1,2,3,4Anna C Vinton-1,5Samuel J L Gascoigne, 1,6Jinlin Chen, 7Liam Lachs1 Department of Biology, University of Oxford, Oxford, OX1 3SZ, UK2 Department of Marine and Environmental Sciences, University of Southern California, Los Angeles, California, USA3 Wrigley Institute for Environment and Sustainability, University of Southern California, Los Angeles, California, USA4 College of Arts and Sciences, University of Maine, Presque Isle, Maine, USA5 School of Biological Sciences, University of Aberdeen, Aberdeen, UK6 School of Life Sciences, Nanjing University, Nanjing, China7 School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, UKAbstract To conserve species biodiversity alongside expected climate changes, it is vital to understand how species respond to changing temperatures. However, plastic adaptations to changing environments can come with tradeoffs, and we lack an understanding of how vital rates correlate within and across organisms’ lifetimes in varying environmental contexts. To address this gap, we ran a series of experiments whereinDrosophila melanogaster flies developed in cooler (21°C) and warmer (26.5°C) environments and, once at adult stages, were exposed to a range of temperatures from 12°C to 32°C to quantify the thermal sensitivity of fitness traits. We then developed a demographic model to investigate individual-scale thermal dependencies scaled to population-level outcomes. Our study revealed a complex interplay between developmental temperature, temperature-dependent adult survival, and fecundity. While flies that developed in cooler conditions initially showed superior demographic rates, our demographic model indicated that these benefits did not translate into higher relative fitness in terms of population growth rate due to a slowing of developmental time. Overall, our findings demonstrate that temperature can have contrasting effects on different traits, such that enhancement of specific vital rates may not necessarily translate into long-term population benefits. This highlights the need for more comprehensive insights on the thermal sensitivity of fitness related traits across life stages.KeywordsThermal adaptation, Drosophila melanogaster, Temperature response, Demographic modeling, Climate change, Developmental plasticity, Population dynamics, Life history trade-offs, Thermal tolerance, Evolutionary ecology, Phenotypic plasticity, DevelopmentIntroductionGlobal climate change poses significant challenges for ectotherms, whose body temperatures and physiological processes are directly influenced by environmental conditions. This vulnerability to temperature fluctuations makes ectotherms particularly important for understanding and predicting biological and ecological impacts of climate change. Among ectotherms, insects represent an especially critical group for study, as they are experiencing widespread population declines (Deutsch et al., 2008; Halsch et al., 2021) and exhibit well-characterized thermal performance curves (TPCs) that describe how their physiological processes and fitness components vary across temperatures. These TPCs typically show reduced performance at both high and low temperature extremes with an optimal range in between, though the shape and position of these curves can vary among species, traits, and past thermal environment (Kingsolver et al., 2015; Sinclair et al., 2016, Kellermann et al., 2019).Understanding the mechanisms of thermal adaptation in ectotherms requires examining how temperature affects multiple aspects of their life history across different life stages. Thermal performance can vary not only across species but also within an organism’s lifetime, with developmental stages often showing different thermal optima and sensitivities compared to adult stages (Bowler & Terblanche, 2008; Kingsolver & Huey, 2008). This developmental thermal plasticity can have profound implications for how populations respond to changing thermal environments, as conditions experienced early in life may influence adult performance in complex and sometimes counterintuitive ways (Angilletta, 2009; Sgrò et al., 2016). Recent research has increasingly focused on quantifying these carry-over effects between life stages, revealing that developmental conditions can shape adult thermal tolerances, metabolic rates, and reproductive capacities through various physiological and molecular mechanisms (Colinet & Hoffmann, 2012; Salachan & Sørensen, 2022).The effects of developmental temperature on adult fitness have been extensively studied, with numerous experiments testing competing hypotheses about thermal adaptation (Klepsatel et al., 2019; Klepsatel et al., 2019; Klepsatel, 2023; Gibert et al., 2001; Sanghvi, et al., 2022; Santoset al., 2021). The Beneficial Acclimation Hypothesis (BAH) suggests that exposure to warm temperatures during development should enhance heat tolerance in adults, particularly when they encounter similar warm conditions (Wilson & Franklin, 2002). This hypothesis has been tested across various fitness components in multiple studies, with mixed results ranging from support to context-dependent findings (Geister et al., 2007; Gibert et al., 2001; Woods et al., 2002; Terblanche, 2003). Conversely, the Optimal Development Temperature Hypothesis