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. Author manuscript; available in PMC: 2025 Feb 1.
Published in final edited form as: J Anim Ecol. 2024 Jan 5;93(2):171–182. doi: 10.1111/1365-2656.14041

A simulated natural heatwave perturbs bumble bee immunity and resistance to infection

Kerrigan B Tobin 1, Rachel Mandes 1, Abraham Martinez 1, Ben M Sadd 1,*
PMCID: PMC10922385  NIHMSID: NIHMS1953906  PMID: 38180280

Abstract

  1. As a consequence of ongoing climate change, heatwaves are predicted to increase in frequency, intensity, and duration in many regions. Such extreme events can shift organisms from thermal optima for physiology and behavior, with the thermal stress hypothesis predicting reduced performance at temperatures where the maintenance of biological functions is energetically costly. Performance includes the ability to resist biotic stressors, including infectious diseases, with increased exposure to extreme temperatures having the potential to synergize with parasite infection.

  2. Climate change is a proposed threat to native bee pollinators, directly and through indirect effects on floral resources, but the thermal stress hypothesis, particularly pertaining to infectious disease resistance, has received limited attention. We exposed adult Bombus impatiens bumble bee workers to simulated, ecologically relevant heatwave or control thermal regimes and assessed longevity, immunity, and resistance to concurrent or future parasite infections.

  3. We demonstrate that survival and induced antibacterial immunity are reduced following heatwaves. Supporting that heatwave exposure compromised immunity, the cost of immune activation was thermal regime dependent, with survival costs in control but not heatwave exposed bees. However, in the face of real infections, an inability to mount an optimal immune response will be detrimental, which was reflected by increased trypanosomatid parasite infections following heatwave exposure.

  4. These results demonstrate interactions between heatwave exposure and bumble bee performance, including immune and infection outcomes. Thus, the health of bumble bee pollinator populations may be affected through altered interactions with parasites and pathogens, in addition to other effects of extreme manifestations of climate change.

Keywords: bumble bee, heatwave, immunity, pollinator health, thermal stress hypothesis

Graphical Abstract

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Extreme climate events are predicted to continue increasing, affecting organisms directly and altering biotic interactions. Under a naturally-relevant heatwave regime, immunity and infection resistance in bumble bees is impaired. Thus, bumble bee health may be directly affected by climate change, but also indirectly through modified interactions with infections.

Introduction

Extreme temperature events from climate change are considered a major ongoing and future threat to species and ecosystems (Spooner et al., 2018). Warm days are expected to increase in frequency and intensity (Frich et al., 2002), and heatwaves, prolonged periods of temperatures above the long-term average, continue to become more frequent, more intense, and longer lasting (Perkins-Kirkpatrick & Lewis, 2020). As such trends are predicted to continue (Lau & Nath, 2012; Perkins-Kirkpatrick & Gibson, 2017), further understanding of the impact of heatwaves on organisms becomes critical. Heatwaves have been shown to have a multitude of effects, including on development (Carter et al., 2018), survival (Anderson & Bell, 2011; García-Robledo et al., 2016), reproduction (Sales et al., 2018), and on population (Zhang et al., 2015) and community dynamics (Sentis et al., 2013). However, we know relatively little about mechanisms underlying responses to extreme climatic events, including heatwaves, and their interactions with other abiotic and biotic stressors (van de Pol et al., 2017).

Threats from climate change (Thomas et al., 2004) and infectious disease (Daszak, 2000) are among the greatest challenges to biodiversity. Beyond their individual effects, changes to the thermal environment may alter interactions between host organisms and their pathogens and parasites, with knock-on consequences for individual, population, and community health (Lafferty, 2009). Increased temperatures have been shown to decrease parasite pathology in some systems (Paull & Johnson, 2014), yet they may lead to increased disease transmission in others, with changes in long-term host-parasite dynamics (Altizer et al., 2013; Elderd & Reilly, 2014). Disease dynamics may also show context dependent shifts under warming (Stewart et al., 2018) and changes in disease dynamics under different environmental temperature regimes can result from changes to host immunity (Greenspan et al., 2017). Though higher temperatures can influence immunity both positively and negatively (Thomas & Blanford, 2003), effects may depend on the immune component investigated (Palacios et al., 2020). High temperatures per se can even be used against infection; for example, locusts that are provided with the opportunity to thermoregulate to fever levels during fungal pathogen infection having increased fitness (Elliot et al., 2002). With such diverse outcomes, there is a need for investigations directly addressing the consequences of stressful thermal events, such as heatwaves, on immunity and infection in species with critical ecosystem functions.

