Does seasonal variation in the corticosterone response affect the nutritional ecology of a free-ranging lizard?

Abstract

1. Challenging events in the environment that are both predictable (e.g. seasonal patterns in breeding activities) and unpredictable (e.g. predator encounter) are known to induce a glucocorticoid response that facilitates metabolic requirements during the challenge.

2. Given its role in mobilizing energy, glucocorticoid levels can influence the nutritional ecology of an individual by shifting dietary intake or retention patterns, but this relationship has not been tested in free-ranging vertebrates.

3. Using a tropical lizard species (Psammophilus dorsalis) as a model system, we tested whether the elemental composition of dietary intake and excretion (faecal samples) varies with stress-induced corticosterone levels in males and females across different seasons. From free-ranging lizards in the wild, we measured levels of stress-induced corticosterone and glucose in blood and determined diet composition from gut-flushing. Elemental composition of the diet was determined by analysing the carbon and nitrogen content of identified prey Orders caught from the wild. We also collected faecal samples and estimated their elemental composition.

4. We found that stress-induced corticosterone levels varied across seasons, with the lowest levels during the breeding season for both males and females. Despite high variation in corticosterone responsiveness, lizards did not shift the elemental composition of their diets and maintained an intake carbon:nitrogen ratio of 4.56. We did, however, find a negative correlation between stress-induced corticosterone levels and faecal elemental composition, suggesting selective retention of both carbon and nitrogen in individuals that have higher corticosterone responsiveness.

5. This study highlights the interplay between corticosterone responsiveness and nutritional ecology, challenging the existing links in literature and illustrating how free-ranging animals, such as lizards, adjust the elemental composition of excretion and not dietary intakes as a potential strategy to modulate natural physiological and ecological challenges.

1. Introduction

Diet of many animals varies across space and time in ways that suggest active selection and not random choice. Food resources can vary in nutritional content, ease of acquisition, and spatial or seasonal availability, and therefore, can influence foraging strategies and diet preferences of animals (Hou et al., 2018; Kohl et al., 2015; Mamou et al., 2019; Razeng & Watson, 2015). According to optimal foraging theory, animals are expected to forage in ways that optimize their rate-of-energy-intake, thereby maximizing fitness (Pyke et al., 1977; Sih & Christensen, 2001). However, it has become increasing clear that an optimization of diet should also explicitly consider the macronutrient requirements necessary to fulfil the nutrient and energetic demands of individuals, which can vary depending on life history stage or in response to challenges (Lihoreau et al., 2015; Raubenheimer et al., 2009; Simpson & Raubenheimer, 2012, 1993).

Animals are subjected to a wide range of challenging events in the wild, involving but not limited to, reproduction, resource competition and predation risk (Cockrem, 2013; Thaker et al., 2009; Wingfield & Sapolsky, 2003). Responding to such events are typically within the reactive scope of the individual and elicits an elevation of circulating glucocorticoid hormones (Del Giudice et al., 2018; Romero et al., 2009; Sapolsky et al., 2000). Glucocorticoids act as a key metabolic regulator and is involved in mobilizing glucose to support essential energetic demands for allostasis (Bonier et al., 2009). When elevated in response to challenging events, glucocorticoids increase glucose metabolism resulting in greater energy expenditure, which is utilized by essential organs such as the brain, muscles and the heart, thereby enabling the animal to combat the challenging period (Koolhaas et al., 2011; Sapolsky et al., 2000). Although corticosterone is known to directly modulate glucose metabolism in response to energy-intensive challenges (e.g. Millanes et al., 2024), the relationship between plasma corticosterone and glucose can vary depending on other physiological and environmental factors including diet, body condition and life history stage (Deviche et al., 2016; Jimeno & Verhulst, 2023; Millanes et al., 2024; Neuman-Lee et al., 2019; Remage-Healey & Romero, 2001). Overall, challenging situations that are energy-intensive requires the reallocation of energy towards urgent survival functions while restricting non-urgent growth and reproductive functions (Crespi et al., 2013; Jimeno & Verhulst, 2023; Wingfield & Sapolsky, 2003).

