Effects of short-term heat stress on the thermal tolerance of western corn rootworm (Coleoptera: Chrysomelidae)

Abstract

Insect responses to warming temperatures are determined partly by their physiology, which is influenced by genetic factors and plasticity induced by past temperature exposure. The effect that prior high temperature exposure has on insect thermal tolerance is complex and depends on the degree of heat stress experienced; high heat exposure may allow for individuals to tolerate higher temperatures through hardening or may reduce an individual’s capacity to withstand higher temperatures through accumulated heat stress. In this study, we assessed how short exposures to high temperatures and a laboratory colony’s geographical origin affected the critical thermal maximum (CTmax) of western corn rootworm (Diabrotica virgifera virgifera LeConte), an economically important pest. Despite a wide latitudinal range of source populations, western corn rootworm colonies did not differ in their CTmax. Regardless of colony origin, we found that exposing western corn rootworm to higher temperatures resulted in lower CTmax, which suggests that heat stress accumulated. This study highlights how western corn rootworm experiences heat stress at temperatures near the temperatures they experience in the field, which may have important and currently unknown implications for its behavior.

Introduction

Warming temperatures and an increasingly variable climate are changing the dynamics of insects worldwide (Wagner 2020, Harvey et al. 2023). Rising temperatures may be stressful for insects and have been implicated in population declines of some taxa, but warmer temperatures have also been associated with eruptions of some economically important pest species (Harvey et al. 2023). Consequently, an insect’s physiological response to elevated temperatures, which can vary within a species as the result of recent temperature exposure and evolutionary adaptation to local climate, is increasingly important in shaping the effects of climate change (Huey et al. 2012, Sunday et al. 2012).

The effect of temperature on insect fitness is characterized by a left-skewed curve, where fitness increases with temperature up to an optimum before rapidly descending as an organism approaches its critical thermal maximum (CTmax; Kingsolver and Huey 2008, Sinclair et al. 2016). CTmax should therefore be a useful metric for comparing the thermal tolerance of different insect species and assessing the potential threat that global warming has on them. Yet, CTmax varies among individuals of the same species (as in ants and mosquitos; Bujan, et al. 2020b, Oliveira et al. 2021), muddling the predictive power of certain models. One cause of this variation is underlying genetic variation because different populations across a species’ range can show modest genetic variation in thermal tolerance (Sørensen et al. 2001, Orlinick et al. 2024). Additionally, thermal tolerance may be influenced by prior exposure to different temperatures, which can have negative or positive impacts on CTmax via thermal stress or hardening, the upregulation of heat shock proteins (Terblanche et al. 2007, Schulte et al. 2011, Sgrò et al. 2016).

Theory predicts that heat stress begins to accumulate above a critical threshold (Tc), such that higher temperatures or longer exposures may result in lower CTmax (Ørsted et al. 2022). This has been used to explain differences in CTmax as a function of the rate at which temperature was increased during CTmax assays (Terblanche et al. 2007, Kingsolver and Umbanhowar 2018). Yet despite the crucially important role of Tc in determining the thermal ecology of insects, empirical estimates remain rare (Kingsolver and Umbanhowar 2018, Ørsted et al. 2022). Alternatively, short exposures to high temperatures may also prime individuals to experience future high temperatures through a process collectively known as hardening (Willot et al. 2017). Although evidence suggests the effects of acclimation tend to result in relatively small increases in thermal tolerance, hardening may play an important role in mitigating the deleterious effects of warming on insects (Sgrò et al. 2016, Willot et al. 2017). Thus, short exposure to high temperatures may increase CTmax. Despite the importance of physiology in determining insect responses to climate change, studies on the thermal tolerance of insects, especially those characterizing factors associated with intraspecific variation, are still limited for many important pest species.

Western corn rootworm [WCR, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae)] is a univoltine holometabolous insect pest that accounts for billions of dollars in damage annually to corn (Zea mays L.) (Gray et al. 2009). After overwintering as eggs, the larvae hatch and feed on the roots of corn and other grasses (Branson and Ortman 1970), which can cause significant damage to the host plant by reducing photosynthetic rates and slowing water uptake (Kahler et al. 1985, Riedell 1990). After 3 larval instars and a pupal stage, the rootworm emerge as adults which feed on above ground vascular and reproductive tissues of corn and other grasses (Ludwig and Hill 1975, Moeser and Vidal 2005), mate, and lay eggs in the soil where eggs will overwinter. As with many insects, their development rate is temperature dependent (Davis et al. 1996, Hibbard et al. 2010, Davis et al. 2014). Temperature also appears to play a major role in shaping their range (Hemerik et al. 2004), which has expanded from the central United States to the east coast and to over 20 countries in Europe (Kim and Sappington 2005, Aragón and Lobo 2012). Given the large economic impact of WCR, its expanding range, and high genetic diversity (Kim and Sappington, 2005), it is essential to understand how climate change may contribute to changes in WCR dynamics. Most previous work on the thermal ecology of WCR sought to understand how temperatures within their permissible range affect development (Davis et al. 1996, Hemerik et al. 2004, Wilstermann and Vidal 2013), yet we are only beginning to understand its physiological capacity to tolerate high temperatures.

