J Exp Zool. 2020;1–13. wileyonlinelibrary.com/journal/jez © 2020 Wiley Periodicals LLC
Received: 1 April 2020
Revised: 12 June 2020
Accepted: 25 June 2020
Thermal variability during ectotherm egg incubation: A
synthesis and framework
Melanie D. Massey
|Jeffrey A. Hutchings
Department of Biology, Dalhousie University,
Halifax, Nova Scotia, Canada
Flødevigen Marine Research Station, Institute
of Marine Research, Bergen, Norway
Department of Natural Sciences, University of
Agder, Kristiansand, Norway
Melanie D. Massey, Department of Biology,
Dalhousie University, 1355 Oxford Street,
Halifax, NS B3H 4R2, Canada.
Natural Sciences and Engineering Research
Council of Canada, Grant/Award Number:
CGSD3 ‐534491 ‐2019
Natural populations of ectothermic oviparous vertebrates typically experience
thermal variability in their incubation environment. Yet an overwhelming number of
laboratory studies incubate animals under constant thermal conditions that cannot
capture natural thermal variability. Here, we systematically searched for studies that
incubated eggs of ectothermic vertebrates, including both fishes and herpetofauna,
under thermally variable regimes. We ultimately developed a compendium of 66
studies that used thermally variable conditions for egg incubation. In this review, we
qualitatively discuss key findings from literature in the compendium, including the
phenotypic effects resulting from different patterns of thermally variable incubation,
as well as the ontogenetic persistence of these effects. We also describe a physio-
logical framework for contextualizing some of these effects, based on thermal
performance theory. Lastly, we highlight key gaps in our understanding of thermally
variable incubation and offer suggestions for future studies.
developmental plasticity, diel fluctuations, seasonal variability, temperature fluctuations,
Variability in temperature is a key feature of natural environments.
For ectotherms, this variability typically results in a wide range of
experienced body temperatures due to diurnal, seasonal, and sto-
chastic thermal fluctuations. Ectotherms have consequently adapted
to perform biological functions over ranges of inconstant tempera-
tures, with the nature of performance changing between populations,
and among and within life stages (Bowler & Terblanche, 2008;
Du, Warner, Langkilde, Robbins, & Shine, 2010; Sunday, Bates, &
The embryo life stage is particularly sensitive to temperature,
owing to the relatively small body size of embryos (Gillooly, Brown,
West, Savage, & Charnov, 2001). Differences in thermal incubation
regimes influence myriad biological functions both during develop-
ment and cascading into later ontogeny. Long‐term effects of the
incubation environment include changes to locomotor performance
(reviewed in Booth, 2006), growth (Booth, Burgess, McCosker, &
Lanyon, 2004), survival (Parker & Andrews, 2007; Warner &
Shine, 2008), behavior (reviewed in Deeming, 2004), reproductive
traits (Jonsson, Jonsson, & Finstad, 2014), and, for organisms with
temperature‐dependent sex determination, the outcome of sex
(Bull & Vogt, 1979; Valenzuela & Lance, 2004).
Given the importance of the embryonic thermal environment to
producing variation that impacts ecological and evolutionary
dynamics in ectotherms (Moczek et al., 2011; Sultan, 2007), it is
unsurprising that considerable attention has been devoted to thermal
incubation experiments. Widespread discoveries of temperature‐
dependent sex determination (TSD) in reptiles in the 1970s began a
snowballing fascination with the vertebrate embryonic thermal en-
vironment (Bull, 2004), ultimately encouraging further research on
other elements of embryonic development and physiology. A recent
review by Warner, Du, and Georges (2018) found that nearly 75% of
803 studies on reptile incubation focused on the effects of tem-
perature, and the number of publications on the effects of embryonic
thermal environment has been steadily rising since 1969.
Yet, under the basic assumption that the eggs of ectothermic
vertebrates typically experience natural daily and seasonal variation
in temperature, the vast majority of incubation studies continue to
employ constant temperature incubation conditions (Bowden,
Carter, & Paitz, 2014; Noble, Stenhouse, & Schwanz, 2018). Although
constant temperature incubation experiments have inarguably led to
important insights regarding the influence of developmental tem-
peratures on many phenotypic measures, it is difficult to interpret
these findings with respect to thermal fluctuations. One reason for
this confusion is that expected phenotypic effects, based on the
means of thermal variance, often poorly align with observed effects
under thermal variation (Bowden et al., 2014; Bull, 1985; Dowd,
King, & Denny, 2015;Gutzke&Bull,1986;Schulte,Healy,&Fangue,
2011; Schwarzkopf & Brooks, 1985). As a result, our understanding of
the ecological relevance of fluctuating temperatures is limited (Pearson
&Warner,2016), necessitating that we broaden our knowledge of how
thermal variation affects organisms, and integrate these findings with
the larger body of work on constant temperature incubation.
In the present review, we provide the first comprehensive
compendium of incubation experiments that employ thermally
variable regimes to incubate the eggs of all ectothermic verte-
brates, including both herpetofauna and fishes. Notably, previous
work on the topic has focused largely on reptiles (Noble,
Stenhouse, et al., 2018), so we aimed to include additional insights
from both fishes and amphibians, taxa that are frequently
excluded from the broader discussion of egg incubation. Within
the compendium, we also provide summaries of important findings
from each study, report the temperature regimes used, and whe-
ther ecologically or physiologically relevant data informed the
study. Ultimately, our goal for this compendium of studies is to act
as an informative reference for authors studying thermal varia-
bility, and to facilitate taxonomic crosstalk between reptile,
amphibian, and fish researchers.