predicts that development at intermediate temperatures (around 21-25°C for D. melanogaster) should maximize adult performance across all temperatures by allowing for optimal tissue development (Klepsatel et al., 2019). Recent work continues to test these competing frameworks (Ramniwas, 2020; Jerbi-Elayed et al., 2021; Min et al., 2021; Sanghvi et al., 2021; Malod et al., 2024), though few studies connect individual-level responses to population dynamics.These competing hypotheses are further complicated by potential trade-offs between different vital rates. What constitutes an optimal temperature may differ for survival versus reproduction (Marshall, 2010; Partridge, 1998; Lind et al., 2013). For instance, temperatures that maximize female fecundity might reduce male fertility (Iossa 2019, David et al., 2005), while conditions that speed development could compromise adult survival (Sibly & Atkinson, 1994; Petavy et al., 2001). Such trade-offs create complex relationships between temperature and overall fitness that cannot be understood by examining single traits in isolation.Although individual-level thermal responses are important, ultimately population persistence depends on how these responses scale up to affect population dynamics. Previous research has extensively documented how temperature affects individual life history traits (Cotter et al., 2010; Gascoigne et al., 2022), but rarely connects these effects to population-level outcomes (Villellas et al., 2015). This represents a crucial knowledge gap, as individual benefits may not necessarily translate into population-level advantages when all vital rates are considered together in demographic models. Even studies that attempt to integrate multiple thermal responses, such as fecundity, survival, and developmental speed into composite fitness measures (Overgaard et al., 2014), often lack explicit age structure in vital rates when making assumptions about population growth and persistence (Maclean et al., 2019).Drosophila melanogaster serves as an ideal model organism for investigating thermal responses due to its rapid life cycle, well-documented biology, and extensive use in previous thermal biology research (McDonald et al., 2018; Halim, 2020; Kapali et al., 2022). Studies have thoroughly characterized how adult flies respond to different temperatures, with optimal performance typically occurring between 21-25°C for most fitness components (Miquel et al., 1976; Berrigan & Partridge, 1997; Klepsatel et al., 2019). However, these thermal responses can vary significantly among different life stages and physiological processes, creating complex patterns of temperature-dependent performance (AL-Saffar, 1995; Hoffmann & Harshman, 1999; Vinton et al., 2023).To address these gaps, our study has three main aims: First, we investigate how developmental temperature affects adult thermal performance curves, testing whether warm development temperatures provide benefits specifically at warm adult temperatures (BAH) or if intermediate developmental temperatures maximize performance across all adult temperatures. Second, we examine potential trade-offs between vital rates, testing whether optimal temperatures differ for survival versus reproduction. Finally, we use demographic modeling to determine how individual-level thermal responses scale up to affect population dynamics, testing whether developmental temperatures that maximize individual performance also lead to larger population sizes and higher growth rates. This approach integrates age-structured vital rates into demographic models to project population dynamics through time, allowing us to quantify how individual-level plasticity translates into population-level outcomes.MethodsFig M1. Experimental design for thermal adaptation study inDrosophila melanogaster. Eggs were collected and assigned to one of two developmental temperatures (21°C or 26.5°C), as flies were unable to develop in 12°C and 32°C. Upon adult emergence, three females and three males from each developmental treatment were transferred to experimental chambers across a range of final temperatures (12°C to 32°C). We measured developmental time from egg to adult emergence, along with adult survival and reproductive rates at each final temperature to parameterize our demographic model.Study system and stockFor our study, we used the Dahomey wild-type strain of D. melanogaster (Wigby & Chapman, 2004). This strain was maintained in large, outbred populations characterized by overlapping generations and was consistently housed in a controlled environment at 25°C with a 12:12 light-dark cycle, within a non-humidified room. The base population was reared on standard Lewis medium (Lewis, 1960).Experimental designIn our study, we examined the impact of early-life stage and final temperature on vital rate Thermal Performance Curves (TPCs) across two pre-adult temperature treatments (21°C and 26.5°C) and four different final temperature conditions (12°C, 21°C, 26.5°C, and 32°C) (see Fig M1). The characteristics (breadth, critical limits, etc.) of a TPC have been shown to correlate with the natural distribution of species, making laboratory experiments that quantify this breadth informative for understanding natural populations (Schulte, 2011; Chen, 