Assessing the upper thermal limit constraining the physiological performance of Callinectes sapidus embryogenesis under climate warming scenarios

63 Assessing the upper thermal limit constraining the physiological performance of Callinectes sapidus embryogenesis under climate warming scenarios Ángela Rodríguez-Ruiz1,2 , Gustavo F. de Carvalho-Souza1, Inma Herrera3, Ignacio González-Gordillo2, Enrique González-Ortegón1 1 Institute of Marine Sciences of Andalusia, Spanish National Research Council (ICMAN-CSIC), Campus Universitario Río San Pedro, 11519, Puerto Real, Spain 2 Department of Biology, Marine Research Institute (INMAR), University of Cadiz, Puerto Real Campus, Puerto Real, Spain 3 Grupo de Investigación en Biodiversidad y Conservación (BIOCON), Instituto Universitario ECOAQUA, Universidad de Las Palmas de Gran Canaria (ULPGC), Telde, Spain Corresponding authors: Ángela Rodríguez-Ruiz (angela.r[email protected]); Enrique González-Ortegón (e.gonzalez.or[email protected]) Copyright: © Ángela Rodríguez-Ruiz et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Embryonic development represents a vulnerable life stage in marine organisms, yet its role in shaping the invasion success of non-native species under climate change remains understudied. In this study, we assessed the upper thermal sensitivity of embryogenesis of the blue crab Callinectes sapidus, a globally invasive species, by quantifying their physiological responses across a temperature gradient relevant to projected climate warming scenarios. Using Electron Transport System (ETS) activity as a proxy for aerobic metabolism, we evaluated respiration, egg size, hatching time, and larval morphology in brooding eggs incubated at 22 °C, 24 °C, 26 °C, and 28 °C. Elevated temperatures induced increased ETS activity, indicating heightened metabolic stress, and were associated with reduced egg size and earlier hatching of malformed, non-viable larvae. Within the Oxygenand Capacity-Limited Thermal Tolerance (OCLTT) framework, we identified a physiological pejus range (24–26 °C) beyond which embryonic performance declined. These results suggest that moderate warming may accelerate development and facilitate invasion, but extreme temperatures constrain aerobic capacity and compromise larval viability. Our results highlight embryogenesis as a potential bottleneck for blue crab recruitment under future warming, with implications for predicting the invasive potential of marine species. Key words: Callinectes sapidus, ecophysiology, ETS, heatwaves, larval ecology, maternal effects, thermal stress Introduction Phenological windows have been suggested as a part of a warning system enabling more targeted programs for monitoring invasive species (Giménez et al. 2020). Warming coastal waters have likely contributed to the recruitment and northward expansion of the invasive blue crab Callinectes sapidus Rathbun, 1896, beyond its historical range at Cape Cod, resulting in the establishment of permanent populations in new areas (Johnson 2015; Taylor et al. 2022; Crane et al. 2024). The American blue crab C. sapidus (Brachyura, Portunidae) is native to the Atlantic Academic editor: Paula Chainho Received: 31 January 2025 Accepted: 12 June 2025 Published: 7 October 2025 Citation: Rodríguez-Ruiz Á, de Carvalho-Souza GF, Herrera I, González-Gordillo I, González-Ortegón E (2025) Assessing the upper thermal limit constraining the physiological performance of Callinectes sapidus embryogenesis under climate warming scenarios. In: Anastácio P, Ribeiro F, Chainho P (Eds) Invasions in Aquatic Systems. NeoBiota 102: 63–79. https://doi.org/10.3897/ neobiota.102.148122 NeoBiota 102: 63–79 (2025) DOI: 10.3897/neobiota.102.148122 Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 64 NeoBiota 102: 63–79 (2025), DOI: 10.3897/neobiota.102.148122 Ángela Rodríguez-Ruiz et al.: Temperature effects on Callinectes sapidus early development coast, ranging from Nova Scotia, Canada, to northern Argentina, including the Gulf of Mexico (Squires 1990). Outside its original distribution, the species has established invasive populations in various regions of Africa, Asia and Europe (Nehring 2011). In the Iberian Peninsula, the first record of the species occurred in the Tagus Estuary in 1978 (Gaudencio and Guerra 1979). Since then, it has rapidly spread along the Spanish coasts, reaching the Guadalquivir Estuary in the Gulf of Cadiz by around 2017 (González-Ortegón et al. 2020). The primary vector for the introduction of C. sapidus is thought to be ballast water releases, which can transport planktonic larvae during uptake (Nehring 2011). Considering that larval development in C. sapidus lasts 37–69 days, the timeframe allows for