Here, we quantified the heat tolerance of adult WCR using individuals from laboratory-maintained colonies reared from source populations that collectively represent much of the species’ range in the United States. We initially measured how CTmax varied across 6 colonies from 5 states, spanning a range of over 2,500 km and 7 ° in latitude. We predicted that WCR thermal tolerance would decrease with increasing latitude of the source population. We then tested whether exposure to high temperatures during a short preassay period increased (via hardening) or reduced (via stress) CTmax. We predicted that slight warming would increase CTmax through hardening, while higher temperatures would reduce CTmax through the accumulation of heat stress. By measuring CTmax across a range of temperatures, we expected to see a distinct shift in this relationship as the exposure temperature crossed Tc. Lastly, we predicted that the relationship between temperature exposure and latitude would depend on the geographic source of WCR colonies, with lower latitude individuals better adapted to high temperature, thus having a stronger hardening response and a higher Tc.

Materials and Methods

Geographic Populations and Rearing

The WCR colonies used in this experiment have been maintained in the laboratory at the USDA-ARS North Central Agricultural Research Laboratory in Brookings, SD for up to 30 yr (Table 1). Methods for rearing WCR in the lab have been in development for over 50 years (Georce and Ortman 1965, Branson and Johnson 1973) and studies show that WCR are well suited to lab rearing. They exhibit high survival and fecundity rates and maintain a similar degree of genetic diversity to wild populations for at least 20 generations under standard rearing protocols (Kim et al. 2007, Li et al. 2014). Yet even after several generations in the lab, WCR colonies still may reflect their source populations and lab rearing for at least 1 generation is recommended for comparing populations to minimize the impact of the natal parental environment on the phenotypes of their offspring (Li et al. 2014).

WCR colonies were collected from geographically distinct populations from across their US range and were maintained in the lab under constant conditions. Field collections of the individuals used to start the colonies occurred from 1995 to 2013 in Kansas, Nebraska, Pennsylvania, South Dakota, and Wisconsin. Since collection, the colonies have been maintained under optimal conditions for WCR development. From these colonies, we reared WCR using methods following Huynh et al. (2021). Briefly, we incubated eggs for 10 d in moist soil. Then, we added 40 ml presoaked corn kernels, 175 ml soil, 60 ml of water, and ca. 500 (487 ± 3, mean ± SE) viable eggs into small plastic containers (13 × 11 × 7 cm). Across all life stages, WCR were exposed to constant temperature (25 °C) and humidity (60%) within an environmental chamber (PGW40, Conviron, Winnipeg, CA) and soil moisture was inspected visually every 2 to 3 d to ensure rootworm did not desiccate.

At 350 degree days (DD) (roughly half of WCR development time; Hibbard et al. 2008), we transferred WCR to new containers with ample amounts of 1-wk-old corn for successful development to adulthood. At 550 DD (typical WCR emergence is at 700 DD; Hibbard et al. 2008), we placed the containers into emergence cages (34 × 25 × 10 cm), which consisted of a dark box with a clear collection tube. We turned on lights in environmental chambers to draw adult WCR into collection tubes. Emergence tubes included an agar water source to limit the effects of dehydration on adult WCR.

Critical Thermal Limits

During emergence, we removed all emerged adult WCR every 1 to 2 d and conducted CTmax assays on a subset of post-teneral adults. We measured CTmax using a dynamic ramping assay as in Roeder and Daniels (2022). We placed individual adult WCR in 1.5 ml microcentrifuge tubes into a prewarmed EchoTherm IC20 heating/chilling dry bath (Torrey Pines Scientific, Carlsbad, CA, USA). Cotton balls were added to the top of the microcentrifuge tubes to remove access to thermal refugia in the caps during assays. We evaluated whether individuals had lost muscle control (ie reached their CTmax) and increased the temperature by 1 °C every 10 min (ramping rate = 0.1 °C/min) until all individuals lost muscle control, as indicated by an inability to show a righting response.

