Drawing the line: Linear or non-linear reaction norms in response to adult acclimation on lower thermal limits
Graphical abstract
Introduction
For ectotherms, adaptation to environmental temperature is a major determining factor for species’ distributions (Angilletta, 2009). As such, thermal biology, both evolved and plastic acclimation responses of thermal limits in particular, are often used to infer adaptation and predict responses to climate variability and capacity to accommodate climate change (Deutsch et al., 2008, Gunderson and Stillman, 2015, Sunday et al., 2011). Studies investigating thermal tolerance generally find greater variation in both evolved and plastic differences at lower limits as compared to upper limits (Araujo et al., 2013, Sunday et al., 2012, Sørensen et al., 2016a). Moreover, it is typically found that cold tolerance is tightly correlated to environmental temperatures (Addo-Bediako et al., 2000, Kellermann et al., 2012, Overgaard et al., 2014, Sunday et al., 2011). Specifically, CTmin assayed by dynamic ramping assays showed a strong correlation with annual minimum temperature and latitude among a range of Drosophila species (Andersen et al., 2015).
The inferences of thermal limits clearly depend on the ecological relevance and the accuracy of the estimates. Therefore, the methods for measuring thermal limits have received a lot of attention in the literature (e.g. Jørgensen et al., 2019, Sinclair et al., 2015). Methodological effects of heat tolerance assays have been widely explored for upper thermal limits in small insects. Particularly, the choice of ramping rate and knock-down temperature, and the interpretation of the differences in results obtained has been widely discussed (Jørgensen et al., 2019, Overgaard et al., 2012, Santos et al., 2011, Terblanche et al., 2007). Increased ramping rates leads to higher CTmax while lower knock-down temperatures leads to longer knock-down times, however, underlying physiological mechanisms suggest that this could be interpreted as differences in rates of heat damage accumulation rather than true variations in upper thermal limits (Jørgensen et al., 2019).
Contrary to the attention to methodological effects on upper thermal limits, only very few studies have used multiple ramping rates for estimating lower thermal limits. These studies find disparate effects of methodology on lower thermal limits (Allen et al., 2012, Chown et al., 2009, Kelty and Lee, 1999, Terblanche et al., 2007). For example, Chown et al. (2009) found that CTmin measured as cessation of movement in D. melanogaster decreased (increased cold tolerance) with decreasing ramping rates. This result points to a beneficial acclimation effect during exposure to slowly decreasing temperatures. In contrast, the same study found the opposite effect in the ant, Linepithema humile, with slower rates of cooling leading to increasing CTmin (decreased cold tolerance). This might be interpreted as chill damage accumulation at lower ramping rates as more time is spent at sub-lethal temperatures. The generality of these results and whether the differences are affected by methodology remain unresolved. However, it is clear that methodological considerations apply for estimates of acclimation capacity. Reaction norms for acclimation responses are often assumed to be linear in ectotherms (e.g. Gunderson and Stillman, 2015, van Heerwaarden et al., 2016). For developmental acclimation in drosophilids, linear reaction norms are well documented for both upper and lower thermal limits (Schou et al. 2017) making linear reaction norms for adult acclimation a reasonable assumption. However, a recent study of adult acclimation in D. melanogaster revealed complex interactions between acclimation temperature and CTmax or time to knock-down. Specifically, decreasing ramping rates and knock-down temperatures led to increasingly non-linear acclimation responses (Salachan et al., 2019).
In addition, there is an increasing awareness on the potential discrepancy between responses to constant laboratory conditions and fluctuating thermal regimes (FTR) (Reviewed by Colinet et al., 2015). This applies for both insect management or storage, where FTRs are applied to relieve stress from the low storage temperature to increase shelf life (Enriquez et al., 2020), and for fluctuations within benign thermal conditions, which seem to induce unique acclimation potential (Salachan and Sørensen, 2017, Sørensen et al., 2016b). Studies using fluctuating temperature regimes have reported that while estimates of CTmax are most often increased by fluctuations (e.g. Bozinovic et al., 2011, Manenti et al., 2014, Overgaard et al., 2011), the response to fluctuations seen for CTmin is less pronounced (Bozinovic et al., 2016, Salachan and Sørensen, 2017, Terblanche et al., 2010) and likely dependent on the exact regime applied.