plausible transoceanic transport, although secondary dispersal is also possible (Nehring 2011). The species’ successful establishment can be attributed to several ecophysiological and developmental traits, such as the larvae’s high resistance to abiotic and biotic conditions (Anger 2006; Morais et al. 2019), enabling adaptation to a wide range of environmental factors. The larval ecology of C. sapidus is of particular interest, as adult population recruitment depends on the survival of larvae and juveniles to replenish the parental stock (Sandifer 1975), acting like a population bottleneck. Larval survival is a vulnerable life stage heavily influenced by environmental factors, particularly seawater temperature and salinity (Costlow and Bookhout 1959; Costlow 1967; Rosenberg and Costlow 1976; Rumrill 1990; Anger 2006). While numerous studies have been conducted on the biology and ecology of C. sapidus in its native range, most research has focused on its commercial importance and the environmental conditions in its original habitats (Olmi and Orth 1995; Daly et al. 2021). However, there is a notable lack of studies addressing the effects of environmental changes in invaded regions such as the Gulf of Cadiz. The Gulf of Cadiz is a temperate and warm ecosystem enriched by river discharges that inject nutrients and trace metals which stimulates primary and secondary production (Prieto et al. 2009; González-Ortegón et al. 2019), thus creating a potentially suitable environment for the development of invasive species like C. sapidus. The Gulf of Cadiz connected to the Mediterranean Sea through the Strait of Gibraltar, facilitates a two-layer water exchange: a surface inflow from the Eastern North Atlantic and deeper outflow of Mediterranean saline waters (Sánchez-Leal et al. 2017). This hydrodynamic regime, along with significant anthropogenic transformation along the coast, including major ports like Algeciras and extensive aquaculture infrastructure (González-Ortegón and Moreno-Andrés 2021), may have facilitated the westward expansion of the invasive Atlantic blue crab Callinectes sapidus from the Mediterranean into the Atlantic. Genetic evidence supports a single invasion event into the Mediterranean followed by secondary spread into adjacent Atlantic waters (González-Ortegón et al. 2022). The potentially less extreme upper thermal limits in the Atlantic region may offer more favourable conditions for the species’ establishment and survival. This is consistent with the invasive success of C. sapidus, which generally exhibits wider thermal tolerance and may possess adaptive advantages over native species such as Carcinus maenas (Anger 2006; Nehring 2011). Laboratory experiments suggested that C. sapidus requires high temperatures (above 21 °C) for optimal larval development (Hill et al. 1989; Bembe et al. 2017). Combined with the warm sea temperatures of the Gulf of Cadiz, this raises the question of how these conditions will affect its development and expansion in the 65 NeoBiota 102: 63–79 (2025), DOI: 10.3897/neobiota.102.148122 Ángela Rodríguez-Ruiz et al.: Temperature effects on Callinectes sapidus early development region. While most studies focus on estimates of critical thermal maxima (CTmax) or lethal limits (LT50) when exposed to thermal stress, insights on the intermediate physiological constraints are more relevant in ecological studies, especially in organisms with critical life stages as crustaceans. Exploring the animal Oxygenand CapacityLimited Thermal Tolerance (OCLTT) through their physiological performance, could provide valuable information about their climate responses in a more realistic scenario of gradual rising temperatures (Pörtner et al. 2017). In this sense, the objective of the present study is to assess the upper thermal limit constraining the embryogenesis performance of C. sapidus, as the global warming context in the Gulf of Cadiz could act as a bottleneck to population recruitment. Performance is explored through interlinked parameters characterising embryonic thermal sensitivity and aerobic window, as the Electron Transport System (ETS) activity, egg size development, hatching success, and larval morphology. ETS activity assays, a potential indicator of respiration, is a common proxy for planktonic respiration (Packard 1971; Herrera et al. 2017), and has proven effective for estimating metabolic rates in crustaceans (Simčič and Brancelj 2004; Simčič et al. 2014; Ruiz-Delgado et al. 2019; Herrera et al. 2024). Exploring ETS activity on brooding eggs of C. sapidus exposed to different temperatures could provide insights into variations on the metabolic dynamics during embryonic development under warming conditions. Egg size