We first conducted these thermal tolerance trials for 10 individuals from each colony (total n = 60) with assays starting at 35 °C to evaluate differences in heat tolerance across colonies. Then, to assess the interactive effects of colony geographic origin and cumulative heat stress, we exposed new sets of WCR to a range of temperatures from 30 to 40 °C (10 levels, 31 °C omitted; n = 6 WCR per colony and temperature; total n = 360) for 1 h and then ran thermal tolerance assays starting at 35 °C as above.

Statistical Analyses

First, we evaluated whether WCR CTmax was associated with the geographic origin of the lab reared colonies. We anticipated that CTmax was negatively associated with latitude. Therefore, we fit a linear model comparing CTmax to latitude using the lm function in the stats package in R (v. 4.2.2; R Core Team, 2022). We averaged CTmax within each colony and weighted this by the inverse of the colony standard error to account for uncertainty in colony-level CTmax estimates. Only the WCR for which we performed a standard dynamic ramping assay (ie that did not experience elevated preassay temperatures) were used for this initial comparison.

Next, we assessed whether preassay temperature exposure affected CTmax for each geographic population. First, we used a linear model to assess the effect of temperature, source population treated as a categorical variable, and their interaction on CTmax_. As before, we fit linear models to average CTmax weighted by the inverse of their standard errors using the lm function. We assessed significance using type II Wald F-tests using Anova in the car package (v. 3.1-1; Fox and Weisberg 2019). If exposure temperature crossed Tc_, we would expect a change in the relationship between CTmax where exposure to increasing temperatures would have weak effects on CTmax below Tc_, followed by a more rapid decrease in CTmax with increasing temperatures above Tc. We tested for a distinct change in the relationship between preassay exposure temperature and CTmax (ie a breakpoint) using Davies’ test for a change in slope (Davies 1987) using davies.test in the segmented package (v. 1.6-1; Fasola et al. 2018).

Results

We found no evidence for population differences in thermal tolerance among WCR colonies. Using our standard dynamic ramping assay, there was no evidence that populations collected from higher latitudes had lower CTmax values (Fig. 1; β = −0.01 ± 0.04; t = −0.336, df = 4, P = 0.75). Similarly, there was no evidence for a colony effect or differences between colonies in the effect of increasing exposure temperature on CTmax (F_5,47 = 2.00; P = 0.10 and F_5,47 = 1.78; P = 0.13, respectively).

However, we found evidence that higher exposure temperatures were associated with higher heat stress. Despite our predictions, our Davies’ test did not find statistical support for a change in the relationship between preassay exposure temperature and CTmax (ψ = 34.4 °C, F = 0.41, P = 0.81), which suggests the data are best fit by a linear model. For every 1 °C increase, we observed a 0.12 ± 0.04 °C reduction in CTmax (Fig. 2, F_1,47 = 16.91, P < 0.001). Although the majority survived the 1 h elevated temperature treatment, 6 individuals (< 2%) were unresponsive following the 1 h exposure to temperatures before the thermal tolerance assay began; this exclusively occurred in WCR who were exposed to temperatures above 35 °C (2 at 36, 3 at 39 and 1 at 40 °C), which suggests that while temperatures were stressful, they were not lethal to most individuals. Similarly, only individuals exposed to temperatures above 35 °C had CTmax below 39 °C, although this was still rare, occurring in less than 5% of WCR.

Discussion

Insect physiological responses to warming temperatures depends on the complex interplay between plasticity and genetics. In this study, we evaluated the effect of both the geographic origin of WCR colonies and a short period of elevated temperature on WCR thermal tolerance. We found no differences in CTmax between colonies. However, warmer preassay exposure temperatures reduced CTmax, suggesting that heat stress accumulates in WCR. Additionally, our study design allowed us to test for the presence of a discrete threshold at which temperatures become stressful (Tc), but which we were unable to locate using breakpoint analyses.

