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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 physiological 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. HIGHLIGHTS • We present and describe a comprehensive compendium of studies that incubate ectothermic vertebrate eggs under thermally variable regimes. • We explain why thermal variability can influence embryonic and posthatching phenotypes in significant ways.
J Exp Zool. 2020;113. © 2020 Wiley Periodicals LLC
Received: 1 April 2020
Revised: 12 June 2020
Accepted: 25 June 2020
DOI: 10.1002/jez.2400
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.
Funding information
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,
thermal variability
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, &
Dulvy, 2011).
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. Longterm 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
temperaturedependent 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 longlasting 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.
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
temperatureand 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 onehalf 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 onethird of development (Dufaure & Hubert, 1961) stages
2533 (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 crossreferenced 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 crossreferencing 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
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.
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 (ac). (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
average air temperatures during development was always equal at
maturity for a given plant species. This thermometric constantre-
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 &
Newman, 1956).
Contemporarily, heat units are often measured in growing
degreedays (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
). Although
traditional GDD approaches are still widely used in agriculture and
food industries, there are longstanding 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:
|− |
0.5 30
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
Figures 13.
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
experienced (P
(T)), rather than a function of the average
temperature experienced (P(T
)). If we calculate P(T
), we
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 = 2733°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
proach P(T
). For example, if we incubate the organism at
2127°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 2127°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
between P
(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
of the thermal performance curve. T
represents the
average temperature of 15°C, and P(T
) represents the
performance expected at a constant T
[Color figure can
be viewed at]
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
represents the
average temperature of 30°C, and P(T
) represents the
performance expected at a constant T
[Color figure can
be viewed at]
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
represents the
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-
mum (T
), 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
minimum (CT
) 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
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-
tween P
(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.
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 averageprovides 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
and 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
should accelerate
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
and 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
around T
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
hotor coldrelative 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
and 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]
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 timedependent effects are available
(Kingsolver & Woods, 2016), TPCs can represent a useful avenue for
explaining or predicting the effects of thermally variable incubation.
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 largebodied 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 longincubating 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
nearconstant 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
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
twiceweekly heat shocks, but not by onceweekly 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) heatshocked 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 wholeorganism 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 significantlyand perhaps, persistentlyaffect 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 temperaturesensitive
periods for sex determination under TSD (Webb, Beal, Manolis, &
Dempsey, 1987), but they can also be applied to developmental studies
investigating timesensitive effects of temperature during development.
Thus far, critical windows of physiological sensitivity to tem-
perature have been identified by temperatureswitching 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 temperatureswitch experiment that embryonic metabo-
lism during the latter half of development was not dependent on
embryosprevious 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 temperatureswitched 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.
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; PoloCavia & GomezMestre, 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 welldescribed
for numerous traits, but what happens when eggs are incubated at
fluctuating temperatures?
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 1215 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
time of testing (112115 d) represent a significant portion of the
lifespan of zebrafish, suggesting that their persistent physiological
plastic response to fluctuating temperatures can affect longterm
fitness. Likewise, longterm morphological differences arising from
incubation regime have been detected in other fishes. Nathanailides,
LopezAlbors, 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 (510°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 longterm
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 traitfocused
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 coldincubated hatchlings
had significantly higher survival and growth rates in situ than did
warmincubated 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 79 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.
Longterm 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, KyriakopoulouSklavounou, & 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.
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 populationlevel 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 populationlevel 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,VerdúRicoy, Siliceo,
&Salvador,2012; Du, Shou, & Shen, 2005; Shine & Harlow, 1996;
Shine, Elphick, & Harlow, 1997) have estimated significant clutch
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
muchneeded insights about the resilience of ectothermic verte-
brates to climate change.
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 datathat is, data
that are empirically anchored in what the organism is likely to
experience under its own, often populationspecific, natural
conditionsto 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 shortlived
organisms with fast lifehistories, 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 changedriven 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
and ontogeny.
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.
Melanie D. 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;113.
... This includes the main abiotic factor responsible for driving physiological reactions: temperature. Developmental temperature affects nearly every organismal trait, including sex determination, developmental rate, morphology, performance and behaviour (Booth, 2006;Colinet et al., 2015;Noble et al., 2018a;Massey and Hutchings, 2020). As a result, understanding the nature of this organism-environment interaction during early development is of fundamental importance in evolutionary ecology (Refsnider and Janzen, 2010). ...