Lower thermal limits have been widely used for ecological predictions and interpretation of thermal adaptation. However, the effects of thermal regimes (constant or fluctuating) and acclimation temperatures on lower thermal limits remain understudied. Here, we investigated adult acclimation at five constant and four fluctuating temperature regimes to evaluate reaction norms for lower thermal limits. We measured CTmin at four ramping rates in order to determine how ramping rate affect the lower thermal limits. Assuming beneficial acclimation we predict that acclimation to lower temperatures will lead to progressively lower CTmin (increased cold tolerance) irrespective of temperature regime, and that fluctuating regimes will lead to a gain of low temperature tolerance equivalent to the lowest temperature reached during the fluctuation. However, the effect of fluctuations likely is influenced by the crossing of certain threshold temperatures (amplitude and mean temperature dependent) and time spent at these temperatures. For the amplitude used here (±4°C) fluctuation is expected to have a minor contribution as compared to the effect of mean temperature (Salachan and Sørensen, 2017). Given the effect of ramping rates on upper thermal limits (Salachan et al., 2019) we expect that lower ramping rates will lead to increasingly non-linear reaction norms. We expect that lower ramping rates will lead to higher CTmin estimates (decreased tolerance) if responses are driven by damage accumulation as found for the ant, Linepithema humile (Chown et al., 2009) or to lower CTmin estimates (increased tolerance) if responses are driven by rapid cold acclimation during the assays as reported for D. melanogaster (Chown et al., 2009, Kelty and Lee, 1999) irrespective of acclimation treatment. To our knowledge, this is the first study to explicitly test how ramping rates, temperature variability and acclimation temperatures interact to affect the tolerance estimate and acclimation responses of lower thermal limits.
Section snippets
Experimental animals
A population of Drosophila melanogaster collected in Odder, Denmark, in 2010 was used as experimental subject for all the experiments (Schou et al., 2014). The population was maintained at laboratory conditions, 25 °C, with a 12/12 h light/dark cycle on standard Drosophila oatmeal-sugar-yeast-agar medium. All experimental flies were raised under controlled density by transferring 40 ± 3 freshly laid eggs into 7 ml food vials. Flies from several vials were mixed (to randomize potential vial
Results
We investigated the effect of treatment, acclimation temperature, and ramping rate on the lower thermal limits of D. melanogaster. We found a significant effect of treatment (constant or fluctuating), mean acclimation temperature and their interaction (Fig. 1, Table 1). Due to the significant interaction, the effect of acclimation temperature on CTmin was analyzed for each treatment separately. Importantly, neither ramping rate nor any interaction including ramping rate contributed
Discussion
Lower thermal limits are important metrics for estimating ecologically relevant local adaptation and adaptation potential in ectotherms. Furthermore, the study of lower thermal limits and the associated physiological mechanisms have provided some mechanistic understanding of cold tolerance (Overgaard and MacMillan, 2017, Sinclair et al., 2015, Teets and Denlinger, 2013). The inferences of such studies rely on ecologically relevant methods and, often, on the assumption of a linear reaction norm
Conclusion
In this study, we explicitly tested how ramping rates, temperature variability and acclimation temperatures interact to affect the acclimation responses as measured by lower thermal limits. To our surprise ramping rates had no impact on CTmin estimates, constant temperature acclimation produced non-linear reaction norms and fluctuating thermal regimes produced linear reaction norms. These results all point to a strong interaction between methodology and an insect’s underlying physiology to
CRediT authorship contribution statement
Jesper Givskov Sørensen: Conceptualization, Methodology, Formal analysis, Visualization, Writing - original draft, Writing - review & editing, Funding acquisition. Marius Løssl Winther: Conceptualization, Investigation, Data curation, Visualization, Writing - original draft, Writing - review & editing. Paul Vinu Salachan: Conceptualization, Methodology, Writing - review & editing. Heidi Joan MacLean: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review &
Acknowledgements
We thank Trine Bech Søgaard and Annemarie Højmark for excellent help in the fly lab, and two anonymous referees for helpful comments. This work was supported by a grant from Aarhus University Research Foundation to JGS.
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