Heatwaves increase larval mortality and delay development of a solitary bee

Heatwaves are expected to increase in frequency, intensity and duration due to climate change. For organisms like insects with discrete development, sensitivity may differ among life stages. Thermal sensitivity is of particular concern for species like bees that provide critical ecosystem services. Although social bees moderate nest temperatures through worker behaviour, solitary bees do not thermoregulate their nests, making immobile developing offspring especially vulnerable to such extreme events. We studied the effects of heatwaves on larval development in the solitary bee, Osmia lignaria, an important orchard pollinator and model species for solitary bee biology. We used a factorial design to assess the impacts of heatwave temperature and duration on larval mortality and development rate. Larvae were exposed to heatwaves under realistic diel temperature regimes, with daytime maxima of 31 or 37°C for 4 or 7 days at the beginning of development. Heatwave temperature strongly affected larval mortality. Exposure to 37°C heatwaves increased larval mortality by 130%, but the cooler 31°C heatwaves did not significantly impact mortality. Heatwave duration did not impact larval mortality. Larval development time also was affected by heatwave exposure. Compared with the no‐heatwave‐control, bees in the 31°C heatwave developed faster, and bees in the 37°C heatwave developed slower. Our study reveals the importance of stage‐specific effects of extreme events and suggests that the timing and maximum temperature of projected heatwaves may be more detrimental to populations than heatwave duration.