We found no evidence for population-level differences in CTmax or differences in the ways colonies responded to prior temperature exposure. While some studies in other taxa show slightly higher CTmax in populations at lower latitudes (Vorhees et al. 2013, Orlinick et al. 2024), other studies on intraspecific variation in CTmax in arthropods show no evidence for geographic variation in CTmax along environmental gradients, including across latitude (Bozinovic et al. 2014, Terblanche et al. 2006, Pallarés et al. 2024a, 2024b). This lack of local adaptation may be due to hard physiological limits on CTmax, which is often more phylogenetically constrained and less variable than lower thermal limits (Hoffmann et al. 2013, Diamond and Chick 2018, Bujan et al. 2020a). In WCR the lack of local adaptation may also be associated with high gene flow between populations or limited time for local adaptation due to their rapid range expansion (Kim and Sappington 2005). Alternatively, long-term rearing may have reduced preexisting population-level differences in thermal tolerance. Rearing conditions have been shown to alter offspring thermal performance in insects, even over short periods (Diamond et al. 2018). The colonies used in this experiment were maintained under the same conditions, including constant temperature (25 °C), for 10 to 29 generations. Furthermore, despite their suitability for rearing in the laboratory, there is evidence that rearing can influence WCR phenotypic traits, including reduced activity and increased body size (Li et al. 2014). Therefore, lab colonies may plausibly differ from wild collected individuals from their source populations in thermal tolerance, especially given apparent tradeoffs between fitness and thermal tolerance (Angilletta 2009). At this point, no direct comparisons of CTmax can be made to individuals from the wild populations used as the source of the lab colonies. Such comparisons may prove useful for disentangling the impacts of rearing conditions and field acclimation on thermal tolerance in the future.

Consistent with theory, higher temperature exposures diminished CTmax, which suggests that heat stress accumulates in WCR. However, we did not find a clear breakpoint or narrow range at which temperatures become stressful. This suggests that our tested range (30 to 40 °C) is above or is sufficiently near Tc that a breakpoint could not be detected and within the linear range for heat stress accumulation (Terblanche et al. 2007, Kingsolver and Umbanhowar 2018, Ørsted et al. 2022). Future studies attempting to estimate Tc could, therefore, consider a much wider temperature range when possible, potentially even incorporating values well below the optimal temperature for development, Topt. Additionally, no preassay exposure temperature improved CTmax over control. Therefore, we found no evidence for heat hardening in the CTmax of adult WCR. While hardening is a relatively common response to elevated temperatures among insects, not all species and not all life stages show plasticity in their CTmax (Weaving et al. 2022, Sepúlveda and Goulson 2023, Gonzalez et al. 2024). This lack of hardening capacity may reflect biological differences between different taxa or life stages. The baseline CTmax of some species may be near physiological limits, such that they are unable to elevate their CTmax, or there may be life stage-specific differences in hardening capacity, often related to mobility or innate CTmax (Bowler and Terblanche 2008, Moghadam et al. 2019). As a consequence, many studies find no evidence of heat hardening for at least some life stages (Moghadam et al. 2019, Zhao et al. 2017, Muluvhahothe et al. 2023, Mutamiswa et al. 2023, Burton et al. 2020). The methodology used to assess heat hardening also varies widely between studies in how temperature exposure was applied and the duration of exposure (Bowler 2005). For example, many studies return species to their rearing conditions for several hours prior to thermal tolerance assays (eg Kingsolver et al. 2016). This short respite from stress may allow for cellular repair from the heat stress and time to induce a hardening response (Dahlgaard et al. 1998, Bahrndorff et al. 2009, González-Tokman et al. 2020), but Sørensen et al. (2019) found the largest heat hardening response in the Arctic seed bug (Nysius groenlandicus) immediately following exposure to elevated temperatures without any respite period. The exact methods that best characterize the capacity for heat hardening will depend on the species and exact questions being addressed.

Warming temperatures place insects and other ectotherms nearer to their CTmax and potentially into the range where heat stress begins to accumulate. Our study found that adult WCR may be limited in their physiological capacity to respond to heatwaves or warming and may show limited genetic variation in CTmax between populations. While their CTmax remains far above the temperatures experienced in their current and expanding range, our study showed that even short exposures to temperatures well below their CTmax can cause physiological stress as evidenced by a reduced capacity to tolerate high temperatures. How this physiological heat stress alters WCR fitness and behavior remains unknown, but shifting behavior in response to heat stress may alter their impact on corn production across the United States and abroad.

Acknowledgments

We thank Chad Nielson for providing corn rootworm eggs and for sharing his rearing expertise. This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the US Department of Energy (DOE) and the US Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-C0014664. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

Author contributions

Jamieson C. Botsch (Conceptualization [equal], Formal analysis [lead], Investigation [lead], Methodology [lead], Writing—original draft [lead], Writing—review & editing [equal]), Jesse D. Daniels (Investigation [equal], Methodology [supporting], Writing—review & editing [supporting]), and Karl A. Roeder (Conceptualization [equal], Investigation [supporting], Methodology [equal], Writing—review & editing [equal])

Conflicts of interest. None declared.

References