Insects numerically and functionally dominate many ecosystems, yet studies of how they are affected by exposure to multiple stressors, including climatic extremes and infectious diseases, are limited (Kaunisto et al., 2016). Insects, along with other largely ectothermic organisms, may be particularly sensitive to a changing thermal environment (Paaijmans et al., 2013). Bumble bees, key social insect pollinators in natural and managed ecosystems (Cameron & Sadd, 2020; Morandin & Winston, 2006), may be particularly vulnerable to warming temperatures. They have been documented to be negatively affected by climate change (Kerr et al., 2015; Soroye et al., 2020) and heatwaves (Martinet, Zambra, et al., 2021; Rasmont & Iserbyt, 2012). Moreover, investigations of variability in gene expression responses of bumble bees under heat stress indicates limited potential for adaptation to increasing temperatures (Pimsler et al., 2020). Bumble bees are considered heterothermic, and their thermal physiology has received considerable research attention (Oyen et al., 2016). They can regulate temperature to some extent at both individual (Heinrich, 1975, 1976) and colony (Heinrich, 1974; Weidenmüller et al., 2002) levels. Despite these thermoregulatory abilities, bumble bees will still experience high and low temperatures under normal conditions (Heinrich, 1976), including thermal extremes under heatwaves (Rasmont & Iserbyt, 2012).

Increasing frequencies of extreme temperature events, such as heatwaves, will mean that organisms will be subjected to a variety of sublethal consequences for their health and fitness, including the potential for modulation of immunity and host-parasite interactions (Little & Lazzaro, 2009). Even though critical thermal limits of organisms are important to delimit predicted distributions and responses to climate change, the relative significance of sublethal effects will have immediate consequences in the face of ongoing climate change. Individual bumble bees have a critical maximum temperature within the range of 44.6-55.0°C (Hamblin et al., 2017; Oyen & Dillon, 2018) and heat stupor can set in within hours of exposure to 40°C (Martinet et al., 2015). However, in temperate regions bumble bees will rarely encounter such temperatures, but heatwaves that impose temperatures outside those regularly encountered are likely to induce thermal stress (Paull et al., 2015). This thermal stress could directly compromise individual bumble bee health and colony fitness through effects on behavior and physiology, but also indirectly by affecting interactions with pathogens and parasites. Immunity may be compromised due to resource-based trade-offs with investment into an energetically extensive thermal stress response (Sinclair et al., 2013), but a direct association between the heat shock response, involved in cellular resistance to heat stress (Feder & Hofmann, 1999; Sørensen et al., 2003), and immunity could also play a role. In honey bees, increased heat shock protein expression has been associated with a reduced ability to upregulate antimicrobial peptides (AMPs) (McKinstry et al., 2017). Conversely, AMP activation curtails the heat shock response (McKinstry et al., 2017). This illustrates a mutually antagonistic relationship between the immunity and the response to the thermal environment that could mediate interactions between thermal stress and infection outcomes.

We tested the thermal stress hypothesis in relation to adult bumble bee immunity and infection in the face of an ecologically relevant stimulated heatwave. The control thermal regime simulated long-term average July temperatures in Central Illinois, while the naturally relevant heatwave was modelled on a composite of three real heatwaves in the same area from 2011, 2012, and 2013 (Figure 1A) and was imposed for 5 days in accordance with the International Panel for Climate Change heatwave definition (IPCC, 2007). Below surface soil temperatures were used to represent the temperatures that adult bees could encounter within their subterranean nests. If bees leave the nest to forage, these temperatures will be conservative, but at such temperatures bumble bee foraging is greatly reduced (Kwon & Saeed, 2003). Due to feasibility of laboratory replication, a non-declining species, Bombus impatiens (Cameron et al., 2011), was used. We predicted that heatwave exposure would itself reduce bee survival. A series of experiments (Figure 1B) were also carried out combining thermal regime treatments (heatwave or control) with non-pathogenic immune induction or infections with a bumble bee gut parasite Crithidia bombi. Importantly, the non-pathogenic immune induction approach allows us to disentangle changes in host immune investment or activation from pathogenic effects of infection. We predicted that, relative to the control thermal regime, (i) heatwave exposure would impair immunity, (ii) costs of immune activation (Sadd & Schmid-Hempel, 2009) would be reduced or absent in heatwave exposed bees due to decreased investment into immunity, and (iii) resistance to parasite infection would be reduced in heatwave exposed bees.