Because of its role in mobilizing energy, glucocorticoids and nutrition are inherently linked (Hawlena & Schmitz, 2010b; Simpson & Raubenheimer, 2012). If corticosterone levels are chronically elevated in response to persistent or repeated disturbances, the additional energy requirements are expected to deplete available and stored carbohydrates and lipids (i.e. carbon-rich compounds) (Hawlena & Schmitz, 2010b). Depleted energy reserves need replenishment. This hormonally mediated adaptive response can therefore create the need for animals to direct foraging efforts towards carbon-rich food resources, which are high in carbohydrates or lipids (Hawlena & Schmitz, 2010b; Simpson & Raubenheimer, 2012). In laboratory rodents, studies have shown a bidirectional link between corticosterone and diet, such that, rodents with high corticosterone levels showed greater weight gains attributed to higher caloric utilization of carbon-rich diets (Moles et al., 2006; Patterson & Abizaid, 2013), and prolonged ingestion of high fat diets induce elevated corticosterone responses (McNeilly et al., 2015), Overall, to deal with the energetic requirements of challenging periods, animals can either consume more carbon-rich or retain more carbon-rich resources from existing food sources (Dalton & Flecker, 2014; Simpson & Raubenheimer, 2012).

Animals can anticipate predictable challenging events, such as breeding activity, and are known to modulate corticosterone levels at diurnal and seasonal cycles (Schwabl et al., 2016; Tokarz et al., 1998). Physiological demands in the breeding season differ from that of non-breeding seasons and are directly involved in facilitating reproductive processes (Hau et al., 2010; Tokarz et al., 1998). Studies have shown that although an increase in baseline levels of corticosterone is adaptive during the breeding phase as it facilitates the greater metabolic requirements, prolonged high levels of corticosterone during the breeding season can negatively impact reproductive success of both sexes (Romero, 2002; Romero-Diaz et al., 2019; Schoenle et al., 2021). Therefore, animals are expected to modulate their corticosterone responses to additional challenges, especially during the breeding season to attain maximum reproductive fitness in the long run (Moore et al., 1991; Romero-Diaz et al., 2019). What is unknown is whether the natural seasonal variation in corticosterone levels, when modulated for breeding activities are also concomitant with changes in the macronutrient composition of diets. For wild animals that are balancing multiple challenges while ensuring reproductive success, appropriate dietary nutritional composition is essential for gaining maximum benefits.

Here, we test whether the nutritional ecology of free-ranging lizards is correlated with differences in their stress-induced corticosterone levels (hereafter, stress-induced CORT). We use the tropical lizard species, Psammophilus dorsalis, as our model system because this species has a distinct breeding season and a relatively short life span, which means that there is high pressure to avoid incurring costs to reproduction (Deodhar & Isvaran, 2017; Radder et al., 2006). Psammophilus dorsalis is also an insectivorous ambush predator and has a broad dietary breadth (Balakrishna et al., 2016) and therefore can potentially modulate their diet to meet different energetic or nutritional requirements. We first quantified seasonal and sex-specific variation in stress-induced CORT as a measure of the reactive scope of individuals to acute challenges that would naturally occur in their environment. We predicted that both sexes will exhibit reduced stress-induced CORT in the breeding season to minimize the potential negative impacts of high corticosterone levels on reproductive processes. We then correlated stress-induced CORT to circulating glucose levels in plasma, with the prediction that glucose would be elevated along with CORT levels (as per Jimeno & Verhulst, 2023; Millanes et al., 2024; but also see Deviche et al., 2016). To determine whether stress-induced CORT and nutrition are linked, we quantified the diet composition of both sexes and across seasons and estimated the nutritional composition of prey eaten in terms of elemental carbon and nitrogen content. Although elemental carbon and nitrogen are not directly equivalent to carbohydrates and proteins (Wilder et al., 2019), elements can be good proxies for these macronutrients in arthropod Orders (Reeves et al., 2021). We predict that the diet of lizards with higher stress-induced CORT will have greater carbon content than those with lower CORT, due to a foraging bias for carbon-rich resources to meet their energetic requirements. Individuals with higher stress-induced CORT are expected to have overall higher energy expenditures. Since macronutrient requirements can also be modulated post-ingestion, we also estimated faecal elemental composition in both sexes and predicted that individuals with higher stress-induced CORT should have lower levels of carbon in the faecal waste, assuming greater retention of carbon to support higher energy expenditures overall. Our study attempts to employ a more nuanced approach towards understanding foraging decisions, testing the hypothesis that predictable metabolic challenges and nutritional ecology are coupled in the natural free-ranging animals.