We also sought to critically review several key concepts relating
to the effects of thermal variation during incubation on embryos,
hatchlings, and later life stages of ectothermic vertebrates, through a
qualitative discussion of a selection of studies found in the com-
pendium. First, we present a basic thermal performance framework
for interpreting the phenotypic results of incubation under thermal
variation, integrating what is known from constant temperature
work. Next, we discuss findings from thermal variability studies not
directly comparable with constant temperature work. Importantly,
we also discuss the long‐lasting effects of the thermal environment
during incubation through developmental plasticity. We finish by
discussing other biological or environmental sources of phenotypic
variation, and present avenues for future research.
2|SUMMARY OF LITERATURE
We conducted a systematic literature search, using the ISI Web of
Science Core Collection database for vertebrate incubation studies in
which variable temperature regimes were used in the artificial in-
cubation of eggs. We applied combinations of synonymous terms for
“temperature”and “fluctuation,”and narrowed our search to the
embryo life stage. The resulting advanced search query was: TS =
(incubation AND [fluctuating OR variable OR seasonal] AND
temperature* AND [embryo OR egg*]). The 561 results were then
organized by “Relevance,”and included articles indexed until
February 28, 2019. We examined abstracts for each study and fol-
lowed these exclusion criteria:
(i) We excluded studies for which the study organism was not an
ectothermic vertebrate, and for which egg incubation was not
artificial (i.e., in natural nests).
(ii) We excluded studies that were unable to incubate eggs for
nearly the entire incubation period, specifically those that were
unable to begin artificial incubation of eggs 72 hr or more after
(iii) We excluded studies of viviparous squamates (e.g., those that
incubated the mother rather than eggs), and squamates that
undergo approximately one‐half or more of development in‐
utero. Specifically, we imposed a cutoff of embryo oviposition at
an average Dufaure and Hubert (1961) stage of 35; the majority
of squamate species oviposit embryos when they have under-
gone one‐third of development (Dufaure & Hubert, 1961) stages
25–33 (Blackburn, 1995; Shine, 1983).
(iv) We excluded any articles that were not written in English.
(v) We excluded studies in which the focal measurements were of
sex ratios, as incubation studies focusing on temperature‐
dependent sex determination are already the most heavily
reviewed in the egg incubation field (While et al., 2018).
If these conditions were met, or if it was ambiguous whether
these conditions were met, we further analyzed the full text of the
article, removing studies that did not meet all conditions.
We cross‐referenced our results with literature found in a recent
review, as well as results from the Reptile Development Database
(Bowden et al., 2014; Noble, Stenhouse, Riley, et al., 2018), to capture
additional studies that did not appear in our search; as a result, we
added eight studies to our pool from the Reptile Development
Database (Noble, Stenhouse, Riley, et al., 2018).
From these studies, we recorded species, the temperatures
used in incubation regimes, as well as, the nature of thermal
variability. We documented whether ecologically relevant nest
temperatures for the population were recorded or cited and
whether physiologically relevant temperatures (i.e., the thermal
minimum, optimum, or maximum for development rate) were
mentioned. We also reported a brief summary of the effects of
thermal variability for each study.
Using the search parameters and cross‐referencing materials
above, we collated 66 studies (58 from the Web of Science search
and 8 from Noble, Stenhouse, Riley, et al., 2018;thecompendium
can be found in Table S1). Most studies focused on squamates,
followed by fishes, testudines, and anurans, with a single paper on
MASSEY AND HUTCHINGS
urodeles (Figure 1), and spanned 48 species. Several types of fluc-
tuating regimes were employed, and we classified them into six
patterns (Figure 2). The most common was diel fluctuations in
temperature, characterized by sinusoidal thermal fluctuations
around a stationary mean (Figure 2a). Among other thermal regimes
used were seasonal shifts in temperature, characterized by a
changing thermal mean throughout incubation, in the absence of
diel fluctuations (Figure 2b); ambient regimes, characterized by
stochastic diel changes in temperature (Figure 2c); combinations of
variable regimes (e.g., Figure 2d); and heat shocks, which involved
brief exposures to high temperatures (Figure 2e). We termed the
last category of variability “idiosyncratic,”characterized by large
changes from one constant temperature to another at different
points during incubation (Figure 2f). The magnitude of thermal
variability ranged from 3°C to 20°C.
We also noted whether studies investigated or cited data relevant
to the thermal physiology of the study organism (specifically: upper or
lower temperatures for successful development, developmental ther-
mal performance curves, critical thermal minima, maxima, and/or
optima, and temperatures that result in high or low mortality and/or
embryonic growth [from previously published or pilot experiments], at
the species level) and the thermal ecology of the study population
(natural nest temperatures from the focal environment, at the popu-
lation level). Only approximately one in three studies (23/66) reported
or cited data regarding thermal physiological parameters for their
study species. In contrast, most studies (43/66) reported or cited
natural nest temperatures for their study population.
It is important to note that that technical reports, government
reports, and conference proceedings not published in books (“gray”
literature) were not found in our search, and may yet represent
valuable sources of information on thermal variability and egg in-
cubation; thus, we acknowledge that our search design precluded
these works from inclusion in Table S1.
3|THE FALLACY OF THE AVERAGE:
NONLINEARITY OF THERMAL
PERFORMANCE CAN RESULT IN
DIFFERENCES BETWEEN CONSTANT AND
VARIABLE TEMPERATURE INCUBATION
There is a long history of approaches to estimating organismal per-
formance under thermal variability, which have been used to inter-
pret the phenotypes produced under inconstant temperatures.