Figure 1: Methodological overview of the effects of exposure to a simulated heatwave (A) on individual bumble bee health parameters, including immune and infection outcomes (B).

Figure 1:

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Five-day experimental thermal regimes (control and heatwave) were imposed after all bees had been acclimated to control conditions for 48 hours (A). All bees were subsequently returned to control conditions. A series of experiments (B) measured survival (i), constitutive and induced immunity (ii), the cost of an immune response for survival under heatwave and control conditions (iii), and infection outcomes following experimental trypanosomatid exposure prior to heatwave initiation (iv) or following the heatwave treatment conclusion (v). Numbers in parentheses represent the number of source colonies providing workers for each response.

Materials and methods

Overview.

Each of the five experiments (Figure 1B) utilized a different set of bumble bees, however the bees, parasites, and microbes used were maintained according to the same methods across experiments.

Bumble bee maintenance and parasite culture.

Bombus impatiens workers in the longevity and immunity studies were from lab-reared colonies from field-caught queens collected with the permission of the ParkLands Foundation (www.parklandsfoundation.org) from the Mackinaw River Study Area (Lexington, IL., U.S.A.). The cost of immunity and infection experiments used workers from commercial colonies (Koppert Biological Systems, Howell, Michigan, USA). All colonies were confirmed free of common infections by observing fecal samples from a subset of workers and the queen under phase contrast microscopy (400x total magnification). Colonies were maintained under standard laboratory conditions at 26±1.5°C under red light illumination and provided with honeybee-collected pollen (Brushy Mountain Bee Farm, North Carolina, USA) three times a week and sugar water (1g cane sugar:1mL boiled tap water with 0.1% cream of tartar) ad libitum. Newly-emerged adult worker bees were isolated and held individually with constant access to sugar water, with experiment specific treatments and thermal regimes implemented as described below.

Two C. bombi strains previously isolated from wild bumble bee populations were used in live infection experiments. Strain AK08.052 (lab specific ID) was isolated from Alaska in 2008 and strain IL16.075 was isolated from Central Illinois in 2016. Following previous methods (Salathé et al., 2012), each strain was derived from a single parasite cell, confirmed as C. bombi, and are maintained in a frozen strain bank at −80°C. Strain stocks were thawed as needed to inoculate fresh FP-FB media (Salathé et al., 2012) and grown following the standard procedure at 27°C to have viable C. bombi cells available for experimental exposures. Each C. bombi culture was diluted with sugar water to 1,000 cells per μL before being mixed in equal proportions to form the cocktail C. bombi inoculum.

Thermal regime treatments.

Experimental bees were exposed to either a control or heatwave thermal regime based on 10.16cm deep soil temperature trace data from Champaign, Illinois, USA (Illinois State Water Survey, www.isws.illinois.edu). The control regime simulated long-term average July temperatures (1996-2016) in Central Illinois, with diurnal cycles of 10 hours at 29°C and 14 hours at 24.5°C. All freshly emerged adult bees were initially kept under this regime for 2 days, before heatwave treatments were imposed. The control regime bees were returned to the control conditions as described above. The heatwave regime was modelled on a composite of three real heatwaves from 2011, 2012, and 2013, with 10 hours at 32.5°C and 14 hours at 28°C for 5 days. The heatwaves to be simulated were identified using a custom script in R (R Core Team, 2020) to identify 5 day windows when the air temperature continuously exceeded the long-term maximum average by at least 5°C, as per an accepted heatwave definition (Intergovernmental Panel on Climate Change (IPCC) (IPCC, 2007)). Maximum and minimum 10.16cm deep soil temperatures from the top three independent heatwave periods were averaged to provide the regime parameters. Following the administration of heatwave treatments (control or heatwave), all bees were returned to control conditions. Temperature treatments were performed in two incubators, with regimes switched at least once between incubators during each experiment, and incubator identity included in analyses to control for individual incubator effects.