2. Materials and Methods

2.1. Study species

Psammophilus dorsalis, commonly known as Indian Rock Agama, is found in semi-arid habitats of peninsular India which are characterized by rocky boulders, sheet rocks and scrub vegetation (Figure 1). This species has a short life span of 1–2 years in the wild and shows sexual size dimorphism where males are larger in size than females (Radder et al., 2006). In most parts of their distribution, they are active during the months of February to October, with the breeding season typically occurring between April to August (Deodhar & Isvaran, 2017; Radder et al., 2006).

Figure 1.

Male of Psammophilus dorsalis and one of their natural habitats in Kolar, Karnataka, India (Photo credits: Anuradha Batabyal for the lizard and Avik Banerjee for the natural habitat).

2.2. Capture and handling

Lizards (P. dorsalis) of both sexes (N = 230) were captured by lassoing from sites in and around Bengaluru, Karnataka, India in 2021. These sites include Avathi, Kuduregere and Kolar, which are a minimum of 10 km apart from each other and represent relatively undisturbed habitats with rocky outcrops and shrub vegetation. Lizards were sampled in three different seasons: (i). Pre-breeding season (late February—early March, N = 51, males = 31, females = 20), (ii) early breeding season (April, N = 60, males = 32, females = 28) and (iii) late breeding season (late July, N = 54, males = 28, females = 26). All lizards were adults and females were not visibly gravid during sampling. From these lizards, we collected stress-induced blood samples, gut-flush contents and faecal samples (detailed below).

We also measured the body size of all lizards as snout to vent length (SVL). All measurements and sample collections were obtained within 2 h of capture, except for faecal sample collection which required a subset of lizards to be brought back to the lab for a maximum of 48 h. After measurements, all lizards had a single toe clipped with a sterile dissecting scissor at their interphalangeal joint, which was then treated with an antiseptic agent betadine, before the animals were released (Ferner, 2007). Toe clipping permanently marked sampled individuals, thereby allowing us to avoid recapture of individuals across seasons.

2.3. Blood sampling and quantification

Blood samples (~50–200 μL) were collected from the retro orbital sinus of each lizard (N = 230) using heparinized microcapillary tubes, following a 30-min capture-restraint protocol (Hau et al., 2015; Sapolsky et al., 2000; Thaker et al., 2009). The 30-min stress protocol included the time spent catching the lizard in the wild. To eliminate the potential effect of prolonged chasing, any lizard that was chased for more than 5 min was not included in the study. Upon capture, lizards were constrained in cloth bags and placed in a cooler with ice packs that ensured constant temperature exposure for all lizards. To account for potential circadian patterns in glucose and corticosterone levels, sampling was done throughout the diurnal active period of the lizards at different time points, that is, 0800, 1000, 1200, 1400 and 1600 h.

Because it is difficult to capture and collect blood samples from lizards within 3–4 min (Tylan et al., 2020), we could not obtain blood samples that would allow for measurement of ‘baseline’ hormones and glucose levels. Therefore, hormone measurements reported here are the stress-induced levels, obtained from a standard 30-min capture-restraint protocol, which represent maximum corticosterone levels that the animal can mount in response to an acute stressor.