dynamics over incubation time may be an indicator of development rates, which coupled with hatching time and larval morphology, provides valuable information about larval fitness and survival success under warming conditions. Methods Egg collection and experimental design Eggs masses of Callinectes sapidus were reared in the laboratory to assess the effects of increased seawater temperature on embryonic development prior to larval hatching. Eggs were obtained from six ovigerous females (referred as F1, F2, F3, F4, F5 and F6) at similar stages of embryonic development. These ovigerous females were manually collected by a local fisherman in a shallow inlet of the Guadalquivir Estuary (Sanlucar de Barrameda, Gulf of Cadiz, Spain) during August 2023. Genetic analysis was performed to determine the haplotype of each female, in order to explore whether the existence of possible outliers in the statistical analysis could be attributed to genetic differences between them. Two different haplotypes of C. sapidus coexist in the studied area: CSWM1, predominant in Spanish Atlantic coast; and CSWM2, predominant in Spanish Mediterranean coast (González-Ortegón et al. 2022). In our study, F1, F2, F3, F5 and F6 belonged to CSWM1 haplotype, while F4 belonged to CSWM2. Each egg mass was removed from the female abdomen and its volume was evenly divided among four temperature treatments in individual containers (1000 ml): 22 °C, 24 °C (defined as control), 26 °C, and 28 °C. These temperatures were selected based on the (i) available bibliography which settle the lower thermal limit for the successful embryonic development (> 21 °C) (Jivoff et al. 2007; Bembe et al. 2017), (ii) the mean summer water temperature at the sampling site of ovigerous females when brooding and spawning season occur (24 °C), and (iii) the IPCC RCP8.5 projected warming scenarios of +2 °C and 66 NeoBiota 102: 63–79 (2025), DOI: 10.3897/neobiota.102.148122 Ángela Rodríguez-Ruiz et al.: Temperature effects on Callinectes sapidus early development +4 °C for the end-of-century (Pörtner et al. 2019). One group was kept at a controlled temperature, while the others were gradually exposed to different experimental temperatures (22 °C, 26 °C, and 28 °C). After 24 hours at 24 °C (T0), they were gradually transferred to 22 °C and 26 °C (T1) over a 24-hour period, while another group, previously at 26 °C, was transferred to 28 °C one day later over a 24-hour period (T4), allowing the eggs time to adapt to the three new temperatures (Fig. 1). Filtered seawater (35 of salinity) was renewed daily, to prevent fungal, bacteria and other microorganism’s growth in the egg mass. Embryonic development During the experiment, a sample of eggs per treatment and female was collected for embryonic developmental analysis, in order to test the possible effects of temperature on egg size over time. Samples were stored in 2 mL Eppendorf tubes containing seawater at -20 °C. For each sample, the major diameters of twenty randomly selected eggs were measured under a stereo microscope (SMZ25/18, Nikon Instruments Inc.) using the NIS-elements Imaging Software v. 5.21.00. Thus, the median egg size (µm) and standard deviation were calculated. Images were captured at a scale of 250 µm. Electron Transport System (ETS) assay Egg samples (~15 mg) from each treatment and female were collected daily to measure respiratory ETS activity (in µL O2 · h−1 · mg prot−1), reflecting cellular-level changes during embryonic development. Samples were preserved in Eppendorf tubes, frozen in liquid nitrogen (-196 °C), and stored at -80 °C prior to analysis. The ETS assay followed the method of Packard (1971), modified by Owens and King (1975), and adapted for microplate readings by Ruiz-Delgado et al. (2019). Eggs were homogenized in 0.5 mL of ice-cold homogenizing buffer solution (20 mM Trizma base, pH 7.8, Sigma-Aldrich) using an ultrasonic homogenizer (UP2005 Hielscher) set to 1 cycle at 25% amplitude for 60 seconds. The homogenate was centrifuged at 3 °C for 10 minutes at 5,000 rpm (Eppendorf Centrifuge 5417R), and the supernatant was used for the ETS assay. In a microplate assay, 60 µL of supernatant (in duplicate) was incubated with 180 µL of substrate solution (0.1 M phosphate buffer, pH 8.5, containing NADPH 30 mM and NADH 1.76 mM, Sigma-Aldrich) or without substrate (control, containing only 180 µL of phosphate buffer, pH 8.5). Then, 60 µL of INT solution (0.2%, 4 mM, pH 8.5) was added to each sample, and absorbance was measured at 490 nm over 8 minutes using a microplate reader (BioTek Synergy H1) and Gen5 3.10 software. ETS activity was corrected for in situ temperature using an activation energy of 15 kcal·mol−1 (Packard 1971) and the Arrhenius equation to calculate in situ ETS activity (units: µL O2 · h−1). To calculate ETS activity per unit biomass, protein biomass (mg protein) was determined using the bicinchoninic acid (BCA) method described by Smith et al. (1985). For this, 25 µL of each sample and standards were incubated with 200 µL of BCA working solution for 30 minutes at 37 °C, and absorbance was read kinetically at 562 nm. ETS activity was then normalized to protein content and expressed as ETS activity per unit protein (µL O2 · h−1 · mg prot−1). 