Heatwave exposure and longevity.

Bees assigned to the survival group were isolated with sugar water ad libitum and survival was tracked daily during and after heatwave exposure.

Heatwave exposure and immunity.

At the end of the heatwave treatment, bees were either left naïve to measure constitutive immune parameters or given a non-pathogenic immune challenge of an injection of heat-killed Arthrobacter globiformis bacteria into the hemocoel to assess induced immunity, using established protocols (see (Czerwinski & Sadd, 2017)). After a further 24 hours, hemolymph was collected. 5μL of extracted hemolymph was added to 20μL of sodium cacodylate buffer (0.01 M Na-Cac, 0.005 M CaCl2). Hemolymph samples were split for assaying phenoloxidase activity and protein, diluted 1:20, and antimicrobial activity, diluted 1:5 in a tube prelined with 1-phenyl-2-thiourea (PTU, Sigma-Aldrich, St. Louis, MO, USA) to inhibit melanization. Samples were snap frozen and stored at −80°C until assayed.

Humoral antimicrobial activity, mediated mostly by AMPs, is responsible for the targeted elimination of microbes (Bulet et al., 1999; Gillespie and et al., 1997) and its lasting induced response functions against persistent pathogens (Haine et al., 2008). As expression of antibacterial activity is largely induced in bumble bees (Barribeau et al., 2016; Korner & Schmid-Hempel, 2004), the prevalence of measurable antibacterial activity was assessed in naïve non-immune challenged bees, but the quantifiable response compared in immune challenged. The antimicrobial activity of the bee hemolymph against A. globiformis was measured through a zone of inhibition assay (Czerwinski & Sadd, 2017).

Phenoloxidase is a key enzyme in the production of cytotoxins and the melanization response in insect immunity (González-Santoyo & Córdoba-Aguilar, 2012). The activity of phenoloxidase in the bee hemolymph was assayed using a spectrophotometric well-plate assay, detecting a change in absorbance over time through the action of hemolymph phenoloxidase (Czerwinski & Sadd, 2017). Total hemolymph protein was also assayed as the available protein pool can determine resources available for immune investment (Lee et al., 2008). Total hemolymph protein was measured using a Pierce™ BCA Protein Assay Kit. 200μL of working reagent was added to 25μL samples in duplicate and incubated at 37°C for 30 min in darkness before absorbance measures at 562 nm. Following blank subtraction, protein per sample was calculated based on bovine serum albumin standards.

Cost of immunity: survival after immune activation and heatwave exposure.

After 48 hours under the control thermal regime, bees were assigned one of three immune challenge treatments (naïve, sham injected, or heat-killed bacteria injected). As above, the injection of heat-killed Arthrobacter globiformis bacteria into the hemocoel acted as a non-pathogenic immune challenge to focus on costs associated with host immune investment without the possibility of the confounding effect of a live pathogenic infection. Following their immune challenge treatment, bees were subsequent placed into the heatwave or control regime, and after 5 days all individuals were held under control conditions. Survival was checked daily following the initiation of the heatwave treatment.

Heatwave exposure and the outcome of an ongoing parasite infection.

After 48 hours under the control thermal regime, bees were starved for 2 hours, and orally administered 10,000 cells of the C. bombi cocktail using standard methods (Tobin et al., 2019). Bees were returned to individual containers and placed into their respective heatwave treatments. At 5 days post-parasite exposure, prevalence of transmitting cells in the feces was determined by microscopically screening feces. At day 8 following exposure, bees were frozen and infection loads quantified by qPCR (Tobin et al., 2019). We verified DNA sample quality using a μDrop plate in a MultiSkan GO plate reader (ThermoFisher, Waltham, MA, USA) and quantified infection using a QuantStudio3 Real-Time PCR Machine (ThermoFisher). Genome copies per sample were calculated based on a standard curve produced using gBlock gene fragments (Integrated DNA Technologies, Coralville, IA, USA) of the C. bombi PCR product. We further normalized to the relative copies of the B. impatiens 5 C-actin gene (based on another gBlock standard curve) to account for differential DNA extraction efficiencies between samples. We ran each DNA sample in duplicate, and reran any duplicates with calculated coefficients of variation above 0.20 to achieve an acceptable coefficient of variation, and then averaged across replicates.