INTRODUCTION
Global climate change is characterised by warming temperatures but also by increased frequency, duration and intensity of extreme climate events (Fischer & Knutti, 2015;Perkins et al., 2012).Among documented temperature changes, short and excessively hot conditions known as 'heatwaves' are particularly concerning (Stillman, 2019).
Heatwaves represent at least two distinct and dramatic stressors for many organisms-their inherent high temperature and their rapid fluctuation between background conditions and temperature peaks.As a result, heatwaves and the associated temperature variability they produce may be more harmful to organisms than mean temperature increases alone (Colinet et al., 2015;Harris et al., 2018;Paaijmans et al., 2013;Vasseur et al., 2014).Failure to consider extreme heat and variation may lead us to underestimate the impacts of climate change and reduce our ability to predict organismal responses to projected environmental conditions (Maxwell et al., 2019;Paaijmans et al., 2013;Vasseur et al., 2014).
In addition, the discrete life stages of many insects (egg, larva, pupa and adult) can differ in tolerance to high temperatures and rapid temperature fluctuation (Abarca et al., 2019;Chiu et al., 2015;Klockmann et al., 2016;Zhang, Chang, et al., 2015).This may be especially true of taxa whose immature stages are constrained within a nest and thus are less able to behaviourally regulate internal temperature.In such cases, larvae (and pupae) cannot escape the heat by moving to cooler microhabitats during heatwaves and instead rely only on innate physiological thermotolerance (González-Tokman et al., 2020;Klockmann et al., 2016).
Immature stages are additionally vulnerable to heatwaves because they are susceptible to temperature-dependent changes in developmental rate that directly or indirectly affect adult performance and fitness.Heatwaves can delay larval development (Kingsolver et al., 2021), which may intensify other stressors such as parasitism risk and additional heat stress if the larval stage extends further into warm parts of the year (Farzan & Yang, 2018;Goulson et al., 2015).
Delayed development could reflect a nonlinear thermal performance curve, where insects (and other ectotherms) develop slower at temperatures above optimum (Kemp & Bosch, 2005).Delayed development may also be due to high temperatures degrading food quality or oxidative damage from heat stress (Hemberger et al., 2023;Miao et al., 2020;Vanderplanck et al., 2019).Moderate heat stress may have opposing immediate effects, for example, increased development rate, that nevertheless lead to similar negative fitness outcomes for subsequent life stages.Insects develop faster at higher temperatures, which could result in smaller adults-potentially leading to decreased fecundity and longevity (Kingsolver & Huey, 2008).The effect would be magnified for populations that reproduce only once per year, for which a single heatwave could have marked effects on the next-generation's population size and fitness (Denny, 2017).
In addition to general sensitivity of immature stages, heatwaves may differentially impact specific juvenile stages and influence survival and fecundity of adult insects (Kingsolver et al., 2021;Zhang et al., 2019;Zhang, Chang, et al., 2015).Patterns among stages appear to vary, supporting the need for more careful investigation.In many cases, early instars are more vulnerable to mortality and changes in development rate than later instars (Blanckenhorn et al., 2014;Kingsolver et al., 2021;Klockmann et al., 2017;Zhang, Chang, et al., 2015); however, later instars may also show particular sensitivity.Two examples from moths illustrate the complexity of responses: in one study, Manduca sexta survival was most reduced by heatwave exposure in the middle of larval development (Kingsolver et al., 2021), but in another, Plutella xylostella exposed to heatwaves later in development had lower adult fecundity than those experiencing heatwaves earlier in development (Zhang, Chang, et al., 2015;Zhang, Rudolf, & Ma, 2015).Regardless of the exact sensitivity among specific larval stages, consequences like decreased longevity and fecundity in later life stages show that full recovery is often not possible (Kingsolver et al., 2021;Martinet et al., 2021;Zhu et al., 2019).
Heatwaves may be particularly detrimental to bees, especially those that nest in cavities above-ground where buffering to direct radiant heat and air temperature is limited.Bee larvae are confined within the nest for all of development, so they cannot seek thermal refuge during heatwaves (Hamblin et al., 2017).Although well-known social species like honey bees and bumble bees can actively thermoregulate brood (Stabentheiner et al., 2010;Weidenmüller, 2004), the vast majority of bee species are solitary (Danforth et al., 2019) and rely only on nesting substrate and nest placement for temperature regulation.In solitary species, the pollen and nectar provision on which larvae feed is also provided en masse prior to oviposition, so the provision itself may be affected by a heatwave.For example, higher temperature may cause desiccation, or altered microbial growth, which in turn could affect larval development.
Understanding the impacts of heatwaves on bee development and survival is particularly critical because of the vital role they play in the pollination of crops and wild plant communities (Garibaldi et al., 2013;Ollerton, 2017;Potts et al., 2016).Seventy percent of modern crops benefit from animal pollination, most of it supplied by bees; and over 80% of wild plant species are animal-pollinated-here too bees are dominant (Klein et al., 2007;Ollerton et al., 2011).In addition, there is increasing evidence for declines of bees across different regions (Biesmeijer et al., 2006;Cameron et al., 2011;Powney et al., 2019).Changing climate and related stressors like heatwaves are strongly associated with species responses (Chown & Nicolson, 2004;Janousek et al., 2023;Wen et al., 2022).Despite the identified need for greater understanding, significant gaps in our knowledge remain for the effect of heatwaves on solitary bee species, especially impacts on larval stages, which may be particularly sensitive and present at times of year when heatwaves are prevalent.
To begin to address such gaps, we used a factorial design to explore the effects of experimentally applied heatwave temperature and duration on the larval development and mortality of the solitary cavity-nesting bee Osmia lignaria (Say).Based on our general understanding of thermal performance curves in insects, we expected heatwaves to increase mortality in a non-linear way, such that (1) moderate temperature heatwaves (which fall close to temperature optima) might accelerate development and have modest impact on larval mortality, whereas (2) higher temperature heatwaves (above temperature optima) would delay development and increase mortality (Chown & Nicolson, 2004).We also expected heatwave duration to act additively with temperature, such that longer, hotter heatwaves would affect survival and development rate more than shorter, cooler ones.

Study species
To evaluate how heatwaves impact bees, we used the solitary bee O. lignaria (Megachilidae), a species that has been the subject of previous thermotolerance research (Pitts-Singer et al., 2014;Kemp, & Bosch 2005).O. lignaria is a vernal species native throughout North America with a subspecies O. lignaria propinqua occurring west of the Rocky Mountains.Wild populations occur across a range of elevations from 100 to $2000 m (Guisse & Miller, 2011).Population densities are highest in mid-elevation foothills.In the United States, O. lignaria is managed for spring blooming crops like almond, plum and sour cherry (Kemp & Bosch, 2005) throughout Northern California, parts of the Pacific Northwest and the Intermountain West.Adults emerge in early spring (February-April) and are active for about 5 weeks.
Females construct their nests as a linear series of brood chambers within preexisting tunnels.They mass-provision brood with pollen and nectar and lay a single egg atop each provision before sealing the chamber with a mud partition (Torchio, 1989).Eggs hatch after $3 days, and individuals progress quickly through a series of four larval instars (Bosch & Kemp, 2000).The fifth and the final instar accounts for the majority of the larval lifespan during which individuals consume most of their provision, then spin a cocoon in which they pupate; adults eclose in late summer and overwinter within the cocoon.Larvae develop well between $20 and 30 C, with mortality and delayed development outside this range (Kemp & Bosch, 2005).
In nature, nests often occupy abandoned beetle tunnels in wood, but females will readily accept holes drilled into wood blocks.These holes can be lined with paper straws to facilitate removal and manipulation of nests for experiments (Stuligross & Williams, 2020;Williams, 2003).Bees for this experiment were sourced from local populations along the western slope of the Sierra Nevada between 350 and 975 m elevation.