... Much of our insight into thermal developmental plasticity has been generated through experiments conducted at constant developmental temperature (Booth, 2006;Bowden et al., 2014;Colinet et al., 2015;Booth, 2018;Noble et al., 2018a;While et al., 2018;Massey and Hutchings, 2020). However, in the wild, developmental temperature is rarely constant, varying across both short and long time periods (Colinet et al., 2015;Booth, 2018;Bowden and Paitz, 2018). ...
... Given these important developmental effects of embryonic temperature, the thermal microenvironment chosen for oviposition can have significant impacts on offspring phenotype. Unfortunately, we still have a limited understanding of how phenotypes are affected by fluctuating developmental temperatures across ectothermic taxa (Bowden et al., 2014;Colinet et al., 2015;Booth, 2018;Noble et al., 2018a;Massey and Hutchings, 2020). ...
Full-text available
During the vulnerable stages of early life, most ectothermic animals experience hourly and diel fluctuations in temperature as air temperatures change. While we know a great deal about how different constant temperatures impact the phenotypes of developing ectotherms, we know remarkably little about the impacts of temperature fluctuations on the development of ectotherms. In this study, we used a meta-analytic approach to compare the mean and variance of phenotypic outcomes from constant and fluctuating incubation temperatures across reptile species. We found that fluctuating temperatures provided a small benefit (higher hatching success and shorter incubation durations) at cool mean temperatures compared with constant temperatures, but had a negative effect at warm mean temperatures. In addition, more extreme temperature fluctuations led to greater reductions in embryonic survival compared with moderate temperature fluctuations. Within the limited data available from species with temperature-dependent sex determination, embryos had a higher chance of developing as female when developing in fluctuating temperatures compared with those developing in constant temperatures. With our meta-analytic approach, we identified average mean nest temperatures across all taxa where reptiles switch from receiving benefits to incurring costs when incubation temperatures fluctuate. More broadly, our study indicates that the impact of fluctuating developmental temperature on some phenotypes in ectothermic taxa are likely to be predictable via integration of developmental temperature profiles with thermal performance curves.
... The generality of constant temperature experiments has also widely been criticized due to the possibility of treatments imparting unintended pathologies, especially under stressful conditions (Massey & Hutchings, 2020). Natural organisms have evolved to respond to changing environments and consequently are expected to perform better when a stressor is applied at a natural time scale rather than through chronic exposure (Angilletta Jr & Angilletta, 2009;Colinet et al., 2015). ...
... Although approaches that modify constant temperature models to extend their applicability have been developed (e.g., controlling for Stress Response in Figure 6; Kingsolver & Woods, 2016;Koussoroplis et al., 2017), many authors now advocate for the use of more ecologically relevant temperature regimes in experimental biology as a means of generating realistic responses and conclusions (e.g., Massey & Hutchings, 2020;Morash et al., 2018;Taylor et al., 2021). ...
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Ecologists often rely on randomized control trials (RCTs) to quantify causal relationships in nature. Many of our foundational insights of ecological phenomena can be traced back to well-designed experiments, and RCTs continue to provide valuable insights today. Although RCTs are often regarded as the "gold standard" for causal inference, it is important to recognize that they too rely on a set of causal assumptions that must be justified and met by the researcher to draw valid causal conclusions. We use key ecological examples to show how biases such as confounding, overcontrol, and collider bias can occur in experimental setups. In tandem, we highlight how such biases can be removed through the application of the structural causal model (SCM) framework. The SCM framework visualizes the causal structure of a system or process under study using directed acyclic graphs (DAGs) and subsequently applies a set of graphical rules to remove bias from both observational and experimental data. We show how DAGs can be applied across ecological experimental studies to ensure proper study design and statistical analysis, leading to more accurate causal estimates drawn from experimental data. Although causal conclusions drawn from RCTs are often taken at face value, ecologists are increasingly becoming aware that experimental approaches must be carefully designed and analyzed to avoid potential biases. By applying DAGs as a visual and conceptual tool, experimental ecologists can increasingly meet the causal assumptions required for valid causal inference.