Heatwave exposure and resistance to a subsequent parasite infection.

Post heatwave treatment, exposure to the C. bombi cocktail and measurement of prevalence of transmission at 5 days and infection load at 8 days post-exposure were performed as above.

Statistical analyses.

Analyses were performed in R 4.2.0 “Vigorous Calisthenics” for Mac OS X (R Core Team, 2020). Mixed Effect Cox Proportional Hazard models were fit with the coxme package (Therneau, 2020) and Linear and Generalized Linear Mixed Effect models with the lme4 (Bates et al., 2015) and glmmTMB (Brooks et al., 2017) packages. For each response variable, potential distributions were assessed for model fit and adherence to assumptions. Interaction terms included in initial models (see details below for each experiment) were removed when not significant to aid in the interpretation of the component main effects. Colony was included as a random effect in all models and body size was included as a covariate to account for size-dependent variation in the measured traits (e.g., Calhoun, et al., 2021; Tobin, et al., 2019). Estimated marginal means and their confidence intervals for levels of model terms were from extracted with the package emmeans (Lenth, 2020). Longevity and the cost of immunity data were analyzed with a Mixed Effect Cox Proportional Hazards model. For longevity, the maximal model included additive fixed effects of incubator identity (thermal regimes were given in two incubators, with regimes being switched between incubators), body size (as determined by wing radial cell measurements ( Müller et al., 1996)), and thermal regime (control or heatwave). For cost of immunity, a model was fitted with the same terms, plus immune challenge treatment (naïve, sham injected, or immune challenged) and the interaction of immune challenge treatment and thermal regime.

Hemolymph protein and phenoloxidase activity were analyzed with Linear Mixed Effect models. Incubator identity, body size, thermal regime, and immune challenge state (naïve or challenged) were included as main effects, and an interaction between thermal regime and immune challenge state. Degrees of freedom were corrected using the Kenward Roger approximation, which has been shown to produce acceptable Type I error rates even for smaller samples (Luke, 2017). A Fisher’s exact test was used to compare a binomial response (zone/no zone) of antibacterial activity between thermal regimes for the naïve group, because the naïve group had few measurable zones of inhibition. Induced antibacterial activity (converted to tetracycline units) in the immune challenged group was analyzed with a linear mixed effects model including incubator identity, body size, and thermal regime as main effects.

Normalized parasite infections for groups exposed before and after the heatwave were analyzed with Generalized Linear Mixed Models using a negative binomial distribution with a linear parameterization (Hardin et al., 2007) and a log link function to account for overdispersion. Models were fitted with incubator identity, body size, and thermal regime as main effects. To overcome false convergence warnings from the default optimizer it was set as method="BFGS". In the parasite exposed before heatwave data, two extreme outliers influencing model fit were removed, but this did not affect the resulting conclusions. The values for both outliers were approximately four times higher than any other sample, with one outlier present in each thermal regime treatment.

Results

Heatwave exposure and longevity.

Exposure to the heatwave regime itself reduced survival of bees relative to control regime individuals (X2=6.349, d.f.=1, p=0.011). The difference was largely due to greater mortality after all bees were returned to the control regime (Figure 2).

Figure 2: The model adjusted proportion of worker bumble bees surviving following heatwave treatment initiation.

Figure 2:

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The grey shaded vertical bar represents the period of heatwave exposure, after which all individuals experienced the control thermal regime.

Heatwave exposure and immunity.

Humoral antimicrobial activity and its induction represents the capacity to respond to and control microbial pathogens. In naïve bees, there was no difference between control (1.9%) and heatwave (5.7%) treatments in detectable antibacterial activity (Fisher’s Exact Test: p=0.351). However, heatwave exposed bees exhibited significantly reduced levels of induced antibacterial activity 24 hours following an immune challenge (F=6.748, d.f.=1, 43.13, p = 0.012; Figure 3).

