DOI: 10.5061/dryad.pzgmsbcs1
title: Data from: Rapid heat hardening in embryos of the lizard Anolis sagrei

description: Adaptive thermal tolerance plasticity can dampen the negative effects of
 warming. However, our knowledge of tolerance plasticity is lacking for
 embryonic stages that are relatively immobile and may benefit the most
 from an adaptive plastic response. We tested for heat hardening capacity
 (a rapid increase in thermal tolerance that manifests in minutes to hours)
 in embryos of the lizard Anolis sagrei. We compared survival of a lethal
 temperature exposure between embryos that either did (‘hardened’) or did
 not (‘not hardened’) receive a high but non-lethal temperature
 pre-treatment. We also measured heart rates at common garden temperatures
 before and after heat exposures to assess metabolic consequences.
 ‘Hardened’ embryos had significantly greater survival after lethal heat
 exposure relative to ‘not hardened’ embryos. That said, heat pre-treatment
 led to a subsequent increase in embryo heart rate that did not occur in
 embryos that did not receive pre-treatment, indicative of an energetic
 cost of mounting the heat hardening response. Our results are consistent
 with adaptive thermal tolerance plasticity in these embryos (greater heat
 survival after heat exposure), but also highlight associated costs.
 Thermal tolerance plasticity may be an important mechanism by which
 embryos respond to warming that warrants greater consideration.
description: Approximately 300 female <em>A. sagrei</em> were
 collected from Miami-Dade County, FL during May of 2022 and transported to
 Loyola University Chicago, where they were housed following established
 protocols. Briefly, females were housed in groups of up to 6 individuals
 per cage with perches and moist potting soil available for egg laying [16,
 37]. Crickets are provided daily as food and enclosures are misted with
 water five or six times per day with an automatic misting system
 (MistKing, Jungle Hobbies Ltd.). Eggs were collected daily between 900 and
 1000 h and were incubated at 27°C, within the natural range of
 <em>A. sagrei</em> nests [16, 38], in a sealed petri dish
 filled with moist vermiculite (-150 kPa)[16]. Given that multiple females
 were housed together, we could not determine the source of each egg.
 However, we assume that each egg was from a different female due to our
 sampling design: <em>A. sagrei</em> lay one egg at a time at
 an interval of 7-10 d, and all eggs used in the study (N = 89) were
 collected over four consecutive days period (June 13-16, 2022). Each egg
 was randomly assigned to one of three groups: Hardened, Not Hardened, or
 Control (see below). Starting on day seven, the heart rate of each embryo
 was measured between 9:00 and 13:00 h every other day using an infrared
 egg monitor (Avitronics, Cornwall, UK)[10]. Heart rates were recorded for
 30 seconds, and if a heart rate could not be found the egg was gently
 rotated up to four times and reassessed [18].
 <em>Experimental Protocol</em>
 Heat tolerance and heat hardening ability were measured on day 12
 (Figure 1). Not all embryos survived to day 12, and we moved forward with
 the following sample sizes: N = 25 Hardened, N = 29 Not hardened, and N =
 28 Control.  Heat tolerance was measured as survival after 1h at 45°C, a
 temperature known to be potentially lethal to developing embryos of this
 species from previous studies [18, 39]. Eggs in the Hardened group
 experienced a heat pretreatment at 39°C for 1 h before exposure to 45°C 2h
 later (Figure 1). The Not Hardened group experienced only the 45°C
 exposure, while the Control group did not experience any elevated
 temperatures. The heart rate of all eggs was measured three times on
 experimental days, corresponding to times before and after heat exposures
 in the groups that received them (figure 1). There was approximately 1.25
 h between the first and second heart rate measurements, and 2 h between
 the second and third heart rate measurements. At the conclusion of data
 collection, eggs were placed back in their chambers at 27°C and raised
 until hatching or egg failure as determined by fungal growth.
 <em>Statistical Analysis</em> We
 tested for differences in mortality between control and treatment groups
 with a G-test. We tested for differences in heart rate change before and
 after heat exposure (i.e., Heart rate 2 – Heart rate1 and Heart rate 3 –
 Heart rate 2; Figure 1) using paired t-tests. Data were analyzed using R
 [40].
description: Data are a .csv file that can be opened with Excel or other
 similar software.