... Another major challenge of generalizing conclusions across acclimation experiments is that the environmental treatments used often do not reflect ecologically realistic conditions. In studies of thermal plasticity, much of what is currently known is derived from studies that employ simplified constant temperature regimes [19] (but see [20]). Although constant temperature experiments have generated remarkable and seminal biological insights [19], they probably reflect evolutionarily novel environments, and their applicability to natural conditions are limited [21,22]. ...
... Interestingly, we note that estimates for spawning success, fecundity, yolk volume and sperm concentrations in our lifetime constant treatment group were similar to previously reported results in wild-type zebrafish held in explicitly optimal constant thermal conditions [69][70][71]. These results suggest that small fluctuations in temperature may have minor impacts, but the magnitude of response is dependent on the magnitude of variability, as others have found [20,22,64]. Ultimately, as the climate changes, we must address both thermal means and magnitudes of variability to accurately predict organismal responses [22,72]. ...
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Human-driven increases in global mean temperatures are associated with con-comitant increases in thermal variability. Yet, few studies have explored the impacts of thermal variability on fitness-related traits, limiting our ability to predict how organisms will respond to dynamic thermal changes. Among the myriad organismal responses to thermal variability, one of the most prox-imate to fitness-and, thus, a population's ability to persist-is reproduction. Here, we examine how a model freshwater fish (Danio rerio) responds to diel thermal fluctuations that span the species's viable developmental range of temperatures. We specifically investigate reproductive performance metrics including spawning success, fecundity, egg provisioning and sperm concentration. Notably, we apply thermal variability treatments during two ontogenetic timepoints to disentangle the relative effects of developmental plasticity and reversible acclimation. We found evidence of direct, negative effects of thermal variability during later ontogenetic stages on reproductive performance metrics. We also found complex interactive effects of early and late-life exposure to thermal variability, with evidence of beneficial acclimation of spawning success and modification of the relationship between fecundity and egg provisioning. Our findings illuminate the plastic life-history modifications that fish may undergo as their thermal environments become increasingly variable.
... Despite using the same incubators in both experimental years, the daily variation in rearing temperatures was lower in 2018 than in 2001 and the mean temperature was slightly higher. Embryonic development in ectotherms is influenced by both mean temperature and fluctuation in temperature in nonadditive ways (Massey and Hutchings 2021). We corrected for the shift in mean temperature analytically, but could not correct for variance. ...
... We corrected for the shift in mean temperature analytically, but could not correct for variance. At moderate temperatures like those used in our experiment, both theory (Liu and Meng 2000;Georges et al. 2005;Massey and Hutchings 2021) and empirical findings (e.g., Niehaus et al. 2006;Arrighi et al. 2013;Hall and Warner 2020) suggest that temperature fluctuations have either no effect or cause accelerated development rates. In our experiments, we find the opposite, embryos in 2001 experience more thermal variability yet slower development that embryos in 2018, making it unlikely that the differences we see are a result of experimental conditions. ...
Environmental change is predicted to accelerate into the future and will exert strong selection pressure on biota. While many species may be fated to extinction, others may survive through their capacity to evolve rapidly at highly localized (i.e. microgeographic) scales. Yet, even as new examples have been discovered, the limits to such evolutionary responses have not often been evaluated. One of the first examples of microgeographic variation involved pond populations of wood frogs (Rana sylvatica). Although separated by just tens to hundreds of meters, these populations exhibited countergradient variation in intrinsic embryonic development rates when reared in a common garden. We repeated this experiment 17 years (approx. 6–9 generations) later and found that microgeographic variation persists in contemporary populations. Furthermore, we found that contemporary embryos have evolved to develop 14% to 19% faster than those in 2001. Structural equation models indicate that the predominant cause for this response is likely due to changes in climate over the intervening 17 years. Despite potential for rapid and fine-scale evolution, demographic declines in populations experiencing the greatest changes in climate and habitat imply a limit to the species’ ability to mitigate extreme environmental change. This article is protected by copyright. All rights reserved
... Durtsche et al. (2021) also suggest an onset of the plastic response to temperature during the embryonic period allowing the adult to better cope with subsequent temperature variations. One possible future study would be to investigate the mechanism underlying the plastic response via trait variations (Toomey et al., 2019(Toomey et al., , 2020(Toomey et al., , 2021 and molecular techniques (Sunde et al., 2019;Massey and Hutchings, 2020) in order to predict how populations will be affected by natural and anthropogenic environmental variability (Oomen and Hutchings, 2015). ...
