DOI: 10.5061/dryad.c59zw3r7p
title: Survival of extreme heat related to circadian cycle and sex in bees

description: 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.
description: <em>Bee maintenance</em> Alfalfa
 leafcutting bees, <em>Megachile rotundata</em>, 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). 
 <em>Temperature treatments</em>
 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). <em>Emergence</em>
 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.
 <em>Starvation resistance</em>
 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 (Χ<sup>2</sup> = 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. <em>Daily
 activity</em> 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.  
 <em>Longevity</em> 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.     <em>Statistical
 analysis</em> 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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