Centuries ago, de Reaumur (1735) described a relationship between
plant development and temperature in which the sum of daily
FIGURE 1 Relative (shaded region) and absolute (label)
frequency taxon appearance in 66 studies incubating ectothermic
vertebrate eggs under thermally variable regimes. Reptiles are best
represented in these studies, followed by fishes. Amphibians are the
least represented group [Color figure can be viewed at
(a) (b) (c)
(d) (e) (f)
FIGURE 2 Different regimes used in studies that incubate ectothermic vertebrate eggs under thermal variability. (a) Sinusoidal fluctuations
on a diel basis. (b) Seasonally changing mean, accomplished by a slow change in temperature over a long period of time. (c) Ambient
temperatures, with stochastic changes in daily temperatures and seasonally shifting means. (d) Any combination of (a–c). (e) A constant mean
with heat shocks applied several times per week. (f) Idiosyncratic, characterized by shifts from temperature to temperature at different stages of
MASSEY AND HUTCHINGS
average air temperatures during development was always equal at
maturity for a given plant species. This “thermometric constant”re-
presented a way of describing the amount of heat energy required
for a plant to reach maturity, and necessarily assumes a linear re-
lationship between growth and temperature. Through time, heat
summation approaches were enhanced, with modifications that take
into consideration biologically informed upper and lower thermal
limits for growth (Arnold, 1959; Boussingault, 1837; Sachs, 1862) and
the inclusion of daily minimum and maximum temperatures (Lindsey &
Contemporarily, heat units are often measured in growing
degree‐days (GDD), which are calculated by subtracting a base value
), representing a temperature at which the process of interest
cannot occur, from a daily average temperature (T
traditional GDD approaches are still widely used in agriculture and
food industries, there are long‐standing criticisms of their underlying
assumptions (Wang, 1960). Notably, rates of biological processes
respond nonlinearly to temperature, especially at thermal extremes
(Sharpe & DeMichele, 1977). Under GDD (and other linear ap-
proaches), temperatures are used as a direct proxy for growth and, as
a result, growth rates at thermal extremes can be overpredicted or
underpredicted. Ruel and Ayres (1999) described this phenomenon
as a consequence of Jensen's inequality, a mathematical principle
stating that the average value of a portion of a curvilinear function
(e.g., the average thermal performance under thermal variability) will
differ from the value expected from the average temperature, de-
pending on the portion's curvature (Box 1).
Box: Jensen's inequality, or the “fallacy of the
average”: why incubation at variable temperatures
sometimes produces different responses than in-
cubation at the average
In this hypothetical example, a biological rate (develop-
ment) has been estimated for a population of organisms
across different constant temperature regimes, and mod-
eled using a gaussian function (for other common functions
used in modeling thermal performance curves [TPCs], see
Angilletta, 2006) with the following equation:
where Dis development rate (stages/day) and Tis the
temperature experienced by the organism (°C). The re-
sulting function modeling thermal performance of devel-
opment in response to temperature is shown in Box
Example 1. If continuous thermal variability (blue box) from
12°C to 18°C around an average of 15°C (T
) is experi-
enced by an organism over a period of 24 hr, Jensen's in-
equality suggests that the realized daily performance will
be the average of the function across the temperatures
(T)), rather than a function of the average
temperature experienced (P(T
)). If we calculate P(T
find an estimate of 0.087 stages for this day. However, if we
take the average of the development rate function from
12°C to 18°C, by taking the average of the integral beneath
the curve, we find an estimate of 0.107 stages for this day.
In this example, taking the function of the average tem-
perature would result in a lower estimation of performance
than taking the average of the function for all Texperi-
enced, which explains why, in this case, constant incubation
at 15°C (T
) should result in lower performance than
thermally variable incubation from 12°C to 18°C.
Example 2. In contrast, if we were to incubate the organism
under the same magnitude of variability (Box Figure 2; red
region), but in the concave portion of the curve (T
30°C, range = 27–33°C, Box Figure 2), we estimate P(T
as 2.00 stages for this day, but P
(T) as 1.92 stages.
Therefore, we expect higher realized performance from
incubation at a constant 30°C.
Example 3. In areas of the curve, which are approximately
linear (Box Figure 3, gray region), we expect P
). For example, if we incubate the organism at
21–27°C with T
=24°C, both P(T
(T) are 1.21
stages. We would therefore expect realized performance to
be similar in constant 24°C and variable 21–27°C regimes
because of the approximate linearity of this region of the TPC.
These generalizations are appropriate for regions of the
TPC that are strictly concave, convex, or linear. However, if
thermal variability occurs across a broad range encom-
passing more than one of these patterns, the relationship
(T) and P(T
) becomes more complex. To
account for Jensen's inequality in these scenarios, P
should be directly calculated by integrating the perfor-
mance function across the temperatures experienced.
Box Figure 1. A hypothetical thermal performance curve
modeled using a gaussian function, with a thermal optimum
at 30°C and a corresponding performance optimum at 2.0
stages/day. The shaded blue region from 12°C to 18°C
represents variable incubation temperatures moving con-
tinuously from 12°C to 18°C over 24 hr, in a convex region
MASSEY AND HUTCHINGS
of the thermal performance curve. T
average temperature of 15°C, and P(T
) represents the
performance expected at a constant T
[Color figure can
be viewed at wileyonlinelibrary.com]
Box Figure 2. In this figure, we use the same thermal per-
formance curve (TPC) as above. The shaded red region
from 27°C to 33°C represents variable incubation tem-
peratures moving continuously from 27°C to 33°C over
24 hr, in a concave region of the TPC. T
average temperature of 30°C, and P(T
) represents the
performance expected at a constant T
[Color figure can
be viewed at wileyonlinelibrary.com]
Box Figure 3. Inthisfigure,weusethesamethermalper-
formance curve (TPC) as above. The shaded gray region from
21°C to 27°C represents variable incubation temperatures
moving continuously from 21°C to 27°C over 24 hr, in an
approximately linear region of the TPC. T
average temperature of 24°C, and P(T
) represents the
performance expected at a constant T
An alternative and a common approach to accounting for the non-
linearity of biological responses (e.g., metabolism, locomotion, and
growth) leverages continuous thermal reaction norms, or TPCs (Huey
& Stevenson, 1979), which illustrate that the rate of biological pro-
cesses increases nonlinearly with temperature to a particular opti-
), after which they descend sharply (Figure 3). These curves
are bounded by critical thermal minima (CT
) and maxima (CT
the minimum and maximum temperatures at which the rate of per-
formance is zero. TPCs are typically developed by measuring per-
formance of animals within one population at a series of constant
temperatures and modeling a continuous reaction norm to fit these
data. Cumulative performance under thermally variable conditions
can then be estimated by integrating the TPC function, using
experienced temperatures (Casagrande, Logan, & Wallner, 1987;
Denny, 2019; Georges, Beggs, Young, & Doody, 2005; Niehaus,
Angilletta, Sears, Franklin, & Wilson, 2012; Rollinson et al., 2018;
Taylor & Shields, 1990; Worner, 1992). We note here that a recent
nonlinear GDD approach has been developed, which closely
resembles a TPC approach (Zhou & Wang, 2018). In contrast to
traditional GDD approaches, a higher degree of accuracy is expected
from using nonlinear approaches because error resulting from
Jensen's inequality is minimized.