Full-text available
In the context of global change, the aim of this study was to investigate the effects of temperature on the survival rate and various variables (development time, morphometric measures, energy values) for both embryos and post-hatch stages until first food intake of northern pike ( Esox lucius ). We applied five constant temperatures (8, 10, 12, 14 and 16 °C) and their effects were analyzed during the entire endogenous feeding period at three stages: hatching, emergence and first food intake. Around 80,000 eggs from five females and five males were used. As expected, the development time was three times longer at 8 than at 16 °C. The survival rate of well-formed post-hatch stages at the end of the endogenous feeding period was similar between temperatures: 76% (10 °C), 73% (12 °C), 72% (14 °C), 72% (16 °C), except at 8 °C (70%). Also at 16 °C, post-hatch stages were larger and had the highest energy value when starting feeding exogenously. These results are in accordance with current models that predict an increase of the distribution area of northern pike in France in coming decades associated with climate change projections (1 to 5 °C increase by 2100 for France).
... In addition, although research in embryo ecology focuses on the impacts of events that occur after conception and before birth or hatching, such research needs to extend data-gathering to the adult stage in order to evaluate the ecological and evolutionary consequences of embryonic responses to developmental environments (Mitchell, Janzen & Warner, 2018) (Fig. 1). Within this framework of embryo ecology, the developmental plasticity of embryos has long been a research field, and has been reviewed in detail elsewhere (Howe, 1967;Braum, 1978;Deeming & Ferguson, 1991;Deeming, 2002Deeming, , 2004Noble, Stenhouse & Schwanz, 2018;Massey & Hutchings, 2021). Therefore, we do not attempt to cover every aspect of embryo ecology in this synthesis. ...
Adaptations of post‐hatching animals have attracted far more study than have embryonic responses to environmental challenges, but recent research suggests that we have underestimated the complexity and flexibility of embryos. We advocate a dynamic view of embryos as organisms capable of responding – on both ecological and evolutionary timescales – to their developmental environments. By viewing embryos in this way, rather than assuming an inability of pre‐hatching stages to adapt and respond, we can broaden the ontogenetic breadth of evolutionary and ecological research. Both biotic and abiotic factors affect embryogenesis, and embryos exhibit a broad range of behavioural and physiological responses that enable them to deal with changes in their developmental environments in the course of interactions with their parents, with other embryos, with predators, and with the physical environment. Such plasticity may profoundly affect offspring phenotypes and fitness, and in turn influence the temporal and spatial dynamics of populations and communities. Future research in this field could benefit from an integrated framework that combines multiple approaches (field investigations, manipulative experiments, ecological modelling) to clarify the mechanisms and consequences of embryonic adaptations and plasticity.
... In some cases (e.g., incubation duration, residual yolk reserves), the direction of the effect of thermal fluctuation varied depending on the amplitude of the fluctuation. These findings fit into a growing body of knowledge clarifying how ther-mal complexity is integrated into developmental processes in reptiles (Les et al., 2007(Les et al., , 2009Warner and Shine, 2011;Bowden et al., 2014;Simoncini et al., 2019;Massey and Hutchings, 2021). Existing models suggest that the relationship between temperature and developmental rate plays a key role in explaining the effects of thermal fluctuations on hatchling phenotypes (Georges et al., 1994(Georges et al., , 2005Bowden et al., 2014). ...