Experimental design
The overall structure of our experiment is a two-way fully blocked design with heatwave temperature (control, moderate heatwave and high heatwave) and duration (short and long) as factors.To obtain bee larvae for our experiment, we began by releasing newly emerged adult O. lignaria into large flight cages at the University of California (UC) Davis Bee Research Facility in the spring of 2020.In each cage, we provided wooden nest blocks with drilled holes and lined with paper straws (7.7 mm internal diameter, 13 cm in length), which we collected and replaced as nests were completed.We provided a consistent mud source in the cages for nesting by watering a patch of open soil.In all cages, we planted the common native wildflowers Phacelia tanacetifolia (Benth.)and Phacelia ciliata (Benth.),both of which are used by O. lignaria and provide high-quality pollen and nectar resources (Boyle & Pitts-Singer, 2017;Williams, 2003).In the unlimited resource environment provided, females completed nests within 3 days of initiation (Tepedino & Torchio, 1982).We collected all newly completed nests each evening throughout the adult nesting period.
This ensured that all eggs within a nest were of relatively uniform age, all laid within 2 days of nest collection.
Upon nest collection, we cut a small window into the paper straw at the position of each brood cell, which allowed us to view development without disturbing the bees (Pitts-Singer et al., 2014; Figure S1).
We randomly assigned collected nests to one of five experimental heatwave treatments and placed them into incubators (Percival I-36 Series Controlled Environment Chambers, 2010, Percival Scientific Inc., Perry, IA, USA) corresponding to their respective heatwave temperature on the night they were collected.We left larvae in the original nest straw to prevent mortality due to transfer or disturbance and wrapped nests in polyethylene food wrap to prevent desiccation.
Because the numbers of offspring vary among nests, our choice created some difference in the numbers of brood cells per treatment.
Moving nests to the assigned treatments at night allowed individual eggs and larvae to experience cooler, nighttime temperatures at heatwave onset before ramping up to the daytime temperature as opposed to suddenly placing them into a high temperature.Bees experienced the heatwave in the first 4 or 7 days after nest collection, encompassing part of early larval development (egg through second instar).Incubator temperatures were monitored throughout the experiment.We recorded larval development stage and mortality daily for all larvae until they finished spinning cocoons.Larval stages recorded were the first, second and fifth instars as they are readily identifiable (Bosch & Kemp, 2000;Torchio, 1989).We also recorded cocoon completion, marking the end of larval development.We weighed all completed cocoons within 10-14 days of cocoon completion and re-weighed them three additional times throughout the following fall and winter to determine the effect of heatwaves on weight loss throughout development.
We stored nests under control environmental conditions after the heatwave period ended until mid-July, when we increased the temperature to 29 C for 4 months to mimic regional summer conditions.
We decreased the incubation temperature to 20 C for fall conditions for 1 month, then overwintered cocoons in an uninsulated outdoor shed at the UC Davis Bee Biology Research facility until the following spring.We then moved the cocoons indoors to the laboratory to monitor adult emergence from cocoons.Cocoons of bees that did not emerge were opened to determine developmental stage at death.Kemp & Bosch, 2005).To elucidate the effects of short and long heatwaves and the potential effects of prolonged heatwave exposure, we established two heatwave durations, 4 and 7 days, to reflect the minimum and maximum length for a single heatwave defined by Cal-Adapt (2021) (Mazdiyasni et al., 2019;Raei et al., 2018).