Figure 3: The influence of prior heatwave exposure on induced antibacterial activity in the hemolymph of immune challenged bumble bee workers.

Figure 3:

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Numbers below confidence intervals represent sample sizes. Individual data points are shown in gray.

Protein levels within the hemolymph can be indicative of resources available for investment into immunity, but despite the reduced antibacterial immune activity, neither immune challenge status (F=0.002, d.f.=1, 93.26, p=0.965), thermal regime (F=0.166, d.f.=1, 94.08, p=0.685), nor the interaction between them (F=0.252, d.f.=1, 92.52, p=0.617) significantly affected total hemolymph protein. Estimated marginal means (±s.e.) of total hemolymph protein were 681 (±57) μg/ml for the control thermal regime and 715 (±58) μg/ml for the heatwave regime. However, body size positively influenced the protein concentration (F=5.388, d.f.=1, 89.50, p=0.023).

Activity of the immune-associated enzyme phenoloxidase in the hemolymph was not affected by immune challenge status (F=0.063, d.f.=1, 96.80, p=0.803), thermal regime (F=0.095, d.f.=1, 98.00, p=0.759), nor the interaction between them (F=0.552, d.f.=1, 95.77, p=0.459). Estimated marginal means (±s.e.) of phenoloxidase activity were 0.56 (±0.034) Δ absorbance/hour for the control thermal regime and 0.52 (±0.034) Δ absorbance/hour for the heatwave regime. However, as for total protein, there was a significant positive effect of body size on phenoloxidase activity (F=6.000, d.f.=1, 75.16, p=0.017).

Cost of immunity: survival after immune activation and heatwave exposure.

The cost of mounting an immune response due to energetic demands or self-reactivity is often manifested in reduced survival in insects (Armitage et al., 2003; Jacot et al., 2004; Moret & Schmid-Hempel, 2000). There was a significant interaction between thermal regime and immune challenge treatment on survival (X2=9.734, d.f.=2, p=0.008; Figure 4). Heatwave exposed bees did not exhibit a cost of immune induction, with similar survival across all immune challenge treatments (Tukey Pairwise Tests, p > 0.999). However, while naïve bees in the control thermal regime had the best survival, those receiving an immune challenge show elevated mortality (Tukey Pairwise Tests, p < 0.001), which is consistent with a cost of induced immunity. Larger individuals also survived longer (X2=14.583, d.f.=1, p < 0.001).

Figure 4: Estimated survival hazards for combinations of heatwave and non-pathogenic immune challenge treatments.

Figure 4:

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Numbers below confidence intervals represent sample sizes.

Heatwave exposure and the outcome of an ongoing and a subsequent parasite infection.

Infection prevalence based on fecal screens at 5 days post-exposure or the sampling of guts at 8 days post-exposure did not differ between the thermal regimes. For 5-day post parasite exposure screens, all individuals had trypanosomatid cells in their feces in the pre-heatwave exposed group and only 3% of individuals (1 heatwave treated bee and 1 control bee) were not positive for infections at the 8-day sampling. In the post-heatwave exposed group, 24% of individuals (5 heatwave treated bees and 13 control bees, Fisher’s Exact Test: p=0.249) were not shedding parasite cells 5 days post parasite exposure, and 12% of individuals (1 heatwave treated bee and 7 control bees, Fisher’s Exact Test: p=0.133) were negative at the 8-day sampling. In the post-heatwave exposed group, four bees did not survive until infection intensity in the gut was quantified. Infection intensities in the bees exposed to the parasite prior to the thermal regime treatment were quantified after 8 days (5 days of thermal regime treatment and 3 days of control conditions), finding no significant difference in infection intensities between control and heatwave individuals (F=0.391, d.f.=1, p=0.532; Figure 5A). In contrast, there was a significant effect of thermal regime on 8-day post-exposure infection of bees exposed to the parasite at the end of the thermal regime treatment (F=21.10, d.f.=1, p < 0.001). Bees experiencing the heatwave prior to parasite exposure had a higher infection load relative to control thermal regime bees (Figure 5B).