67 NeoBiota 102: 63–79 (2025), DOI: 10.3897/neobiota.102.148122 Ángela Rodríguez-Ruiz et al.: Temperature effects on Callinectes sapidus early development Egg hatching time and larval viability Egg mass cultures were maintained under different temperature treatments until larval hatching, which marked the end of the experiment for each sample condition. Hatching time was recorded as the number of days from day 0 (the start of incubation) until larvae appeared. The approximate number of newly hatched larvae was counted for each treatment and female. Observations on the morphological condition and motility of the larvae were documented to assess their viability. This included noting the presence or absence of aberrant zoea, and evaluating their phototactic swimming behavior, according to Jivoff et al. (2007). In this study, aberrant zoea is defined as an individual at a morphological stage similar to prezoea but that is immobile, exhibits sinking behaviour, and subsequently dies. In contrast, under optimal conditions, prezoea molts into the first zoeal stage within the first 3 minutes of life after their release (Costlow and Bookhout 1959; Davis 1965). Statistical analysis The relationship between predictors with the ETS activity (µL O2 · h-1 · mg prot-1) and egg size (µm) of C. sapidus embryos was examined through a Generalized Additive Model (GAM) with lognormal and normal distribution, respectively, based on the distribution of the dependent variables (Zuur et al. 2009). The GAM model was selected as it assumes no functional form between dependent and independent variables and describes both linear and non-linear effects, and was performed on RStudio software v.2024.12.0. A variance inflation factor (VIF) with a threshold of 3 was used to identify possible collinearity between predictors in the data set, before fitting models to the data (Zuur et al. 2010). This analysis indicated no multicollinearity as all explanatory variables had VIF < 3 (see Suppl. material 1: tables S1, S2), so the general form of the GAM was: g(y) ~ α + f1(xi) + εt + εf Figure 1. Experimental design. Schematic diagram of the temperature treatment design and acclimation protocol applied to C. sapidus embryos across the experimental period (T0-T10). Dashed line indicates control at 24 °C. 68 NeoBiota 102: 63–79 (2025), DOI: 10.3897/neobiota.102.148122 Ángela Rodríguez-Ruiz et al.: Temperature effects on Callinectes sapidus early development where g() is the link function, y is the response variable (ETS activity or egg size), f1 is the smooth function for the xi continuous explanatory variable (incubation time), εt is the categorical effect of the temperature treatments, and εf is the random effect of female origin of embryos, capturing its intrinsic variability. Also, due to the unbalance representation of haplotypes, this factor was not included in the GAM analysis. Estimated R2 and explained deviance were used to evaluate the predictive performance of the model. The residuals were graphically evaluated with QQ-plots, histograms, and plots of residual response against fitted values to explore any patterns in the residual errors (see Suppl. material 1: figs S2, S3). Post-hoc analysis using the Tukey test for varying family sizes was performed for identifying significant differences between specific levels of the categorical factor affecting embryo ETS activity and egg size. Results Temperature-dependent variation in egg size A general pattern of decreasing egg size over development time was observed across all temperature treatments (see Suppl. material 1: fig. S1). Under control conditions (24 °C), among all females, eggs at the very early stage of embryonic development had a mean diameter of 273.4 ± 13.8 µm, which decreased to 241.5 ± 12.8 µm just before hatching, a reduction of approximately 11.7%. The global median egg size resulted in 247.59 µm. Smaller egg sizes, below 245 µm, were found under warmer conditions of 26 °C and 28 °C; while larger eggs, above 245 µm, resulted in colder temperatures of 22 °C and 24 °C, reaching the maximum at control conditions of 24 °C (306.40 µm) (Table 1). These results suggest that elevated temperatures negatively