Heatwave treatments
To study the effects of heatwave temperature and duration, we used a crossed heatwave temperature Â duration design.We exposed developing larvae to one of four experimental conditions: a low (31 C) or high (37 C) temperature heatwave ('heatwave temperature') for a short (4 day) or long (7 day) duration, or control (25 C) conditions.Temperatures in each treatment followed a 14:10 h diel cycle with a daily high of 37 C and a low of 22 C for the high temperature heatwave, 31 C and 20 C for the low temperature heatwave and 25 C and 15 C for the control, following, for example, Radmacher and Strohm (2011).Projections of heatwaves indicate that warmer nights will be an important factor in determining their severity (Meehl & Tebaldi, 2004); our heatwave treatments reflect real-world heatwave conditions with a lower amplitude between daytime high and low for the highest temperature heatwave.Brood cell sample sizes for each group were the following: 25 C control (n = 45), 31 C 4-day (n = 33), 31 C 7-day (n = 62), 37 C 4-day (n = 40) and 37 C 7-day (n = 34).
We estimated larval sex from provision size and nest position, where we assumed that larger provisions and positions closest to the nest origin were females (Table S1).Nests remained in the incubator at heatwave temperature for the duration of their assigned heatwave and then were moved to control conditions for the remainder of larval development.

Statistical analysis
To assess the effect of heatwave temperature and duration on larval mortality, larval development and adult emergence, we used a generalised linear mixed model (GLMM) framework using the 'glmmTMB' package (Brooks et al., 2017).All statistical analyses were performed using R (version 4.0.2;R Core Team, 2021).We included heatwave temperature (25, 31 and 37 C) and duration (4 days, 7 days) as fixed effects and nest identity as a random effect to account for maternal effects.Because larvae in the control group experienced no heatwave, we randomly assigned each bee in the control group to one duration treatment.This allowed us to model the interaction of heatwave temperature and duration.We generated 500 such datasets, each with a different random assignment for the control duration.We compared the results of the model from each randomization and found no differences based on the control assignments.The interaction between heatwave temperature and duration was not significant for any response variable, so we removed the interaction term in our final models.Our final model structure was response $ temperature + duration + (1jnest ID).Larval mortality and adult emergence were analysed with a binomial error distribution and logit link, and mortality timing was analysed with a negative binomial error distribution and log link.
We confirmed requirements of distributions with visual inspection of residual plots.p-Values were calculated using likelihood ratio tests comparing models with and without the focal variable.We performed post hoc pairwise comparisons using the Tukey method to determine differences among treatments.To assess differences in survival throughout larval development by heatwave temperature and duration, we conducted a Kaplan-Meier survivorship analysis using the package 'survminer' (Kassambara, 2020).We fit a survival curve with the number of days a larva was alive as a function of heatwave temperature and duration.We right-censored data after 50 days as all bees were cocooned or had died by this point; the exact choice of this cut-off did not qualitatively affect our results (Figure S2).

Larval mortality
Heatwave temperature dramatically affected larval mortality (χ 2 = 48.55,p < 0.001; Figure 1).Larvae exposed to 37 C heatwaves were 385% more likely to die than larvae that were not exposed to a heatwave ( p < 0.001).Larval mortality in the 31 C treatments did not differ from those in the no heatwave control (p = 0.940).Heatwave duration did not significantly influence larval mortality of any treatment groups (χ 2 = 2.36, p = 0.125; Figure 1).
Ninety-one percent of larval mortality occurred within the first 10 days of development, and over 60% of larvae died during the egg and first instar stages across all treatments.Of the larvae that died, those that experienced a heatwave of any temperature died approximately 6 days earlier than larvae in the control group (χ 2 = 79.29,p = 0.001; Figure S3).Even for larvae in the 31 C heatwave, whose overall mortality was fractionally lower than for no-heatwave controls, timing of mortality was earlier (Figures 2 and S3).For larvae experiencing heatwaves, 56% and 91% of mortality occurred during the heatwave period for 4-and 7-day heatwaves, respectively.

Larval development
Heatwave temperature also affected development time.Larvae exposed to 37 C heatwaves took 4.6 days longer to complete development (egg to cocoon completion) than those in the no-heatwave control (p = 0.002) and 6.4 days longer than those in the 31 C heatwave (p < 0.001; Figure 3).Larvae exposed to 31 C heatwaves developed slightly faster (1.8 days) than those in the control, although the difference was not significant (p = 0.138; Figure 3).Heatwave duration did not influence the time to complete larval development (χ 2 = 1.43, p = 0.231; Figure 3).