Figure 5: Infection outcomes for bees exposed to the trypanosomatid parasite Crithidia bombi (A) prior or (B) subsequent to control or heatwave thermal regime treatments.

Figure 5:

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Points represent estimated marginal means of standardized infection intensities and numbers below 95% confidence intervals represent sample sizes.

Discussion

This study adds to our understanding of how environmental variability can lead to variation in immunity and infection outcomes in general (Little & Lazzaro, 2009; Thomas & Blanford, 2003), providing evidence for the thermal stress hypothesis (Paull et al., 2015). More specifically, the results demonstrate potential mechanisms by which climate change, and in particular exposure to heatwaves, may be detrimental to bumble bee health and population viability, even when well below critical thermal limits (Martinet, Dellicour, et al., 2021; Martinet et al., 2015; Oyen et al., 2016). Bees exposed to a naturally relevant heatwave showed decreased survival, compromised antimicrobial immunity, and reduced resistance to a trypanosomatid parasite exposure.

Decreased survival following heatwave exposure supports the thermal stress hypothesis and agrees with other research that has found survival costs due to increased temperatures and heatwave conditions in other systems (Mech et al., 2018; Rukke et al., 2018). It has been suggested that tropical species are at highest risk under warming conditions (Deutsch et al., 2008), but our results suggest that even currently stable, temperate species such as the bumble bee B. impatiens may be susceptible to the sub-lethal consequences of climate change through heatwaves. Many factors have been associated with concerning declines of many bumble bee species (Cameron & Sadd, 2020), including climate change (Kerr et al., 2015; Soroye et al., 2020). It has been proposed that consequences of climate change for bumble bee species could result from direct effects on bee physiology (Rasmont & Iserbyt, 2012) or indirect effects stemming from mismatched floral resources with bumble bee foraging need (Pyke et al., 2016). Recently, it has also been shown that heat shock disrupts male bumble bee reproductive traits (Martinet, Zambra, et al., 2021). Our results show that consequences may not only come about through direct negative effects on survival, but also through altered interactions with biotic stressors like pathogens and parasites.

The result of reduced humoral antimicrobial activity following heatwave exposure is further evidence for a proposed connection between thermal stress and immunity (Sinclair et al., 2013). However, the lack of an effect on the phenoloxidase activity, considered a constitutive immune parameter (Schmid-Hempel & Ebert, 2003), suggests that such effects are immune component dependent (Palacios et al., 2020). Yet, an overall reduced investment into immunity upon heatwave exposure was supported by a cost of immune activation (Sadd & Schmid-Hempel, 2009) being absent in heatwave exposed individuals, but present in control regime bees. A potential mechanistic explanation for these findings may be that the heat stress and antibacterial responses are antagonistic. In fact, the relationship between heat shock and antibacterial pathways has been shown to be mutually antagonistic in honey bees (McKinstry et al., 2017). Despite the exposure temperatures being much higher (35°C control versus 45°C heat shock) and it being in honey bees, a similar relationship could be responsible for the effects in bumble bees experiencing a heatwave in our study. Under this model, immune challenged bees under control thermal conditions upregulate antimicrobial immunity, reaping the benefits for defense under a real infection, but pay a classical cost of immune activation (Armitage et al., 2003; Moret & Schmid-Hempel, 2000) on a benign immune challenge. On the other hand, immune challenged bees entering the heatwave would see a suppression of antibacterial immune expression, consequently showing reduced antibacterial activity, but also not paying the costs for it. Yet, with a live pathogenic infection, this apparent benefit would likely be overturned, as the heatwave exposed bees, with reduced immune function and resistance, would suffer greater costs from infection. However, our study does not directly quantify implications for host fitness of changes in live parasite infection loads, and this would need to be confirmed.

