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.

Approximately 300 female A. sagrei 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 A. sagrei 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: A. sagrei 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].

Experimental Protocol

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.

Statistical Analysis

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].

Data are a .csv file that can be opened with Excel or other similar software.