Post-larval performance and adult outcomes
Overall, 56% of all bees that completed cocoons successfully emerged as adults the following spring.The probability of adult emergence from cocoons did not differ between heatwave temperature (χ 2 = 0.83, p = 0.661; Figure S4) nor duration (χ 2 = 0.33, p = 0.563; Figure S4).Bees surviving to adult emergence lost an average of 0.04 ± 0.004 g (mean ± SE) overwinter, and this weight loss was not influenced by heatwave temperature (χ 2 = 0.24, p = 0.886) nor duration (χ 2 = 0.11, p = 0.739; Figure S5).Of the larvae that successfully made cocoons but did not emerge as adults, most died as larvae or eclosed adults, and one died in the pupal stage (Figure S6).

DISCUSSION
Understanding the impacts of extreme climate events on bee survival and development is key to supporting their diversity and the pollination services they provide (Kremen et al., 2007;Ollerton, 2017;Potts et al., 2010).Although the prevalence of heatwaves continues to dramatically increase as part of ongoing global climate change, their impacts on beneficial insects, particularly during sensitive developmental stages, remain least well explored.Our experiment reveals that the increasing prevalence and severity of heatwaves (Meehl & Tebaldi, 2004) are likely to have dramatic impacts for bee populations, not only through effects on adult performance and behaviour (e.g.Hemberger et al., 2023)  The limited research that exists on heat stress in early life stages has mostly considered social bee taxa for which impacts are buffered by the social context and extended brood care (Vanderplanck et al., 2019).Solitary bees like O. lignaria end brood-care after massprovisioning the egg.As such, the immature stages of solitary bees depend entirely on the ambient nest temperature to determine survival and development.Our study provides a critical look into the impacts of heatwaves on early life stages and demonstrates that the effects may contribute to bee declines and have long-term implications for population persistence in the face of global climate change.