The environment experienced during embryonic development is a rich source of phenotypic variation, as environmental signals have the potential to both inform adaptive plastic responses and disrupt normal developmental programs. Environment-by-embryo interactions are particularly consequential for species with temperature-dependent sex determination, a mode of sex determination common in non-avian reptiles and fish, in which thermal cues during a discrete period of development drive the formation of either an ovary or a testis. Here we examine the impact of thermal variation during incubation in combination with developmental exposure to a common endocrine-disrupting contaminant on fitness-related hatchling traits in the American alligator (Alligator mississippiensis), a species with temperature-dependent sex determination. Using a factorial design, we exposed field-collected eggs to five thermal profiles (three constant temperatures, two fluctuating temperatures) and two environmentally relevant doses of the pesticide metabolite dichlorodiphenyldichloroethylene; and we quantified incubation duration, sex ratios, hatchling morphometric traits, and growth (9-10 days post-hatch). Whereas dichlorodiphenyldichloroethylene exposure did not generally affect hatchling traits, constant and fluctuating temperatures produced diverse phenotypic effects. Thermal fluctuations led to subtle changes in incubation duration and produced shorter hatchlings with smaller heads when compared to the constant temperature control. Warmer, male-promoting incubation temperatures resulted in larger hatchlings with more residual yolk reserves when compared to cooler, female-promoting temperatures. Together, these findings advance our understanding of how complex environmental factors interact with developing organisms to generate phenotypic variation and raise questions regarding the mechanisms connecting variable thermal conditions to responses in hatchling traits and their evolutionary implications for temperature-dependent sex determination.
In nature, organisms are exposed to variable environmental conditions that impact their performance and fitness. Despite the ubiquity of environmental variability, substantial knowledge gaps in our understanding of organismal responses to non-constant thermal regimes remain. In the present study, using zebrafish (Danio rerio) as a model organism, we applied geometric morphometric methods to examine how challenging but ecologically realistic diel thermal fluctuations experienced during different life stages influence adult body shape, size and condition. Zebrafish were exposed to either thermal fluctuations (22 - 32 °C) or a static optimal temperature (27 °C) sharing the same thermal mean during an Early period spanning embryonic and larval ontogeny (days 0 - 30), a Later period spanning juvenile and adult ontogeny (days 31 - 210), or a combination of both. We found that body shape, size, and condition were affected by thermal variability, but these plasticity-mediated changes were dependent on the timing of ontogenetic exposure. Notably, after experiencing fluctuating temperatures during Early ontogeny, females displayed a deeper abdomen while males displayed an elongated caudal peduncle region. Moreover, males displayed beneficial acclimation of body condition under lifelong fluctuating temperature exposure, whereas females did not. The present study, using ecologically realistic thermal regimes, provides insight into the timing of environmental experiences that generate phenotypic variation in zebrafish. This article is protected by copyright. All rights reserved.
Data about the effect of temperature on the duration of embryonic development, or the incubation process, are relatively abundant for octopod cephalopods. Experimental data can be used to fit the thermal time classic model of development in order to predict the duration of the process and the range of temperatures within which development proceeds according to the model and with high survival (permissive range), with potential applications for basic research and production activities. The present work is aimed at fitting this classical model to fourteen data sources dealing with eleven species of octopods, seven of them not being previously analyzed, and discussing the constraints and applications of the model. As a result, it is possible to hold that the thermal time model of development duration suitably fits experimental data in octopods, and can be used to schedule hatching events in a production facility. Nevertheless, the range of temperatures tested in the literature is usually too limited to allow an appropriate estimation of the permissive range, so an extension of the temperature range to the known limits of each species is suggested. In addition, when the model is applied to whole spawnings with culture purposes, it is recommended to set the very first hatchings as the criterion for the end of the incubation. Further research is needed to ascertain if temperature fluctuations within and/or out of the permissive range can have implications for the parameterization of the model in octopod species.
The incubation temperature for embryonic development affects several aspects of hatchling performance, but its impact on the thermal sensitivity of performance attributes remains poorly investigated. In the present study, Trachemys scripta elegans hatchlings from two different latitudinal populations were collected to assess the effects of different incubation temperatures on the locomotor (swimming speed) and physiological (heart rate) performances, and the thermal sensitivity of these two attributes. The incubation temperature significantly affected the examined physiological traits. Hatchling turtles produced at low incubation temperature exhibited relatively higher cold tolerance (lower body temperatures at which the animals lose the ability to escape from the lethal conditions), and reduced heart rate and swimming speed. Furthermore, the effect of incubation temperature on the thermal sensitivity of swimming speed differed between the low- and high-latitude populations. At relatively high incubation temperatures, the high-latitude hatchling turtles exhibited reduced thermal sensitivities of swimming speed than those of the low-latitude ones. Reduced thermal sensitivity of locomotor performance together with high cold tolerance, exhibited by the high-latitude hatchling turtles potentially reflected local adaptation to relatively colder and more thermally-variable environments.