Heatwave compromised antimicrobial immunity, as demonstrated here, will likely have consequences for infections with live bacterial and other pathogens. Induced antibacterial activity aids in clearing bacterial infections that would otherwise persist in the insect host (Haine et al., 2008), meaning that heatwave-exposed bees could have decreased competency to fight and clear persistent bacterial infections. As would be predicted by the reduction in some aspects of immunity following heatwave exposure, resistance to a future infection by the trypanosomatid parasite C. bombi was also reduced following the heatwave treatment. It is known that some pathways involved in antimicrobial responses, as measured in the immune assay of antimicrobial activity, are also involved in the immune response against Crithidia (Barribeau et al., 2014). Interactions between heatwave exposure and infectious disease could be one manifestation of the detrimental effects of climate change on bumble bees, which has been indicated by changes in populations of bumble bee species (Soroye et al., 2020). However, it must be noted that the focus of our study is a non-declining species (Cameron et al., 2011). Interspecific variability in critical thermal limits has been found in bumble bees (Martinet, Dellicour, et al., 2021), and future studies of the interactions between thermal regime exposure and infection across species may elucidate specific susceptibilities to direct and indirect effects of climate change and other stressors (Cameron & Sadd, 2020). Furthermore, work in other bee genera has shown variable changes in infection outcomes upon exposure to higher temperatures (e.g., Martín-Hernández, et al., 2009; Palmer-Young, et al., 2023; Xu & James, 2012). In addition to the differences in thermal physiology of bumble bees and other bees, varied outcomes such as these could stem from differences in how temperature affects the colonization by the parasite on one hand, and the ability of the host to defend itself on the other. This could depend on the parasite or host type in the context of the experienced environment (Thomas & Blanford, 2003), which further supports the need for additional work in diverse bumble bee species and their different parasites and pathogens.

Overall, we demonstrate important consequences of heatwaves for immunity and infection outcomes, with likely detrimental consequences for the bumble bee host. Yet, we also show that the temporal scale and timing of exposure to the heatwave and infection stressors is key to the outcome, as has been suggested for other multiple stressors (Jackson et al., 2021). Temperature and its variation, albeit at lower absolute values (21-29°C), has earlier been shown not to affect infection in the bumble bee-Crithidia system (Tobin et al., 2019). Furthermore, in the current study, in contrast to the decreased resistance in bees exposed to C. bombi post-heatwave treatment, bees exposed to the parasite prior to heatwave initiation showed no differences in infection when they subsequently experienced control or heatwave thermal regimes. It could be that the infection outcome is largely determined before heatwave effects take hold, or alternatively, because both host and parasite go through the heatwave regime, and thus any negative effects on the parasite could mask increased host susceptibility. Although transmitting cells of C. bombi outside of bees have been reported to be robust to extreme temperature exposure (40°C) for 60minutes (Wolmuth-Gordon & Brown, 2023) negative effects of higher temperatures (37°C) on existing C. bombi infections have been reported (Palmer-Young et al., 2019) and temperature may affect symbiont-mediated protection (Palmer-Young et al., 2018). The increased susceptibility to future infections from heatwave exposure, and the temporal-dependence of this effect add to the list of factors that influence infection outcomes in this bumble bee-trypanosomatid system (Sadd & Barribeau, 2013).

Overall, our work demonstrates that, through heatwaves, current climatic conditions may pose risks to organisms either directly, decreasing survival, or indirectly by decreasing immunity, thus leaving them susceptible to increased infection. This highlights the importance of experimental studies of direct effects of climate change (van de Pol et al., 2017), and how the thermal environment may interact with other factors as just one of multiple environmental stressors influencing pollinator health (Szabo et al., 2012; Woodard, 2017).

Acknowledgements

This work was supported by the National Institutes of Health [grant R15 GM129681-01 to B.M.S.], the United States Department of Agriculture [grant 2017-67013-26536 to B.M.S.], the Great Lakes Restoration Initiative through the U.S. Fish and Wildlife Service's Threatened and Endangered Species Template [grant F22AP02271-00 to B.M.S.], and a Phi Sigma Biological Society Weigel grant to K.B.T. Instrumentation used in this project was funded by a National Science Foundation MRI grant [1725199] with B.M.S. as a co-PI.

Footnotes

Conflict of Interest

The authors have no conflict of interest to declare.

Data Availability

Data available from the Dryad Digital Repository https://doi.org/10.5061/dryad.8sf7m0crn (Sadd et al., 2023).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data available from the Dryad Digital Repository https://doi.org/10.5061/dryad.8sf7m0crn (Sadd et al., 2023).

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