Using integration of a TPC to predict performance results in
three general expectations, and assists in understanding why fluc-
tuating temperatures during incubation often do not produce the
same phenotypic effects we see at constant temperatures sharing the
same mean. We walk through examples of these expectations in
Box 1. First, if temperatures fall within a convex region of the TPC
(e.g., near CT
), the realized average performance of an organism
under a fluctuating temperature regime (P
(T)), is expected to be
greater than the performance of the same organism under a constant
temperature sharing the same thermal average (P(T
)). In other
words, we expect lower performance under fluctuating temperatures
than at the constant mean of fluctuation (Box Figure 1). Second, if the
temperatures fall within a concave region of the TPC (e.g., near the
optimum), realized average performance is expected to be lower than
performance at a constant temperature sharing the same thermal
average (Box Figure 2). Last, when variable temperatures fall within
FIGURE 3 A thermal performance curve (TPC), showing that
performance typically increases nonlinearly from a critical thermal
) at which the biological process of interest cannot
occur, to a thermal optimum (T
) at which performance is maximal,
and then descends sharply back to a critical thermal maximum
) at which the biological process can no longer occur. TPCs are
usually constructed by keeping organisms at a range of constant
temperatures, measuring the traits of interest at each temperature,
and then modeling the response curve to temperature
MASSEY AND HUTCHINGS
an approximately linear range of the TPC, realized average perfor-
mance and performance at the constant average temperature should
be similar (Box Figure 3). Within these regions, as the magnitude of
thermal fluctuations increases, we also expect the difference be-
(T) and P(T
) to increase. Of course, the exact relation-
ship between P
(T) and P(T
) will depend on the actual thermal
experience of the organism(s) in question, and can be estimated by
integrating the TPC function across the temperatures experienced.
4|INTERPRETING THERMAL VARIABILITY
Studies incubating ectothermic vertebrate eggs under different
thermal regimes have reported complex findings with regard to the
relative effects of thermally variable incubation and constant tem-
perature incubation. Overall, authors have found that the phenotypic
results of incubation are largely dissimilar between regimes; that is,
variability itself does not represent a single treatment. The “fallacy of
the average”provides a reasonable explanation for why these results
differ in magnitude and direction of phenotypic effects, based on
what might be expected from Jensen's inequality. Specifically, the
fallacy of the average is useful for interpreting differences between
results expected from a constant mean temperature, and the effects
resulting from thermal variation around a mean (e.g., Figure 2a).
For example, Les, Paitz, and Bowden (2009) incubated painted
turtle (Chrysemys picta) eggs at constant temperatures near the lower
and upper limits for successful development (23°C and 31°C; in the
area of CT
, respectively; Figure 4), and regimes fluc-
tuating 3°C around those means. Based on the theory, we expect that
fluctuations around the convex area of CT
developmental traits, and those around the concave area of CT
should decelerate them, relative to constant average temperatures.
Indeed, in Les et al.'s (2009) study, C. picta embryos incubated near
the lower limit experienced enhanced survival and development
rates relative to constant temperatures, whereas those incubated
near the upper limit developed slower and had higher mortality, as
predicted. Similarly, Warner and Shine (2011) found that diel thermal
fluctuations around a warm temperature mean near the thermal
optimum decreased development rate in lizard (Amphibolurus mur-
icatus) embryos, but development rate was enhanced when fluctua-
tions occurred around a cool temperature mean, aligning with
predictions. In a study incubating a lizard (Sceloporus undulatus)ata
constant 28°C, as well as 28 ± 5°C, no difference in development rate
was found (Andrews, Mathies, & Warner, 2000). Given that this
range of temperatures falls between CT
for S. undulatus
(Sexton & Marion, 1974), it is likely that the temperatures experi-
enced fall in an approximately linear portion of the TPC for devel-
opment rate, which explains the lack of difference in incubation
period between the two treatments.
Studies modifying the magnitude of variability around a constant
thermal mean also demonstrate predictions stemming from the fal-
lacy of the average. When eggs of Bynoe's geckos (Heteronotia binoei)
are incubated at a constant mean (32°C), as well as treatments with
fluctuations around that mean (32 ± 3°C, 5°C, and 9°C), increasing
fluctuations result in slower developmental rates, and the
largest fluctuation results in significant mortality (Andrewartha,
Mitchell, & Frappell, 2010). Given that the thermal optimum for devel-
opment lies at 30°C in this species (Kearney & Shine, 2004), we assume
that the temperatures experienced largely fall in the concave portion of
the TPC for development rate in this species, and increasing thermal
variability around a mean of 32°C is expected to produce increasing
reductions in performance than at a constant 32°C. Similarly, in Chinese
pond turtles (Chinemys reevesii) and Chinese softshell turtles (Pelodiscus
sinensis), development rate decreases as the magnitude of fluctuations
increases (Du, Shen, & Wang, 2009;Li,Zhou,Wu,Wu,&
Ji, 2013), supporting the predictions of theory.