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Body temperature affects plants’ and animals’ performance, but these effects are complicated by thermal variation through time within an individual and variation through space among individuals in a population. This review and synthesis describes how the effects of thermal variation—in both time and space—can be estimated by applying a simple, nonlinear averaging scheme. The method is first applied to the temporal variation experienced by an individual, providing an estimate of the individual’s average performance. The method is then applied to the scale-dependent thermal variation among individuals, which is modelled as a 1/f-noise phenomenon. For an individual, thermal variation reduces average performance, lowers the temperature of maximum performance (Topt) and contracts the range of viable temperatures. Thermal variation among individuals similarly reduces performance and lowers Topt, but increases the viable range of average temperatures. These results must be viewed with caution, however, because they do not take into account the time-dependent interaction between body temperature and physiological plasticity. Quantifying these interactions is perhaps the largest challenge for ecological and conservation physiologists as they attempt to predict the effects of climate change.
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Metabolic heating caused by physiological processes during the development of oviparous embryos can raise nest temperatures above those of the surrounding substrate and may be sufficient to increase embryonic growth rates, influence sex ratios of hatchlings with temperature-dependent sex determination, and increase hatching success in seasonal environments. In sea turtles with large clutch sizes, metabolic heating can raise nest temperatures by as much as 6ºC. However, no studies have directly investigated metabolic heating in any species of freshwater turtle. We investigated whether metabolic heating occurs in nests of snapping turtles (Chelydra serpentina) from southeastern Michigan, USA. A temperature logger was placed in the center of eight unaltered snapping turtle nests. A second temperature logger was placed at the same depth in the surrounding substrate 5 cm from the side of the nest chamber. As metabolic heating is more pronounced in nests with larger clutches, we artificially increased the size of two additional nests using donor clutches of 11 and 21 eggs, respectively. Temperatures were recorded at two-hour intervals until after the presumptive hatch date of all nests. We found that there was a significant increase both in mean nest temperature and accumulated heat units for natural and experimental treatment nests during the last third of incubation. Further, in nests with experimentally increased clutch sizes, mean nest temperature was significantly greater than substrate temperature throughout incubation, suggesting that large nests also exhibit a thermal inertia that results in positive heat balance throughout development, at least in the soils studied.
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How temperature influences development has direct relevance to ascertaining the impact of climate change on natural populations. Reptiles have served as empirical models for understanding how the environment experienced by embryos can influence phenotypic variation, including sex ratio, phenology and survival. Such an understanding has important implications for basic eco-evolutionary theory and conservation efforts worldwide. While there is a burgeoning empirical literature of experimental manipulations of embryonic thermal environments, addressing widespread patterns at a comparative level has been hampered by the lack of accessible data in a format that is amendable to updates as new studies emerge. Here, we describe a database with nearly 10, 000 phenotypic estimates from 155 species of reptile, collected from 300 studies manipulating incubation temperature (published between 1974–2016). The data encompass various morphological, physiological, behavioural and performance traits along with growth rates, developmental timing, sex ratio and survival (e.g., hatching success). This resource will serve as an important data repository for addressing overarching questions about thermal plasticity of reptile embryos.
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Precise calculations of growing degree days (GDD) are an important component in crop simulation models and managerial decisions. Traditional methods for calculating GDD assume linear developmental responses to temperature and cannot precisely account for the delay in growth or development at temperatures above the optimal temperature (Topt). A new nonlinear method for calculating GDD was developed. Variations in the prediction of the dates since sowing to various developmental stages and performance measures for describing the accumulation of dry matter by GDD for two widely planted crops (corn and wheat) were used to evaluate the new method in comparison with the traditional methods. The new method predicted the dates of the developmental stages more precisely (date variations reduced by 1 d), and the errors for the predictions of the accumulation of dry matter for winter wheat and corn were smaller. The method was most promising for spring wheat. The new method was more stable and more precise than traditional methods, especially when Topt was lower than the maximum air temperature.
