticity for polyandry. These results indicate evolutionary potential in both traits, but that a shift in mating system would arise through selection on genes associated with polyandry, rather than plasticity. Polyandry improved offspring production after initially mating to a sterile male, but did not fully restore reproductive output relative to fertile monandrous pairs, and mating with heat-stressed males increased female mortality. Heat stress also altered mating behaviour which could impact female mate choice. Together, these findings show that increasing temperatures may shape species’ mating systems and the interplay between thermal ecology and sexual selection under climate change.

RNA sequencing (RNAseq) methodology has experienced a burst of technological developments in the last decade, which has opened up opportunities for studying the mechanisms of adaptation to environmental factors at both the organismal and cellular level. Selecting the most suitable experimental approach for specific research questions and model systems can, however, be a challenge and researchers in ecology and evolution are commonly faced with the choice of whether to study gene expression variation in whole bodies, specific tissues, and/or single cells. A wide range of sometimes polarised opinions exists over which approach is best. Here, we highlight the advantages and disadvantages of each of these approaches to provide a guide to help researchers make informed decisions and maximise the power of their study. Using illustrative examples of various ecological and evolutionary research questions, we guide the readers through the different RNAseq approaches and help them identify the most suitable design for their own projects.

Extensive research indicates that fertilization outcomes are shaped by individual female and male traits that reflect their intrinsic quality. Yet, surprisingly little is known about the influence of interactions between the sexes and their adaptive significance in either externally or internally fertilizing species. Here, we review empirical evidence on how female–male interactions influence each stage of the fertilization process, including sperm transfer, transport, storage, chemoattraction and fertilization. We also address the challenges of examining female–male interaction effects within a realistic biological context and why research in this area lags behind understanding the role of individual sex-specific traits. While relatively little data are currently available to address the interactive effects between the sexes and their impact on the fertilization process, what is presently known suggests that these effects are likely to be more common across the animal tree of life than appreciated. Future research will help identify these interactions, and their understanding can also help to explain the maintenance of genetic variation and inform applied studies of fertility.

Understanding how increasing temperatures influence ectotherm population growth rate is necessary for predicting population persistence. Population growth rate depends on the thermal performance of multiple life-history traits that have different thermal sensitivities. Reproductive traits are considered more thermally sensitive than other life-history traits, such as survival and development rate. Moreover, the thermal sensitivity of reproductive traits can be sex-specific, which may differentially affect population growth. However, research concurrently assessing the sex-specific influence of heat stress on multiple reproductive traits is limited. We investigated the effect of heat stress on pupal survival and reproductive traits in both sexes to determine sex-specific thermal sensitivity and reproductive performance. Individuals of the butterfly Pieris napi were reared at either 22°C or 29°C throughout the larval and pupal stages. The latter temperature reflects the fastest development rate in this population, influencing generation time, a common population growth rate metric. We recorded pupal survival and adult body weight in both sexes. After eclosion, males and females from both treatments were allowed to interact, and mating success, copulation duration, egg production, fertility and male sterility recovery were measured. A subset of mated females was dissected to assess the number and length of fertilising eupyrene and non-fertilising apyrene sperm transferred by males of each treatment. While elevated temperatures reduced pupal survival and resulted in smaller body weights in both sexes, more substantial sex-specific effects on reproductive traits were observed. Mating success was reduced in heat-stressed females but not in males. In contrast, egg production and fertility were unaffected by heat stress in females, while heat-stressed males, despite having longer copulation durations, exhibited near-complete sterility. Male heat-induced sterility was mediated by a disruption to both eupyrene and apyrene sperm production or transfer. Male remating did not recover fertility, suggesting continued negative effects on sperm production. Our results highlight how increasing temperatures affect reproduction, illustrating that temperatures generating optimal performance for non-reproductive traits, like development rate, can negatively and differentially impact sex-specific reproductive fitness. These negative reproductive consequences may impact population persistence, highlighting the necessity to incorporate these findings into future advanced models predicting species' responses to climate warming.