Effects of heatwave temperature
High-temperature heatwaves dramatically increased larval mortality and delayed development in exposed bees.Similar impacts of simulated high temperatures delayed development in O. bicornis and were accentuated by temperature fluctuation (Radmacher & Strohm, 2011).
However, the more moderate temperature heatwave killed substantially fewer larvae and did not delay development.If anything, the moderate heatwave accelerated development slightly.The patterns of mortality and development rate reveal an underlying, non-linear thermal performance profile consistent for other ectotherms (Chown & Nicolson, 2004;Denny, 2017;Roitberg & Mangel, 2016).Indeed, our moderate temperature heatwave may have been very near to a temperature optimum, producing fractionally lower mortality and faster larval development than the lower 'control' temperature.
We suspect that such nonlinear responses to higher temperature with strong negative impacts at the extreme will be similar for solitary bee species in general and in particular for above-ground cavitynesters, which are more vulnerable to thermal variation than groundnesting bees due to greater exposure to solar radiation (CaraDonna et al., 2018;Hamblin et al., 2017;Ostap-Chec et al., 2021).
We posit that the impacts of the high-temperature heatwaves on larval mortality and development were caused by (a) direct impacts of heat through bee physiological responses to thermal stress, (b) desiccation stress from heat-induced humidity changes and (c) indirect changes to provision quality that impaired larval feeding.
Heat shock response is conserved across many insect taxa, including disruption to cell membranes through thermal wounding, denaturation and subsequent loss of function of proteins, and disruption to the electron transport chain through oxidative damage (Denlinger & Yocum, 1998;González-Tokman et al., 2020).Additionally, as ectotherms, insects' metabolic rate is largely controlled by environmental temperature.Increasing metabolic rate at high temperatures can lead to energy depletion, which may be particularly costly for early larval instars that feed much more slowly than the fourth and fifth instar larvae.At stressfully high temperatures, faster metabolism leads to the increased production of free radicals (reactive oxygen species), resulting in oxidative damage in insects and other organisms (Feidantsis et al., 2020;Srivastava & Kumar, 2015).Free radical damage to the electron transport chain reduces cell energy production and can overwhelm cellular protective mechanisms, triggering apoptosis (González-Tokman et al., 2020).Indeed, Drosophila melanogaster exposed to high temperatures during development had a long-term reduction in fat storage due in part to apoptosis, even in flies exposed only to transient thermal stress (Klepsatel et al., 2016).Developmental delay could be caused by the buildup of these stress-induced free radicals or heat-induced energy depletion (Chen et al., 2019;Malmendal et al., 2006).Such buildup may partially explain the higher mortality and slower development we observed in larvae exposed to the highest temperature heatwaves (Apirajkamol et al., 2020;Kingsolver et al., 2021).Much of the work on insect physiological responses to heat characterised development at constant, high temperatures or heat shocks (1-24 h), which differ from heatwave conditions (Mazdiyasni et al., 2019;Raei et al., 2018).Greater understanding of the physiological response to heatwaves, both during and after heatwaves, will increase our predictive power on the long-term effects on insect development and adult fitness.
Our finding that eggs and early instars were the most sensitive to heatwaves suggests that stage-specific or size-related physiological sensitivity may drive differing responses to heat stress.This pattern of early sensitivity is consistent with field studies of the related cavitynesting bee, Megachile apicalis (Spinola), in our study region and Osmia bicornis, an Osmia species found across Europe (Hranitz et al., 2009;Ostap-Chec et al., 2021).The M. apicalis study found significant increases in offspring mortality, likely attributable to heat stress.It also identified high concentrations of heat-shock proteins-a group of chaperone proteins upregulated during thermal stress to protect cells and proteins from damage-especially at the egg stage from nests in high-temperature sites.Laboratory-based studies of prepupal stages of Megachile species showed surprising robustness to heat shocks later in development (Hranitz & Barthell, 2003).Although mechanistic details for the difference are not known, difference in overall size and surface-area-to-volume ratios across life stages might play a role, especially with respect to desiccation.
Our inspections of eggs and larvae also support the idea that desiccation stress contributed to mortality.Many bees that died in the highest temperature heatwave shrivelled and discoloured before death.Water loss and desiccation can reduce fitness and survival from heatwaves in a variety of terrestrial ectotherms (Jørgensen et al., 2022;Klockmann & Fischer, 2017;Stillman, 2019).Our experiments were not set up to examine desiccation tolerance; however, the response to fluctuating humidity during heatwaves is an important topic for further research given the co-occurrence of heatwaves and drought (Leung et al., 2004;Padda et al., 2021;Pierce et al., 2013).
In addition to direct physiological effects, heatwaves altered the consistency of larval provisions through changes to relative humidity leading to evaporative loss.Pollen and nectar provisions are hygroscopic, absorbing moisture from the air and responding to fluctuating humidity (Danforth et al., 2019;May, 1972), and provision consistency can be an important driver of larval survival in sweat bees (May, 1972).We observed some provisions within the hightemperature heatwave treatment dry out and become difficult for larvae to ingest, and others in the same treatment liquified such that early instar larvae sank into them and drowned.It is unclear what caused such contrasting effects, but larvae feeding on the drier provisions were delayed relative to others.
Heatwaves did not affect adult emergence success or body weight the following spring, indicating that individuals that survive heatwaves in early development successfully complete development to adulthood.These results contrast with some previous work on other insects that have followed the effects of heatwaves from early life stages into adults and found reduced adult body mass and fitness (Kingsolver et al., 2021;Klockmann et al., 2017;Moore et al., 2022;Zhang, Chang, et al., 2015).Smaller adult body size in many Hymenoptera correlates with lower thermal tolerance, which could increase future populations' sensitivity to warmer climates (Barrett et al., 2023).Most holometabolous insects, including bees, rely entirely on the fat body reserves accumulated during the larval feeding stage to complete pupation and emerge (Sgolastra et al., 2016), so larvae with extended development may deplete a greater portion of these energy stores during larval development, leaving less for later life stages and inhibiting emergence.It is possible that the lack of carry-over effects to surviving adults in our study is due to our only exposing early instar larvae to heat stress.Real-world heatwaves would likely affect multiple stages simultaneously because adults lay eggs sequentially in the nest (Danforth et al., 2019), and females initiate new nests over several weeks.Stage-specific variation in response to heatwaves demonstrated in other insects (Huang et al., 2020;Kingsolver et al., 2021;Zhang, Chang, et al., 2015;Zhang, Rudolf, & Ma, 2015) merits further investigation in solitary bees.

