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

Data files for manuscript under review titled "Effects of short-term heat stress on the thermal tolerance of western corn rootworm (Coleoptera: Chrysomelidae)".Two .csv files: Metadata and Master Data. The Master Data tab contains all of the information used in the analyses.<b>Abstract from paper under review:</b>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 (WCR, <i>Diabrotica virgifera virgifera</i> LeConte), an economically important crop pest. Despite a wide latitudinal range of source populations, WCR colonies did not differ in their CTmax. Regardless of colony origin, we found that exposing WCR to higher temperatures resulted in lower CTmax, which suggests that heat stress accumulated. This study highlights how WCR experiences heat stress at temperatures near the high temperatures they experience in the field, which may have important and currently unknown implications for its behavior.<br><b>Methods from paper under review:</b><i>Geographic populations and rearing</i>WCR colonies were collected from geographically distinct populations from across their U.S. 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. We incubated eggs for 10 d in moist soil. Then, we added 40 mL pre-soaked corn kernels, 175 mL soil, 60 mL of water, and ca. 500 (487 ± 3, mean ± SE) viable eggs into small plastic containers (13 x 11 x 7 cm). Across all life stages, WCR were exposed to constant temperature (25°C) and humidity (60%) within environmental chambers and soil moisture was inspected visually every 2-3 d to ensure rootworm did not desiccate.<br>At 350 degree days (DD) (roughly half of WCR development time), we transferred WCR to new containers with ample amounts of 1 week old corn for successful development to adulthood. At 550 DD (typical WCR emergence is at 700 DD), 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.<br><i>Critical thermal limits</i>During emergence, we removed all emerged adult WCR every 1-2 d and conducted CTmax assays on a subset of post-teneral adults. We measured CTmax using a dynamic ramping assay. We placed individual adult WCR in 1.5 mL microcentrifuge tubes into a prewarmed EchoThermTM IC20 heating/chilling dry bath. 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 (i.e., reached their CTmax) and increased the temperature by 1°C every 10 minutes (ramping rate = 0.1°C/min) until all individuals lost muscle control, as indicated by an inability to show a righting response.<br>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 hr and then ran thermal tolerance assays starting at 35 °C as above.<br>

本数据集对应待审稿件《短期热胁迫对西部玉米根虫（鞘翅目：叶甲科）热耐受性的影响》的数据文件。包含2个.csv格式文件：元数据（Metadata）与主数据集（Master Data）。主数据集包含本研究分析所用的全部信息。

**待审稿件摘要：**
前期高温暴露对昆虫热耐受性的影响机制复杂，取决于其所经历的热胁迫程度：高温暴露可通过热硬化作用使个体耐受更高温度，也可能因累积热胁迫降低个体的高温耐受能力。本研究针对西部玉米根虫（western corn rootworm, WCR，*Diabrotica virgifera virgifera* LeConte）——一种具有重要经济危害性的作物害虫——评估了短期高温暴露与实验室种群地理起源对其临界热最大值（critical thermal maximum, CTmax）的影响。尽管源种群的纬度分布范围较广，但供试WCR种群的CTmax并无显著差异。无论种群起源如何，将WCR暴露于更高温度后其CTmax均显著降低，这表明热胁迫发生了累积。本研究揭示了WCR在接近田间实际遭遇的高温环境下会受到热胁迫，这一现象对其行为学的潜在影响目前仍未知且具有重要研究价值。

**方法部分：**
#### 地理种群与饲养
西部玉米根虫种群采自其美国分布范围内的多个地理隔离种群，并在实验室恒定条件下饲养。用于建立种群的野外采集工作于1995年至2013年间在堪萨斯州、内布拉斯加州、宾夕法尼亚州、南达科他州以及威斯康星州开展。自种群建立以来，所有供试种群均在适宜WCR生长发育的最优条件下饲养。我们将虫卵置于湿润土壤中孵化10天，随后向小型塑料容器（13×11×7 cm）中加入40 mL预浸泡玉米粒、175 mL土壤、60 mL去离子水以及约500枚（487±3，平均值±标准误）存活虫卵。在各虫态阶段，WCR均饲养于控温控湿培养箱中，温度维持25℃，相对湿度60%，每2-3天目视检查土壤湿度，以避免根虫脱水死亡。
当累积发育温积达到350日度（degree days, DD）时，将WCR转移至新容器中，提供充足的1周龄玉米苗以保障其顺利发育至成虫。当累积温积达到550日度时（常规WCR羽化温积为700日度），将容器移入羽化笼（34×25×10 cm）中，该装置由遮光箱与透明收集管组成。通过开启培养箱灯光诱导羽化的成虫WCR进入收集管。收集管内放置琼脂水基食物以缓解成虫脱水的影响。

#### 临界热极限测定
在成虫羽化期间，每1-2天收集全部羽化的成虫，并选取部分刚完成羽化的成虫开展CTmax测定。本研究采用动态升温法测定CTmax：将单头成虫置于1.5 mL微量离心管中，放入预加热的EchoTherm™ IC20型加热/制冷干式恒温槽中。在离心管顶部塞入脱脂棉以消除虫体在管盖处的热避难行为。以个体丧失肌肉控制能力（即达到CTmax）作为测定终点，每10分钟升温1℃（升温速率为0.1℃/min），直至所有供试个体均丧失肌肉控制能力，表现为无法完成翻正反应。
本研究首先对每个种群的10头成虫开展热耐受性测定（总样本量n=60），测定起始温度设置为35℃，以比较不同种群间的耐热性差异。随后，为评估种群地理起源与累积热胁迫的交互效应，将新一批WCR分别暴露于30℃至40℃的10个梯度温度（剔除31℃，每个种群-温度组合的样本量n=6，总样本量n=360）1小时，随后按照前述方法以35℃为起始温度开展热耐受性测定。