A new method of estimating thermal performance of embryonic development rate yields accurate prediction of embryonic age in wild reptile nests
Introduction
Temperature has a strong effect on rates of physiological processes in plants and ectothermic animals (Gillooly et al., 2002, Kingsolver, 2009). The reaction norm that describes how a performance trait is related to temperature is typically referred to as thermal performance curve (TPC, Fig. 1a) (Huey and Stevenson, 1979). General biochemical principles govern the shape of TPCs in broad taxa (Schoolfield et al., 1981, Sharpe and DeMichele, 1977), such that the shape of this curve is conserved, being Gaussian and left-skewed (Kingsolver, 2009). Yet, TPCs are also under selection, such that their precise shape varies considerably among species and populations.
If the TPC for development rate can be accurately characterized, it can be used as a basis for a development model and applied to estimate developmental milestones under fluctuating temperature conditions (Georges et al., 2005). Indeed, development models of various types have proven very useful in agricultural and forensic sciences (Pedigo, 1996), for example in determining the suitability of different crop strains to local environments, controlling pests, and predicting the timing of flowering in plants and larval instar stages of insects (Got et al., 1997, Manel and Debouzie, 1995). Development models have also proven critical in ecology and evolution, especially in predicting sex ratios of species with temperature dependent sex determination (Georges et al., 1994, Telemeco et al., 2013).
Estimation of TPCs for development rely on accurate characterization of development at constant temperatures (Georges et al., 2005), yet characterization of development rate can be difficult in some taxa. For instance, in ectothermic vertebrates, development rate at a given temperature is often estimated by applying constant temperature throughout the entire incubation period, then measuring time-to-hatch in days (Ewert, 1985, Lang and Andrews, 1994, Niehaus et al., 2012, Shine and Harlow, 1996). This method is likely used because development rate is easy to estimate when rates can be delineated within obvious developmental milestones. However, rates estimated over the entire incubation period may be subject to bias. For example, if egg hatching is environmentally-cued (e.g., by oxygen levels or social factors), then the number of days between oviposition and hatching is not a good proxy for development rate, as embryos are not developing for the entire period they are in the egg (Webb et al., 1986). Environmentally-cued hatching is not uncommon among reptiles (Doody, 2011), amphibians (Mills and Barnhart, 1999), fish (Czerkies et al., 2001), and ectotherms in general (Warkentin and Caldwell, 2009). Alternative methods of estimating development usually rely on enlargement of a single morphological feature across ontogeny, such as head width (Beggs et al., 2000, Georges et al., 1994). Although embryonic enlargement may provide a good estimate of developmental progression under some conditions (e.g. Webb et al., 1986), it is generally recognized that development (passing through life stages) is a process that is distinct from growth (increasing in size), and these processes have different thermal sensitivities (Forster et al., 2011, van der Have and de Jong, 1996). Specifically, development is more sensitive to temperature than growth (Forster et al., 2011), which helps explain why full-term embryos are often relatively small when incubated under relatively warm conditions (Janzen and Morjan, 2002, Van Damme et al., 1992). Given that the size of the term embryo itself may depend on temperature (Janzen and Morjan, 2002, Packard et al., 1984), it follows that embryonic growth expressed as a fraction of term embryo size may be an imprecise method of estimating development rate (Georges et al., 2005). More broadly, estimates of development obtained by measuring enlargement of a morphological feature must be considered a proxy for development, as enlargement is a measure of growth and an indirect measure of development (Forster et al., 2011).
A method that is more strongly rooted in the process of development would allow rates of development to be compared unambiguously across a diversity of environments, including that created by the mother (e.g., egg size). Webb et al. (1983) developed such a method by leveraging a reference series of development stages for embryos of the crocodile Crocodylus porosus, which was quantified at 30 °C. Importantly, the reference series acts to map each distinct developmental stage onto embryonic age in days at 30 °C. The implication is that an embryo arbitrarily collect from an incubation environment of 30 °C could be aged accurately, as developmental stage corresponds to a particular embryonic age, in days, within the reference series. Next, Webb et al. (1983) assumed that temperature would affect rate of development, and introduced a correction factor that allowed a ‘30 °C age’ of embryos to be estimated at different temperatures. For example, an embryo incubated at 33 °C for a given number of days could be collected and dissected to determine developmental stage. Then developmental stage could be converted to the number of days at 30 °C necessary to reach that developmental stage, resulting in a ‘30 °C age’. Herein, we refer to this method of aging embryos with the term “Equivalent Development”, as it maps the development stage of an embryo to its equivalent age at a reference temperature.
The goal of the present study is, first, to apply the concept of Equivalent Development (Webb et al., 1983) in a new way: to create a TPC for embryonic development rate. Given that we modify the methods of Webb et al. (1983), we provide validation of the modified method by testing whether the TPC for Equivalent Development (TPCED) can accurately predict embryonic age and stage in wild nests. We find that our TPCED explains a majority of the variation in developmental age and stage in the wild, performing better than alternative development models. Second, we emphasize some strengths of the Equivalent Development concept, and outline why it may be particularly useful in the field of temperature dependent sex determination.
Section snippets
Methods
The present study is part of a long-term research program on the biology of snapping turtles, which was initiated in 1972 in the Algonquin Wildlife Research Station (AWRS) in Algonquin Provincial Park, Ontario, Canada (45°30′ N, 78°30′ W). Snapping turtles in this population nest from late May through early July, and clutch size varies between 19 and 69 eggs (Armstrong et al., 2017, Edge et al., 2017, Rollinson et al., 2012).
Estimating the thermal performance curve for Equivalent Development (TPCED)
In total, 79 embryos from the constant temperature experiment were staged in 14 experimental runs. Embryonic mortality at 38 °C was 100% (all 4 embryos sampled were dead), and 17% at 34 °C (1 of 6 dead), leaving 74 staged embryos. Dead embryos with zero development rate were not included in our analyses, such that 42 estimates of development rate across 14 set-point temperatures were ultimately collected. We note that two of the 74 embryos in our experiment appeared to experience negative
Discussion
In the present study, we refine a method developed by Webb et al. (1983) to quantify development; here we call this method “Equivalent Development”. We use the principle of Equivalent Development to help estimate a thermal performance curve (TPCED) for embryonic development rate of snapping turtles, and we provide evidence that our TPCED accurately characterizes development rate. Specifically, we demonstrated that development rate estimated at an arbitrary ED20 age (ca. ED2014.0) provides
Acknowledgements
We thank the undergraduate and graduate students who participated on this project, and four anonymous reviewers who significantly improved and clarified this work. The Algonquin Wildlife Research Station provided field support, laboratory space, and accommodations, and Locke Rowe provided additional laboratory space. This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN-2016-06469) Discovery Grants to RJB and NR. The University of Guelph Animal
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Author contributions were equal.