Effects of heatwave duration
We were somewhat surprised that heatwave duration had no significant additional effect on larval mortality or development, regardless of heatwave temperature.Previous studies found that longer heatwaves increased mortality of caterpillars and juvenile aphids (York & Oberhauser, 2002;Zhao et al., 2019) and decreased emergence success of a parasitoid wasp (Moore et al., 2022).Limited past work on bees also showed additive effects of heatwave duration on the growth and survival of bumble bee colonies (Bombus terrestris) (Vanderplanck et al., 2019).This previous work, however, measured colony-level effects rather than impacts to individual bees.For bumble bee colonies, prolonged heat exposure would affect multiple cohorts of offspring, thus even heatwave effects on individual larvae that were duration-independent could act additively among individuals to reduce overall growth and survival of colonies.In our study, much of the mortality caused by high temperature heatwaves occurred within the few days of the heatwave treatment onset, suggesting that O. lignaria eggs and early larval stages are likely to die during extreme heatwaves regardless of the heatwave duration.This may be a primary reason why we saw no effect of our duration treatment on larval mortality.To understand the full implication of heatwaves for bee populations, it would be useful to know whether heatwaves acting on the more robust later larval stages show duration effects in addition to temperature effects.

Conclusion
Simulated heatwaves dramatically increased bee larval mortality and delayed development at temperatures that are well within those observed in current spring heatwaves in the study region.In a broad sense, these impacts indicate that heatwaves could become a major determinant of population persistence of solitary bees in the context of climate change.Given the physiological performance profiles for bees and other insects (Deutsch et al., 2008;Harvey et al., 2020;Kammerer et al., 2021;Kingsolver et al., 2013;Roitberg & Mangel, 2016), heatwave events place bees into the most stressful (if not lethal) conditions and are, therefore, potentially more important than average seasonal temperatures in determining abundance and distribution under changing climate.Model scenarios of heatwave We chose our experimental heatwave temperature and duration based on past and projected heatwave temperatures in Davis, CA(Cal-Adapt, 2021), O. lignaria wild and managed range, and prior larval thermotolerance research(Kemp & Bosch, 2005;Pitts-Singer et al., 2014).The highest heatwave treatment of 37 C is 6 beyond the optimum temperature range of O. lignaria larvae and within the 95th percentile of daytime high temperatures between April and June in Davis, CA, when the bee is historically active and larvae are developing within nests (Cal-Adapt, 2021; Kemp & Bosch, 2005; Scalici et al., 2023).Heatwaves with a 37 C daytime high are expected to increase in frequency and occur earlier in the year under two predicted climate change models via Cal-Adapt for Davis, CA, within the next 100 years (Cal-Adapt, 2021).The lower heatwave treatment of 31 C is within the 75th percentile of daytime high temperatures between April and June in Davis, CA, and within the optimum temperature range for O. lignaria pre-pupal development (under experimental conditions; Survival curves of Osmia lignaria larvae by heatwave temperature (25 C, blue; 31 C, yellow; 37 C, red) and duration (4 days, solid lines; 7 days, dashed lines).P-value generated from log-rank test of Kaplan-Meier survival curves.(b) Illustrations of O. lignaria larval developmental stages aligned with approximate development timing along the experimental day axis.F I G U R E 3 Total number of days from Osmia lignaria egg to cocoon completion (total larval development) by heatwave temperature (25 C, blue circle; 31 C, yellow triangle; 37 C, red square).Letters indicate significant differences ( p < 0.05).Error bars show 95% confidence intervals.F I G U R E 1 Proportion mortality of Osmia lignaria larvae in different heatwave duration and temperature treatments (25 C, blue circle; 31 C, yellow triangle; 37 C, red square).Letters indicate significant differences ( p < 0.05).Error bars show 95% confidence intervals (N = 214 larvae).
but also through lethal and sublethal effects on eggs and developing larvae within bee nests.Exposure to experimental heatwaves during early larval development caused significant mortality and delayed development in the cavity-nesting bee O. lignaria.Although the duration of simulated heatwaves did not affect bee larvae, heatwave temperature strongly influenced both mortality and total developmental time.Over 75% of individuals exposed to a high-temperature heatwave as eggs or early instars died, and the development of survivors was delayed.

Figure S6 .
Figure S6.Life stage of bees which completed cocoons but died within their cocoons before springtime emergence by heatwave temperature (25 C, blue bars; 31 C, yellow bars; 37 C, red bars) (N = 63 bees).