Most studies of thermal tolerance use adults, but early-life stages (e.g. embryos) are often more sensitive to thermal agitation. Studies that examine effects on embryos rarely assess the potential for thermal tolerance to change with ontogeny or how effects differ among sympatric species, and often utilize unrealistic temperature treatments. We used thermal fluctuations from nests within the urban-heat island to determine how thermal tolerance of embryos changes across development and differs among two sympatric lizard species (Anolis sagrei and A. cristatellus). We applied fluctuations that varied in frequency and magnitude at different times during development and measured effects on embryo physiology, egg survival, and hatchling morphology, growth, and survival. Thermal tolerance differed between the species by ~ 2 °C: embryos of A. sagrei, a lizard that prefers warmer, open-canopy microhabitats, were more robust to thermal stress than embryos of A. cristatellus, which prefers cooler, closed-canopy microhabitats. Moreover, thermal tolerance changed through development; however, the nature of this change differed between the species. For A. cristatellus, thermal tolerance was greatest mid-development. For A. sagrei the relationship was not statistically clear. The greatest effects of thermal stress were on embryo and hatchling survival and embryo physiology. Hatchling morphology and growth were less affected. Inter-specific responses and the timing of stochastic thermal events with respect to development have important effects on egg mortality. Thus, research that integrates ecologically-meaningful thermal treatments, considers multiple life-history stages, and examines interspecific responses will be critical to make robust predictions of the impacts of global change on wildlife.
Widely distributed terrestrial ectotherms from the southern European peninsulas show patterns of subdivision (related to isolation in temperate refugia) that allow us to test the relative importance of phylogeographic lineage, population of origin and familial effects as sources of variation for life-history traits. We collected gravid females from 15 geographically separated populations of the lacertid lizard Psammodromus algirus, a widely distributed species with well differentiated eastern and western lineages. We incubated eggs under two treatments of constant (28�C) and fluctuating (28 ± 4�C) temperature, and we examined clutch, population, and lineage effects on several traits of females, eggs, and hatchlings. Incubation time was mainly explained by differences between lineages, but it was also influenced by population and female effects. Within each lineage, incubation was shorter at cooler and wetter sites, and for a given climate it was shorter for eastern than for western populations, suggesting that countergradient variation has evolved independently in the two lineages. Female size, clutch size, and relative fecundity were primarily influenced by inter-population differences, a pattern that seemed attributable to environmental differences in productivity, because mean female size was positively correlated with a gradient of increasing precipitation and decreasing temperature. Clutch size was mainly, but not entirely, dependent on female SVL, suggesting both a proximate effect of local conditions and intrinsic differences among populations. Females from drier and warmer sites produced larger hatchlings. Mean egg mass was mainly determined by familial effects. Eggs incubated at a constant temperature hatched earlier than did their siblings incubated at fluctuating temperatures, a fact that could be explained by considering that in Mediterranean environments developmental rate might increase at a lower speed above average incubation temperature than it does decrease below it.
For many oviparous animals, incubation temperature influences sex through temperature-dependent sex determination (TSD). Although climate change may skew sex ratios in species with TSD, few available methods predict sex under natural conditions, fewer still are based on mechanistic hypotheses of development, and field tests of existing methods are rare. We propose a new approach that calculates the probability of masculinization (PM) in natural nests. This approach subsumes the mechanistic hypotheses describing the outcome of TSD, by integrating embryonic development with the temperature-dependent reaction norm for sex determination. Further, we modify a commonly used method of sex ratio estimation, the constant temperature equivalent (CTE), to provide quantitative estimates of sex ratios. We test our new approaches using snapping turtles (Chelydra serpentina). We experimentally manipulated nests in the field, and found that the PM method is better supported than the modified CTE, explaining 69% of the variation in sex ratios across 27 semi-natural nests. Next, we used the PM method to predict variation in sex ratios across 14 natural nests over 2 years, explaining 67% of the variation. We suggest that the PM approach is effective and broadly applicable to species with TSD, particularly for forecasting how sex ratios may respond to climate change. Interestingly, we also found that the modified CTE explained up to 64% of variation in sex ratios in a Type II TSD species, suggesting that our modifications will be useful for future research. Finally, our data suggest that the Algonquin Park population of snapping turtles possesses resilience to biased sex ratios under climate change.
Scientific interest in developmental plasticity spans many disciplines, and research on reptiles has provided many insights into this field. We highlight these contributions, review the field's history, and introduce the special issue on this topic .