2024). Phenotypic plasticity can result in shifts in the TPC breadth and optimum temperature, making TPCs useful tools for assessing the circumstances under which phenotypic plasticity may enhance the survival of populations in a changing climate.The experimental procedure began with 400 eggs obtained from the stock population maintained at 25°C. Out of these, 200 eggs were placed in one 25mm diameter standard Drosophila vials (VWR International) containing 4 ml of fly medium. These vials were submerged in water baths kept at either constant 21°C or 26.5°C during egg-to-adult development. It’s worth noting that individuals of this species did not progress beyond the pupal stage at extreme developmental temperatures of 12°C and 32°C. To ensure flies experienced a homogeneous temperature set at our target, the vials were submerged in water to a level above the area in which the flies could move freely. The water bath temperature was maintained with a variation of approximately ±0.5°C around the set temperature. Relative humidity levels were maintained within the range of 80% to 90%.Virgin adults were collected within 7 hours after eclosion. After determining the sex of individuals over ice to immobilize them, 3 males and 3 females were allocated to a vial with fresh fly medium. Subsequently, 5 vials from each of the two developmental temperature groups were allocated to each of the four adult temperatures (12°C, 21°C, 26.5°C, and 32°C) to measure vital rates. Daily survival rates were recorded and deceased flies were removed from the vials. Additionally, fecundity was measured every 3 days. The day before egg counts, flies were transferred to new vials with a set small amount of yeast paste. After 24 hours of laying eggs, surviving flies were transferred to new vials and placed back into their respective water baths. Numbers of eggs were immediately recorded and the egg vials were also placed back to the same water baths as their parents. The developmental time of these emerging offspring was measured by the number of days between the oviposition of eggs and the eclosion of the first adult of the next generation.Estimating vital ratesTo quantify the impacts of developmental and adult temperatures on adult survival, fertility and overall fitness, we (1) statistically estimated vital rates with respect to age and (2) coerced these vital rates into a structured population model.Through daily assessments of survival and fertility assays performed every three days, we estimated a suite of vital rates. These vital rates include: probability of emergence, development time, adult survival and fecundity (i.e., number of eggs oviposited per female). Whilst probability of emergence and development time only vary across developmental temperatures (i.e., 21°C and 26.5°C), adult survival and fertility vary across all combinations of developmental and final temperatures (i.e., 12°C, 21°C, 26.5°C, and 32°C) in a fully factorial manner, resulting in eight estimates of adult survival and fertility across treatments.Egg to adult offspring viability was calculated by dividing the number of eclosing adults by the number of eggs laid in the fertility assay. Development time was calculated as the number of days between the oviposition of eggs and the eclosion of adults. Adult survival was estimated in two steps. First, a Cox regression was used to model the survivorship (lx, where x denotes ages) of a cohort (i.e., combination of developmental and final temperatures) across the adults’ lifespan. Second, survivorship was converted to daily estimates of survival (px); px = lx / lx-1. Likewise, adult fertility was also estimated in two steps. First, a quasi-poisson generalized linear model (GLM) was used to regress the number of eggs per female with respect to age across all combinations of developmental and final temperatures. These GLMs had the functional form:number of eggs per female ~ intercept + β1 x age + β2 x age2 + error ,to allow for curvilinear relationships of fertility with respect to age (Sanghvi 2024).Demographic model: Construction and analysisTo infer fitness from vital rates (e.g. , survival, growth, reproduction), we constructed matrix population models. Matrix population models (MPMs) are stage/age/size structured population models that project populations across discrete timesteps (i.e ., t\(\rightarrow\) t+1; Caswell 2001). In turn, for all combinations of developmental and final temperature, we constructed MPMs which predictD. melanogaster population dynamics structured by age (in days) across daily timesteps. To construct these MPMs, the following vital rates were included: egg-adult survival (i.e. , expected proportion of eggs successfully eclosing as adults), developmental time (i.e. , duration from initial egg oviposition to adult eclosion), adult survival (i.e. , expected adult survival as inferred from cox regression), adult fertility (i.e. , expected number of eggs produced per female per day). Following the construction of these MPMs, the associated fitness was calculated as the real part of the dominant eigenvalue of the MPM (i.e. , lambda) which is often used as a proxy of fitness (Cubaynes 2022). The constructed MPMs and their associated code are available in the supplementary online materials.