These experiments demonstrating the fallacy of the average
show that variability should not be considered a monolithic treat-
ment; the phenotypic response to variability appears to depend on
the range of temperatures experienced, relative to the thermal
sensitivity of the trait and organism in question. An existing diffi-
culty in understanding the results of thermal variability experi-
ments lies in the fact that thermal reaction norms for
developmental and developmentally influenced traits are typically
poorly characterized (Noble, Stenhouse, et al., 2018; While
et al., 2018), resulting in a lack of physiological context when in-
terpreting results. Future work would benefit, at the least, from
qualitative knowledge of whether incubation temperatures are
“hot”or “cold”relative to the thermal sensitivity of the trait and
study organism (Les et al., 2009), or by incorporating empirical
frameworks describing thermal reaction norms derived at constant
temperatures for the traits in question (Georges et al., 2005;
Massey, Holt, Brooks, & Rollinson, 2018).
FIGURE 4 A schematic representing fluctuating temperature
treatments (blue boxes) in a study on eggs of Chrysemys picta.CT
the temperatures at which development cannot proceed,
bound the curve. Temperature fluctuations around 23°C (convex
region of the curve) resulted in faster development rates than those
expected from a constant 23°C. The opposite was true around 31°C
(concave region of the curve). Adapted from Les et al. (2009) [Color
figure can be viewed at wileyonlinelibrary.com]
MASSEY AND HUTCHINGS
It is important to note, however, that TPCs have several limita-
tions (Kingsolver & Woods, 2016; Schulte et al., 2011). A key but
rarely met assumption of TPCs is that thermal histories, both leading
up to and during incubation treatments, do not affect resulting phe-
notypes through acclimation. Although we can eliminate the effect of
the embryo's previous thermal history in the present review, as the
studies discussed herein incubated embryos for the entirety of in-
cubation, there is a possibility that transgenerational plasticity (via
parental effects) or acclimation to temperature during incubation
contributed to additional variation in phenotype. Further, given that
TPCs are typically constructed under constant conditions, they may
not accurately reflect instantaneous performance, especially at
stressful temperatures. For example, Niehaus et al. (2012)incubated
striped marsh frogs (Limnodynastes peronii) under two levels of diel
thermal variation and found that TPCs underpredicted development
rate when thermal variability was high. This finding suggests that de-
velopment occurs more quickly at stressful temperatures when ex-
posure periods are brief (i.e., under daily sinusoidal variation), but
development rates are slowed under chronic exposure to high tem-
peratures (i.e., the conditions under which TPCs are constructed).
Nevertheless, given that TPCs have been able to explain the majority
of variation in development rate in the wild (Rollinson et al., 2018), and
modifications accounting for time‐dependent effects are available
(Kingsolver & Woods, 2016), TPCs can represent a useful avenue for
explaining or predicting the effects of thermally variable incubation.
5|WHAT HAVE WE LEARNED FROM
OTHER THERMALLY VARIABLE REGIMES?
5.1 |Seasonal variation in temperature
In addition to experiencing diel thermal variation, embryos in sea-
sonal environments can experience increasing or decreasing mean
temperatures as incubation progresses (illustrated in Figure 2b).
Seasonal thermal variation is particularly relevant to embryos that
undergo long incubation periods: northern Australian saltwater cro-
codiles (Crocodylus porosus), for example, have a mean incubation
period of 101 days, and mean nest temperatures decrease seasonally
throughout development (Magnusson, 1979; Webb & Cooper‐
Preston, 1989). In combination with environmental sources of heat,
both large‐bodied testudines and crocodilian embryos can also gen-
erate metabolic heat throughout incubation, adding considerable
heat to nests during the latter half of development (Broderick,
Godley, & Hays, 2001; Carr & Hirth, 1961; Ewert & Nelson, 2003;
Massey, Congdon, Davy, & Rollinson, 2019).
Seasonally variable incubation regimes appear to influence em-
bryonic development differently than do stable temperatures, when
thermal means are identical. For example, Shine (2004) simulated
three realistic seasonal incubation scenarios for Bassiana duperreyi
lizards, all with the same thermal mean: stable, increasing throughout
incubation, and decreasing throughout incubation. These three
treatments generated significant differences in developmental
dynamics and hatchling phenotypes, with faster development oc-
curring in both seasonal regimes, and high deformity levels resulting
in poor locomotor performance under seasonally decreasing tem-
peratures (Shine, 2004). These results suggest that seasonal changes
in temperature, in spite of mean temperatures, are sufficient to
generate significant phenotypic variation, and may be of particular
relevance to species that have phenologically or geographically dis-
persed nesting (Shine, 2004).
As many organisms with long incubation periods experience
gradual seasonal thermal changes, it is also possible that embryonic
thermal sensitivities can be locally adapted to seasonality, that is,
thermal sensitivity changes as development progresses. Indeed, in
Arctic char (Salvelinus alpinus), populations that spawn in relatively
warm autumn temperatures and incubate throughout winter appear
to have much higher hatching success and fewer deformities when
warm temperatures are used during early incubation, and subse-
quently fall (Jeuthe, Brännäs, & Nilsson, 2016). In fact, the reversal of
natural seasonal temperatures, that is, moving from cool to warm
incubation temperatures, results in significantly higher mortality and
deformity rate in embryos (Jeuthe et al., 2016). These results high-
light the importance of understanding how natural thermal variability
in study populations relate to the temporal dynamics of development.
For long‐incubating species in seasonal environments, the question of
whether embryonic thermal sensitivities change as development
progresses has yet to be explicitly explored.