affect embryonic development by reducing egg size. GAM analysis showed significant effect of incubation time, temperature treatment, and maternal origin on egg size (see Suppl. material 1: table S1). The model explained a moderate portion of the variability in egg size (R2 = 0.282; deviance explained = 28.7%), with all predictors showing significant effects (p < 0.001). The effect of incubation time on egg size was non-linear and highly significant (edf = 7.37, F = 70.02, p < 0.001). The smooth function in Fig. 2A indicated a sharp decline in egg size during the early stages of embryo development, followed by a period of stabilization and a secondary decline around T8. Confidence intervals around the smooth term suggested a precise estimation of this temporal pattern, with wider intervals in later stages, where data density is lower due to different larvae hatching times. The random effect of maternal origin was significant as well (edf = 4.78, F = 20.71, p < 0.001), indicating that individual females contributed to variability in egg size, justifying its inclusion as a random smoother in the model. Although the genetic background of females could be a factor contributing to the variability in egg size, the presence of outliers was not restricted to a single female (F4 belonged to CSWM2 haplotype), but rather distributed across several individuals. Temperature treatments also significantly affected egg size (F = 39.51, p < 0.001). Compared to the reference level (22 °C), egg size decreased significantly at higher temperatures of 26 °C and 28 °C (estimates of -6.77 µm and -4.17 µm, respectively; p < 0.0001 for both cases), and to a lesser extent at 24 °C (Estimate = -1.73 µm; p = 0.016). The Tukey-adjusted post hoc comparisons confirmed that all pairwise differences were significant, except between 22 °C and 24 °C temperature groups 69 NeoBiota 102: 63–79 (2025), DOI: 10.3897/neobiota.102.148122 Ángela Rodríguez-Ruiz et al.: Temperature effects on Callinectes sapidus early development (see Suppl. material 1: table S1). These results indicated that small increments in temperature beyond the optimal range can influence egg development. The violin plot in Fig. 2B supported these findings, providing a visual summary of both central tendency and data distribution. The boxplots nested within the violins highlight the central tendency of decreasing median egg size with increasing temperature, as well as increasing outlier prevalence at 26 °C and 28 °C, suggesting a stress-related developmental constraint. Electron Transport System (ETS) activity assay Embryos ETS activity, a proxy for potential respiration rates and expressed as log-transformed specific ETS activity, was significantly influenced by incubation time, temperature treatment, and maternal origin (see Suppl. material 1: table S2). The GAM explained 61.4% of the deviance in ETS activity (R2 = 0.584), indicating strong explanatory power of those selected variables and reinforcing the biological relevance of thermal and maternal influences on embryonic metabolic performance. The smooth effect of incubation time was significant (edf = 2.80, F = 33.14, p < 0.001), indicating a non-linear increase in ETS activity throughout the incubation period (Fig. 3A). The fitted smooth term suggested a gradual acceleration of ETS activities over time, with a pronounced increase around T4 (4th day), when Table 1. Egg size (µm) summary by temperature treatment. Median egg size (µm), standard deviation (SD), and range (maximum and minimum values) per temperature treatment. Temperature (°C) Median ± SD (µm) Maximum (µm) Minimum (µm) 22 248.22 ± 13.21 296.06 215.03 24 250.55 ± 17.34 306.40 211.15 26 244.49 ± 12.64 291.90 218.21 28 244.84 ± 13.24 301.26 220.66 Figure 2. Graphical representation of the effects of the incubation time and temperature on C. sapidus egg size (µm). Results of the GAM analysis showing A. Partial effect of incubation time (during 10 days) on egg size, where the black solid line indicates the modelled relationship, and the grey band denotes the 95% confidence interval about the estimated relationship, and B. Violin plot showing the distribution of egg size under tested temperature treatments (22 °C, 24 °C, 26 °C, and 28 °C), where boxplots embedded within the violins indicate the interquartile ranges and central tendency. 