Exposure to extreme temperatures can negatively affect animal reproduction, by disrupting the ability of individuals to produce any offspring (fertility), or the number of offspring produced by fertile individuals (fecundity). This has important ecological consequences, because reproduction is the ultimate measure of population fitness: a reduction in reproductive output lowers the population growth rate and increases the extinction risk. Despite this importance, there have been no large-scale summaries of the evidence for effect of temperature on reproduction. We provide a systematic map of studies testing the relationship between temperature and animal reproduction. We systematically searched for published studies that statistically test for a direct link between temperature and animal reproduction, in terms of fertility, fecundity or indirect measures of reproductive potential (gamete and gonad traits). Overall, we collated a large and rich evidence base, with 1654 papers that met our inclusion criteria, encompassing 1191 species. The map revealed several important research gaps. Insects made up almost half of the dataset, but reptiles and amphibians were uncommon, as were non-arthropod invertebrates. Fecundity was the most common reproductive trait examined, and relatively few studies measured fertility. It was uncommon for experimental studies to test exposure of different life stages, exposure to short-term heat or cold shock, exposure to temperature fluctuations, or to independently assess male and female effects. Studies were most often published in journals focusing on entomology and pest control, ecology and evolution, aquaculture and fisheries science, and marine biology. Finally, while individuals were sampled from every continent, there was a strong sampling bias towards mid-latitudes in the Northern Hemisphere, such that the tropics and polar regions are less well sampled. This map reveals a rich literature of studies testing the relationship between temperature and animal reproduction, but also uncovers substantial missing treatment of taxa, traits, and thermal regimes. This database will provide a valuable resource for future quantitative meta-analyses, and direct future studies aiming to fill identified gaps. We summarise 1600+ papers testing the relationship between temperature and animal reproduction, and identify major taxonomic, geographic and methodological research gaps. This database will provide a valuable resource for future analyses, and direct future studies aiming to fill research gaps.

Frequent and extreme temperatures associated with climate change pose a major threat to biodiversity, particularly for organisms whose metabolism is strictly linked to ambient temperatures. Many studies have explored thermal effects on survival, but heat-induced fertility loss is emerging as a greater threat to population persistence. However, while evidence is accumulating that both juvenile and adult stages heat exposure can impair fertility in their own ways, much less is known about the immediate and longer-term fitness consequences of repeated heat stress across life stages. To address this knowledge gap, we used male Drosophila melanogaster to investigate (i) the cumulative fitness effects of repeated heat stress across life stages, (ii) the potential of recovery from these heat exposures, and (iii) the underlying mechanisms. We found individual and combined effects of chronic juvenile and acute adult heat stress on male fitness traits. These effects tended to exacerbate over several days after brief heat exposure, indicating a substantial fertility loss for these short-lived organisms. Our findings highlight the cumulative and persistent effects of heat stress on fitness. Such combined effects could accelerate population declines, particularly in more vulnerable species, emphasizing the importance of considering reproduction and its recovery for more accurate models of species persistence.

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. Graphical AbstractOverview of the systematic methods (A, C, and D) to simultaneously assay lethal limits and thermal fertility limits or (B and E) thermal sensitivity of fertility. These are most easily applied to laboratory settings but can be used for assessing the fertility of wild-caught animals that have been exposed to natural temperatures.

Climates are changing rapidly, demanding equally rapid adaptation of natural populations. Whether sexual selection can aid such adaptation is under debate; while sexual selection should promote adaptation when individuals with high mating success are also best adapted to their local surroundings, the expression of sexually selected traits can incur costs. Here we asked what the demographic consequences of such costs may be once climates change to become harsher and the strength of natural selection increases. We first adopted a classic life history theory framework, incorporating a trade-off between reproduction and maintenance, and applied it to the male germline to generate formalized predictions for how an evolutionary history of strong postcopulatory sexual selection (sperm competition) may affect male fertility under acute adult heat stress. We then tested these predictions by assessing the thermal sensitivity of fertility (TSF) in replicated lineages of seed beetles maintained for 68 generations under three alternative mating regimes manipulating the opportunity for sexual and natural selection. In line with the theoretical predictions, we find that males evolving under strong sexual selection suffer from increased TSF. Interestingly, females from the regime under strong sexual selection, who experienced relaxed selection on their own reproductive effort, had high fertility in benign settings but suffered increased TSF, like their brothers. This implies that female fertility and TSF evolved through genetic correlation with reproductive traits sexually selected in males. Paternal but not maternal heat stress reduced offspring fertility with no evidence for adaptive transgenerational plasticity among heat-exposed offspring, indicating that the observed effects may compound over generations. Our results suggest that trade-offs between fertility and traits increasing success in postcopulatory sexual selection can be revealed in harsh environments. This can put polyandrous species under immediate risk during extreme heat waves expected under future climate change.

Understanding of how selection can act on traits that improve competitiveness and subsequent paternity has advanced, including the idea that internal and external fertilization presents different environments that may select differentially on ejaculate traits. However, no studies have quantitatively synthesized the intra-specific relationships between these traits and paternity. Therefore, we conducted a meta-analysis across 52 papers to determine which ejaculate traits positively correlate with paternity share and how these correlations vary with fertilization mode. Overall, most ejaculate traits were positively associated with paternity, with the notable exception of sperm length. Sub-analyses on sperm number, sperm length, and sperm velocity revealed no statistical differences between fertilization modes in the relationship between traits and paternity when all effect sizes across species were combined. However, in a sub-analysis on fish species only, we found evidence that sperm velocity may be more important in external fertilizers. We also observed differences in the importance of phylogenetic relatedness and some species-specific differences. Our results suggest that while most ejaculate traits should be under positive directional selection in both internal and external fertilizers, sperm length may be subject to more nuanced selection pressures. Overall, we highlight important patterns of intra-specific relationships between ejaculate traits and competitive fertilization success.