5.2 |Heat shock
Heat shock refers to short exposures to sublethal, high temperatures,
often resulting in increased thermal tolerance upon subsequent
exposures (Bowler, 2005). If an organism experiences temperatures
near the upper range of its thermal tolerance, denatured and ab-
normal proteins are produced, triggering the production of heat
shock proteins (hsps). Hsps, among other cellular changes, ameliorate
negative effects of heat on cellular proteins (Goldberg, 2003;
Hightower, 1980; Parsell & Lindquist, 1993). Ultimately, brief ex-
posures to hot temperatures in adult organisms have been shown to
produce an effect known as “heat hardening”(Alexandrov, 1964)in
which there is a transient increase in thermal tolerance.
For embryos, however, it appears that very brief exposures to
high temperatures (i.e., heat shocks, example illustrated in Figure 2e)
throughout development have minute effects on embryonic and
posthatching phenotypes, at least for many of the traits investigated
so far. For instance, Lim, Manzon, Somers, Boreham, and Wilson
(2017) incubated lake whitefish (Coregonus clupeaformis) under a
near‐constant 2°C regime, with 1 hr temperature spikes to 5°C twice‐
weekly, and found that development proceeded at the same rate as
in constant 2°C treatments. A slight difference in body length
emerged at the prehatch stage, wherein embryos from the heat‐
shock regime were slightly (5%) longer. Another study on whitefish
found similar results, where weekly 1 hr temperature spikes of
varying magnitude (from 2°C to 5°C and from 2°C to 7°C) had no
MASSEY AND HUTCHINGS
effect on incubation period, survival, or size at hatch relative to a
constant 2°C regime (Lee et al., 2016). In future experiments, it may
prove interesting to increase the frequency of heat shocks as body
size of whitefish embryos in these experiments was affected by
twice‐weekly heat shocks, but not by once‐weekly heat shocks of
the same magnitude, suggesting, as others have (Kingsolver &
Woods, 2016; Niehaus et al., 2012), that performance responds
differently as the duration of exposures changes.
The severity of the heat shock relative to the organism's thermal
sensitivity may also play a role in determining whether phenotypic
effects are significant. Overall (1994) incubated canyon lizards
(Sceloporus merriami) at both moderate (31°C) and hot (34°C) con-
stant temperatures with brief daily exposures to 37°C. Heat shocks
accelerated development rate and increased hatchling body size
relative to constant temperatures, but there was low embryo survival
in the hot (34°C) heat‐shocked treatment. In canyon lizards, constant
incubation at 37°C is lethal to embryos, but short exposures to
severe temperatures appear to enhance phenotype (larger body size,
earlier hatch date), increasing mortality only when mean incubation
temperatures are already stressful (Overall, 1994).
There are still many questions that remain unanswered regard-
ing temperature shocks during development. First, to our knowledge,
no experiments have tested the effects of cold shocks on oviparous
ectothermic vertebrate embryos. Given that cold temperatures and
hot temperatures illicit different gene expression responses in adult
ectotherms (Podrabsky & Somero, 2004), cold shocks may result in
different phenotypic responses than those observed as a result of
heat shocks. Next, it is important to note that these studies generally
tested whole‐organism level responses to heat shocks (e.g., body size,
locomotor performance). Given that acute heat shocks to embryos
have been shown to illicit significant transcriptomic changes in ex-
pression of developmental and hsp genes (Bentley, Haas, Tedeschi, &
Berry, 2017; Tedeschi et al., 2015), and that isolated incidents of
thermal stress can transiently affect embryonic heart rates (Hall &
Warner, 2019), it is likely that temperature shocks during develop-
ment can significantly—and perhaps, persistently—affect other traits
that have not yet been tested.
5.3 |Idiosyncratic thermal regimes
We dubbed regimes involving temperature changes at various time-
points during development “idiosyncratic”(Figure 2f). Often, these ex-
periments aim to isolate temperature effects that occur at discrete
developmental stages; for example, to determine temperature‐sensitive
periods for sex determination under TSD (Webb, Beal, Manolis, &
Dempsey, 1987), but they can also be applied to developmental studies
investigating time‐sensitive effects of temperature during development.
Thus far, critical windows of physiological sensitivity to tem-
perature have been identified by temperature‐switching experi-
ments. By changing C. clupeaformis embryos between cool, moderate,
and warm constant thermal regimes at key milestones during de-
velopment, Eme et al. (2015) found that organogenesis represented a
particularly sensitive period through which strong plasticity acts on
heart rate and oxygen metabolism. Ultimately, the significant phy-
siological changes occurring during organogenesis persisted into
hatchlings. Identification of critical windows, such as organogenesis,
may further elucidate mechanisms by which developmental plasticity
(see Section 6 below) occurs.
Other studies that changed temperatures at various timepoints
throughout development have specifically investigated embryonic
acclimation capacity. Booth (1998) found no evidence for metabolic
acclimation in Brisbane river turtles (Emydura signata), showing
through a temperature‐switch experiment that embryonic metabo-
lism during the latter half of development was not dependent on
embryos’previous thermal experience in early development.
Likewise, Angilletta, Lee, and Silva (2006) reported that, although
embryos exhibit metabolic temperature sensitivity throughout
development, S. undulatus embryos did not undergo metabolic accli-
mation after being temperature‐switched during development.
Interestingly, these results directly conflict with the identification of
organogenesis as a plastic or acclimatory window in C. clupeaformis
(Eme et al., 2015), perhaps suggesting that taxonomic differences in
embryonic metabolic acclimation capacity exist.
PLASTICITY: HOW PERSISTENT ARE THE
EFFECTS OF THERMAL FLUCTUATIONS?
Previously, we discussed the effects of thermal variation primarily as
they affect embryos and hatchlings. However, the developmental
environment can continue to shape the phenotype of organisms
beyond the incubation period, via developmental plasticity.
