The pollination services provided by insects have been a crucial part of evolution and survival for many species, including humans. For bees to be efficient pollinators they must survive the environmental insults they face daily. Thus, looking into the short- and long-term effects of heat exposure on bee performance provides us with a foundation for investigating how stress can affect insect pollination. Solitary bees are a great model for investigating the effects of environmental stress on pollinators because the vast majority of insect pollinator species are solitary rather than social. One of the most pervasive environmental stressors to insects is temperature. Here we investigated how a one-hour heat shock affected multiple metrics of performance in the alfalfa leafcutting bee, Megachile rotundata. We found that a short heat shock (1hr at 45ºC) can delay adult emergence in males but not females. Bee pupae were rather resilient to a range of high temperature exposures that larvae did not survive. Following heat shock (1hr at 50ºC), adult bees were drastically less active than untreated bees, and this reduction in activity was evident over several days. Heat shock also led to a decrease in bee survival and longevity. Additionally, we found a connection between starvation survival after heat shock and time of exposure, where bees exposed in the morning survived longer than those exposed in the afternoon, when they would normally experience heat shock in the field. These data suggest that there is an unexplored daily/circadian component to the stress response in bees likely similar to that seen in flies, nematodes, and plants which is constitutive or preemptive rather than restorative. Taken together our data indicates that single heat shock events have strong potential to negatively impact multiple life history traits correlated with reproduction and fitness.

Bee maintenance

Alfalfa leafcutting bees, Megachile rotundata, were purchased from JWM Leafcutters (Nampa, ID) as post-diapause prepupae that were quiescent. To maintain that quiescence until needed, brood cells containing prepupa were stored in an incubator at 6°C in the dark (Percival Scientific, Perry, IA, USA). In preparation for experiments, bees were moved to a 29°C Percival incubator under a long day (15L:9D) photoperiod and 50 ±5% relative humidity. Under these conditions, it took ~20 days for the prepupae to complete their development and emerge as adults. Bees were treated at different life stages and afterward kept in the same 29°C incubator under the same conditions, a standard temperature for this bee species (Tepedino and Parker 1986). 

Temperature treatments

Bees were treated to a single 1-hour heat shock. This heat shock occurred at one of three life stages tested (prepupa, pupa, or adult). Prepupae were treated 19 days prior to adult emergence, a day after the transfer from 6°C to 29°C, while still in the prepupal stage that just finished overwintering. Pupae were treated two days prior to adult emergence to target the pharate adult stage and make it comparable to previous work. Adults were treated within 24 hrs. of emergence from the brood cells. The treatments consisted of a one-hour exposure to 29 (control), 35, 40, 45, or 50°C. Exposures were carried out in EchoTherm (Torrey Pines Scientific, Carlsbad, CA, USA) bench top incubators. A 473 ml cup containing only water was added to the incubator chamber to maintain humidity during treatment but was not a drinking water source for the bees. All bees were treated in 473 ml cups lined with moist paper towels. This water was added to prevent dehydration-induced mortality during the temperature treatment as seen in preliminary studies. Prepupae and pupae were left inside their brood cells during treatment. Once emerged, all adult bees were fed Pro-sweet liquid feed ad libitum (Mann Lake Ltd., Hackensack, MN, USA). Pollen was not provided because in preliminary studies only certain females consumed the pollen provided and it was deemed as a nutritional advantage over males and females that did not consume pollen. Treated bees were only used for one experiment (emergence, starvation resistance, daily activity, or longevity).

Emergence

To monitor the effect of temperature on emergence, brood cells containing one treated bee (prepupa or pupa) were placed individually in 24-well plates (Corning Life Sciences, Corning, NY, USA). Five replicate plates were filled for each treatment (29, 35, 40, 45, and 50°C), and the experiment was performed at least two separate times (10 replicates/treatment). 24-well plates were monitored daily for emergence which normally occurred over an eight-day period with the males emerging in the first few days, a mix of sexes in days 4 and 5, and mostly females towards the end of the emergence period (Pitts-Singer and Cane 2011). Data are presented as percent bee emergence per day by sex.

Starvation resistance

Because overwintering bees might not have immediate access to food upon emergence, we wanted to test the response to temperature in newly emerged bees without access to food. To determine the appropriate number of bees to use and whether density affected bee performance, a series of density experiments were carried out. Three replicates of 15 or 20 bees were placed in 946 ml cages and starvation until death was recorded. The experiment was repeated four times. We found no effect of density on bee performance (Χ² = 1.535, p = 0.2153; Fig. S1C). Three to five groups of adult bees (15 bees/group) were placed in cages in an incubator as described above. Exactly every 24 hrs. mortality was assessed, and dead bees were removed, sexed, and counted. To investigate the effect of time of treatment, an identical experiment was carried out, but bees were treated either in the morning (10 am) or the afternoon (3 pm). Both experiments were performed three separate times using different cohorts of bees. Data are presented as percent survival by day and by sex.

Daily activity

Density experiments were conducted in the activity monitors to determine the effect of density on bee activity and the appropriate number of bees. Five, ten, or twenty bees were placed in each activity monitor and their activity was recorded for three hours during the afternoon, two hours after treatment recovery. There was no effect of density on bee activity (p = 0.3482: Fig. S1B). Groups of ten bees (5 females and 5 males) were placed in drosophila vials (Genesee Scientific, San Diego, CA, USA) after determining that these conditions were neither crowding the bees nor negatively affecting their performance. The open end of the vial was closed with micro mesh and a 3D printed fitted lid that prevented bee escape while allowing adequate airflow. These vials were placed in Drosophila Population Monitors (DPMs; Trikinetics, Inc., MA, USA). Six DPMs ran simultaneously for three control (29°C) and three heat-shocked (1hr @ 50°C) replicates. This temperature was chosen based on previous work showing that performance in this species decreases above 45°C (Barthell et al. 2002) and recent temperature data from out nest boxes (Wilson et al. 2020). The activity was tracked for 24 and 48 hours without food as there was no effect of food availability in activity within the first 48 hours following treatment (p = 0.774; Fig. S1A), and at that point, a new batch of freshly treated bees was placed into the DPMs. There was no mortality recorded in the activity experiments within the first 48 hrs. The experiment was repeated three times using different bee cohorts. Data are presented as total activity/minute/bee.  

Longevity

Groups of ~20 treated (29 or 50°C) bees were placed in 2.37 L containers with food (Pro-sweet soaked cotton in 60mm petri dish bottoms). Three times per week, the bee containers were checked for mortality and the dead bees were removed, sexed, and counted. The food was replaced as needed to ensure a surplus of fresh food. Data are presented as percent survivorship over time by sex.    

Statistical analysis

Emergence data was analyzed using multivariable (one, two, or three-way) ANOVAs with temperature, time, sex, and their interaction as variables where applicable. Bee cohort was used as a random block term in the analysis. ANOVAs were followed with a Tukey’s post hoc analysis. General linear models (GLMs) were used to analyze daily activity, followed by linear contrast analysis to tease out treatment effects. Starvation survivorship and longevity data were analyzed using a proportional hazards model. All statistical analyses were carried out in JMP15.

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