70 NeoBiota 102: 63–79 (2025), DOI: 10.3897/neobiota.102.148122 Ángela Rodríguez-Ruiz et al.: Temperature effects on Callinectes sapidus early development the experimental increase in temperature to 28 °C was performed. This point of inflection indicates a metabolic shift, followed by continued increasing of ETS and widening confidence intervals toward the later stages of development. The effect of maternal origin was also highly significant (edf = 4.75, F = 17.31, p < 0.001), underlining individual female variability in the baseline metabolic activity during embryogenesis. Although the genetic background of females could be a factor contributing to the variability in ETS activity, the presence of outliers was not restricted to a single female (F4), but rather distributed across several individuals. Temperature treatments strongly affected ETS activity of embryos. Compared to the reference level of 22 °C, ETS activities were significantly higher at major temperature regimes of 24 °C (Estimate = 0.39, p < 0.001), 26 °C (Estimate = 0.82, p < 0.0001), and 28 °C (Estimate = 0.79, p < 0.0001). Post hoc Tukey-adjusted comparisons confirmed significant pairwise differences between 22 °C and all other treatments, as well as between 24 °C and both 26 °C and 28 °C (see Suppl. material 1: table S2). ETS activity seemed to become constant at the two highest temperatures, with no significant difference between 26 °C and 28 °C (p = 0.998), suggesting a threshold effect where ETS activity ceases to increase despite additional warming. The Fig. 3B corroborated this pattern with wider distributions and higher medians under warmer conditions, consistent with thermally enhanced metabolic rates. The shape of the violins indicates relatively symmetric distributions, contrasting with the right-skew observed in egg size. Hatching time A general pattern of early larval hatching at higher temperatures was observed (Table 2), which was also confirmed by the photographic analysis of the morphological characteristics on egg development samples (Fig. 4). In all females, early hatching occurred between 6–7 days at the highest temperatures (26–28 °C), while hatching at lower temperatures (22–24 °C) was delayed, taking up to a maximum of 11 days. Figure 3. Graphical representation of the effects of the incubation time and temperature on ETS activity (µL O2 · h−1 · mg prot−1) of C. sapidus embryos. Results of the GAM analysis showing A. Partial effect of incubation time (during 10 days) on ETS activity, where the black solid line indicates the modelled relationship, and the grey band denotes the 95% confidence interval about the estimated relationship, and B. Violin plot showing the distribution of ETS activity under tested temperature treatments (22 °C, 24 °C, 26 °C, and 28 °C), where boxplots embedded within the violins indicate the interquartile ranges and central tendency. 71 NeoBiota 102: 63–79 (2025), DOI: 10.3897/neobiota.102.148122 Ángela Rodríguez-Ruiz et al.: Temperature effects on Callinectes sapidus early development In general, lower temperatures resulted in a higher number of hatched larvae, although there were differences among females. Only a small number of larvae from females F2 and F3 hatched at lower temperatures (22–24 °C), but these larvae exhibited active and phototactic swimming behaviour, similar to those from other females under the same temperature treatments. In contrast, at 26–28 °C, early hatching often produced > 50 larvae initially, but many were aberrant or non-viable. Thus, thermal stress likely induced premature hatching, leading to malformations in larvae morphology (Fig. 5). Table 2. Hatching time. Eggs hatching day per female and temperature treatment. The plus symbol in brackets indicates samplings where massive larvae hatching occurred (> 50 larvae hatched of total egg volume, similar across all temperature treatments and females). The asterisk symbol indicates the presence of aberrant larvae. Female Temperature (°C) 22 24 26 28 18 (+) 7 (+) 6* 6* 27 (+) 6 6* 6* 37 6 6* 6* 411 (+) 10 (+) 7* 0 511 (+) 10 (+) 7 (+) 7 (+)* 69 (+) 8 (+) 6* 7 (+)* Figure 4. Comparative morphological analysis of eggs’ development between temperature treatments. 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ICES Journal of Marine Science 57: 438–451. https://doi. org/10.1006/jmsc.1999.0521 Zuur AF, Ieno EN, Walker N, Saveliev AA, Smith GM (2009) Smart Society: A Sociological Perspective on Smart Living Mixed Effects Models and Extensions in Ecology with R. Springer, 579 pp. https://doi.org/10.4324/9780429201271-2 Zuur AF, Ieno EN, Elphick CS (2010) A protocol for data exploration to avoid common statistical problems. Methods in Ecology and Evolution 1: 3–14. https://doi.org/10.1111/j.2041210x.2009.00001.x Supplementary material 1 Figures and tables of the statistical analysis for both dependent variables (ETS activity on embryos and egg size) Authors: Ángela Rodríguez-Ruiz, Gustavo F. de Carvalho-Souza, Inma Herrera, Ignacio González-Gordillo, Enrique González-Ortegón Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.148122.suppl1