Developmental plasticity is often considered a permanent change to
phenotype as a result of the developmental environment (“irrever-
sible nongenetic adaptation”; Kinne, 1962), with a few exceptions
(e.g., Ligon, Backues, Fillmore, & Thompson, 2014; McKeown,
Thompson, & Cline, 2017; Polo‐Cavia & Gomez‐Mestre, 2017), and
plastic effects of the developmental environment arise from changes
in gene expression that occur during and after development
(reviewed in Beldade, Mateus, & Keller, 2011). At constant tem-
peratures, developmental plasticity to temperature is well‐described
for numerous traits, but what happens when eggs are incubated at
In an elegant experiment in zebrafish (Danio rerio), Schaefer and
Ryan (2006) disentangled the relative contributions of reversible and
developmental plasticity to diel fluctuating temperature regimes to
thermal tolerance. After rearing fish from eggs for 100 days across a
range of constant and fluctuating conditions, the authors acclima-
tized fish to constant temperatures for 12–15 days. They then tested
the thermal tolerance of fish, and determined that the critical ther-
mal maximum, or temperature at which opercular spasms occur, was
significantly higher in fish incubated and reared at fluctuating tem-
peratures when compared with constant temperatures, regardless of
acclimation temperature (reversible plasticity). The ages of fish at the
MASSEY AND HUTCHINGS
time of testing (112–115 d) represent a significant portion of the
lifespan of zebrafish, suggesting that their persistent physiological
plastic response to fluctuating temperatures can affect long‐term
fitness. Likewise, long‐term morphological differences arising from
incubation regime have been detected in other fishes. Nathanailides,
Lopez‐Albors, and Stickland (1995) incubated Atlantic salmon (Salmo
salar) at a constant temperature of 11°C and at natural, seasonal
temperatures that gradually rose throughout incubation (5–10°C).
Although fish from the constant regime hatched sooner and had
larger muscle fibers, by 3 weeks postfeeding, fish from the ambient
thermal regime exhibited significantly faster growth rates and larger
muscle fiber area. Again, these effects have significant ecological
importance: the posthatching growth rates of salmonids are strongly
linked to fitness (Hutchings, 1993).
Several experiments in reptiles have also examined long‐term
responses of traits to variable developmental temperatures, finding
mixed results with respect to the persistence of developmental
plasticity. For example, Pearson and Warner (2016) incubated the
lizard Anolis sagrei at fluctuating temperatures with different thermal
means and found that differences in running performance between
treatment groups at hatching disappeared at 3 weeks. Conversely, in
the lizard B. duperreyi, running performance was consistently higher
in individuals incubated in a warm fluctuating regime for 20 weeks
posthatching relative to those incubated in cold fluctuating regimes,
although morphological differences apparent at hatching disappeared
within 6 weeks (Elphick & Shine, 1998). In a study on Western fence
lizards (Sceloporus occidentalis), some morphological characters (body
size and hindlimb length) persist after incubation, while others
(forelimb and tail lengths) do not (Buckley, Irschick, & Adolph, 2010);
it is possible that body size and hindlimb lengths, which are directly
linked to sprint speed in this species (Mayr, 1956), exhibit a higher
degree of developmental canalization than traits for which links to
fitness are unclear (Buckley et al., 2010), although more trait‐focused
studies are needed to resolve this puzzling inconsistency.
The studies mentioned previously in this section described long‐
term effects measured in captive animals, but how do measures
change when animals are reared in natural habitats? Dayananda,
Gray, Pike, and Webb (2016); Dayananda, Penfold, and Webb (2017)
conducted experiments on velvet geckos (Oedura lesueurii) in which
eggs were incubated at fluctuating temperature regimes reflecting
current (cold) and future (warm) conditions. After releasing hatchl-
ings into the field, they determined that cold‐incubated hatchlings
had significantly higher survival and growth rates in situ than did
warm‐incubated hatchlings 10 months after release. Furthermore,
the authors found differences in posthatching growth rates of geckos
between two release sites, suggesting that posthatching environ-
mental conditions interact with incubation conditions to produce
significant effects on phenotype. Similarly, Andrews et al. (2000)
found that cold fluctuating incubation temperatures resulted in
higher survival 7–9 months posthatch in lizards (Sceloporus un-
dulatus), when compared with high fluctuating incubation tempera-
tures. Interestingly, in this experiment, posthatching growth rates in
the field ultimately were not influenced by incubation regime; it is
possible that the degree of developmental plasticity in response to
temperatures was low for growth rates, or that developmentally
plastic growth rate differences could not be fully realized under
natural conditions (e.g., due to low food availability). Although long‐
term studies under natural conditions are scarce, they raise inter-
esting questions about whether phenotypic differences caused by
incubation regime can be masked by natural conditions, and about
which traits are ultimately relevant to fitness in the wild.
Long‐term changes in gene and protein expression in response
to developmentally variable temperatures have not been widely
explored. Nonetheless, they may represent an interesting avenue
for future research. Recent literature has revealed that, during
acclimation to fluctuating thermal regimes, adult fish exhibit large‐
scale changes in messenger RNA (mRNA) levels for genes that
regulate cell growth and proliferation, molecular chaperones, and
cellular membrane integrity (Podrabsky & Somero, 2004). Further-
more, different genes appear to be activated at fluctuating versus
constant temperature conditions (Podrabsky & Somero, 2004). With
regard to proteins, increases in the concentration of hsp70 mole-
cular chaperones in response to fluctuating temperatures have been
linked to improved thermal tolerance in adult fishes (Coulter, Höök,
Mahapatra, Guffey, & Sepúlveda, 2015; Nakano & Iwama, 2002),
and seasonal changes in HSP levels occur in frogs (Feidantsis,
Anestis, Vasara, Kyriakopoulou‐Sklavounou, & Michaelidis, 2012).
Given the persistent effects of thermal variability during incubation
on ectothermic vertebrate thermal tolerance, morphology, growth
rates, and locomotor performance mentioned herein, it is not dif-
ficult to imagine that developmentally plastic molecular changes
would also be detectable. Future work should leverage established
molecular techniques such as mRNA and heat shock protein assays
to illuminate the proximate mechanisms of developmental plasticity
in response to thermal variability.
7|OTHER SOURCES OF PHENOTYPIC
Although temperature overwhelmingly governs the development of
oviparous ectothermic vertebrates, there are other major factors
that can influence incubation and phenotype of larvae and hatchl-
ings. First, theory predicts that population‐level differences
between the thermal sensitivity of traits should emerge due to
adaptation to local thermal regimes (Huey & Stevenson, 1979), and
indeed this prediction is founded within the studies of thermal
variability herein. For example, Buckley et al. (2010) incubated
S. occidentalis eggs from four populations under variable thermal
regimes and found significant population‐level effects on several
traits relevant to fitness (e.g., body size and hindlimb length). Fur-
ther differences that arise because of differences between clutches
of eggs have also been heavily discussed. Numerous studies in
reptiles (e.g., Andrews et al., 2000;Díaz,Iraeta,Verdú‐Ricoy, Siliceo,
&Salvador,2012; Du, Shou, & Shen, 2005; Shine & Harlow, 1996;
Shine, Elphick, & Harlow, 1997) have estimated significant clutch
MASSEY AND HUTCHINGS
effects on the phenotypic outcomes that were measured, although
the extent to which these differences arise from genetic differences
between families, maternal effects, or combinations thereof have
Recent studies that illustrate significant effects of transge-
nerational plasticity, or the ability of environmental influences
during a parent's lifetime to affect offspring as well, also present
interesting avenues through which to explore the effects of thermal
variability. In fishes, parental acclimation to high temperatures en-
hances growth (Salinas & Munch, 2012) and size (Shama, Strobel,
Mark, & Wegner, 2014) of offspring. Transgenerational effects are
particularly strong at high temperatures, suggesting they may
ameliorate negative physiological consequences of climate change
(Shama et al., 2014). Given predicted rises in both mean and
variability of future temperatures (Meehl & Tebaldi, 2004)andthe
potential of thermal variability to significantly enhance thermal
tolerance (Schaefer & Ryan, 2006), studies that examine transge-
nerational plasticity in a variable temperature context could yield
much‐needed insights about the resilience of ectothermic verte-
brates to climate change.
8|CONCLUSIONS AND FUTURE
It has long been acknowledged that incubation at constant tempera-
tures poorly reflects what organisms encounter in nature (Georges
et al., 2005; Oufiero & Angilletta, 2010; Pearson & Warner, 2016).
Despite this empirical reality, constant temperatures continue to
dominate incubation studies (Noble, Stenhouse, et al., 2018). Variable
thermal regimes experienced during incubation have resulted in un-
anticipated and complex effects, producing phenotypic effects that
differ from what would be expected from constant thermal means
(e.g., Les et al., 2009; Warner & Shine, 2011), and permanently en-
hancing thermal tolerance (Schaefer & Ryan, 2006), morphology
(Buckley et al., 2010), and even growth rates, after release into the
wild (Dayanada et al., 2016,2017).
Existing studies that employ variable temperature incubation
suggest fruitful avenues for future research. First, the field would
benefit significantly from incorporating physiological frameworks
that assist in explaining the phenotypes produced under variable
temperature regimes (e.g., the use of TPCs). Incorporation of phy-
siological knowledge opens up possibilities that integrate our vast
knowledge of incubation under constant regimes with thermal
variability, and is especially important because the majority of ex-
isting studies do not use physiologically relevant data—that is, data
that are empirically anchored in what the organism is likely to
experience under its own, often population‐specific, natural
conditions—to inform the experimental design or analyses. To facil-
itate this integration, authors could aim to quantify thermal reaction
norms for their traits of interest, especially because broadly char-
acterized reaction norms are uncommon in the literature (Noble,
Stenhouse, et al., 2018).
Most of the attention in studies of thermal variation has been,
thus far, devoted to reptiles, and currently there is a relative paucity
of studies on amphibians and noncommercial fish species. This fact is
puzzling, considering that many amphibians and fishes are short‐lived
organisms with fast life‐histories, and are thus predicted to experi-
ence strong developmental plasticity to temperature (Shine
et al., 1997). Researchers could consider directing future studies of
fluctuating temperature incubation toward taxa that might be ideal
for informing a generally applicable predictive framework for un-
derstanding organismal responses to climate change‐driven thermal
fluctuations. In particular, tropical stenotherms with narrow margins
of thermal functionality may be more heavily impacted by climate‐
driven increases in thermal variation, and thus are prime candidates
for further investigation (Dowd et al., 2015).
Importantly, more studies of proximate mechanisms that lead to
phenotypic changes under variable temperatures are needed. Con-
stant temperature studies have already utilized genetic tools such as
transcriptomics to explain thermal acclimation (Scott & Johnston,
2012), and studies in adult organisms have investigated gene ex-
pression changes in response to variable thermal regimes in adult
organisms (Podrabsky & Somero, 2004). However, there is currently
a considerable gap of knowledge about the mechanisms that impart
phenotype under thermal variability.
Taken together, these recommendations suggest the promise of
exciting future work on thermal variability. The breadth of studies
incubating ectothermic vertebrates under thermally variable condi-
tions is much more robust than previously acknowledged, and these
studies have significantly advanced our understanding of natural
thermal regimes and their relationship to organismal development
We thank Njal Rollinson, Austin Lloyd, and Michael Foisy for pro-
viding their insights during the writing of this manuscript. We also
thank two anonymous reviewers, whose helpful comments greatly
improved the quality of our manuscript. Funding was provided by the
Natural Sciences and Engineering Research Council of Canada
(Canada Graduate Scholarship) and the Province of Nova Scotia
(Nova Scotia Graduate Scholarship) to Melanie Massey.
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Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Massey MD, Hutchings JA. Thermal
variability during ectotherm egg incubation: A synthesis and
framework. J Exp Zool. 2020;1–13.
MASSEY AND HUTCHINGS