Blood glucose levels (mg/dL) were immediately measured in the field using hand-held glucometer (Bayer/Ascensia) (Grenot et al., 2000; Sparkman et al., 2018), and the rest of the blood sample was kept on ice and transported to the laboratory within 10–12 h of collection. In the laboratory, plasma was extracted from the blood samples after centrifugation and stored at −20°C until further hormone analyses. Corticosterone levels were quantified from the plasma samples using an enzyme immune-assay kit (Arbor Assays) that has been optimized for this species (Batabyal & Thaker, 2019). We used a plasma volume of 4ul with a dilution ratio of 1:100 for the assays. All samples were run in triplicate and a laboratory standard of known concentration was run in duplicate in each assay. The coefficient of variation (CV) within plates ranged between 0.17 and 13.7 (mean ± SD = 5.95 ± 1.57) and the CV across plates was 9.47 ± 1.53 SE (calculated from the laboratory standard).

2.4. Gut flushing and faecal sample collection

To determine the diet of lizards, wild-caught individuals (N = 51 pre-breeding season; N = 61 early breeding season, N = 54 late breeding season) were gut-flushed at the field sites using a standard protocol for lizards and frogs (Legler & Sullivan, 1979; Solé et al., 2005). Gut-flushing was done within 2 h of capture, following the blood sampling (Secor & Faulkner, 2002). Gut-flushed samples were stored in 50 mL vials in 70% ethanol (Balakrishna et al., 2016) and transported to laboratory for storage until further analysis.

To obtain faecal samples, a separate set of lizards (N = 60, males = 29, females = 31 from early breeding season only) were wild-caught and transported to the laboratory within 6 h of capture, where they were housed for a maximum of 48 h. Lizards sampled during the early breeding season were randomly assigned to either gut-flushing or faecal sample group. A separate set of lizards was used because gut-flushing could have significant effects on faecal volume, elimination frequency and nutrient content. During captivity, lizards were housed in individual terraria that were lined with tissue paper substratum and provided with ad-libitum water but no food. Lizards in captivity were allowed to bask under 100 W incandescent bulbs during active hours. The first faecal sample, which represents waste from wild-obtained diet, was collected and stored in 5 mL vials in −20°C until further analysis.

2.5. Dietary analysis

In the laboratory, gut-flushed samples were thoroughly inspected under magnification using a hand lens and microscope (Leica M205 C stereomicroscope, fitted with Leica MC 120HD camera and LAS Montage software). All invertebrate remains (>99% of the samples) were identified to the taxonomic level of Order. We calculated the frequency of occurrence (%) and relative abundance (%) of each prey Order, across seasons and sites (Loveridge & Macdonald, 2003). The frequency of occurrence and relative abundance was calculated as per Equations (1 and 2) as follows:

$$\text{Frequency}\text{of}\text{occurrence}\text{of}\text{prey}\text{Order}\text{in}\text{diet}\text{(Occurrence}\%)=\frac{S(100)}{n},$$ (1)

where ‘S’ is the number of diet samples having each prey Order and ‘n’ is the total number of diet samples.

$$\text{Relative}\text{abundance}\text{of}\text{prey}\text{Order}\text{in}\text{diet}(\text{Diet}\text{RA}\%)=\frac{R(100)}{N}\text{, }$$ (2)

where ‘R’ is the total number of prey items of individual Order and ‘N’ is the total number of prey items of all Orders.

Shannon’s Diversity Index (H) was used to calculate prey diversity in the diet of P. dorsalis across all seasons (and sites) (as per Equation 3), as it accounts for both the abundance and the evenness of all taxonomic groups, such that higher values of H indicate a more diverse diet (Nordberg et al., 2017),

$$\text{Shannon’s}\text{Diversity}\text{Index}(H)=-\sum _{i=1}^{s}pi(\ln (pi)),$$ (3)

where ‘S’ is the total number of taxonomic groups encountered and ‘pi’ is the proportion of the taxonomic group i compared to the total number of taxonomic groups.

2.6. Prey sampling and elemental content of diets

To estimate the elemental content of diets, we captured prey specimens from the field sites belonging to the identified prey Orders from the gut-flushed samples in April of 2022. Prey items were sampled from all the three sites, where lizards were captured from in the previous year, using sweep nets and the hand-picking method. Sweep net (15-inch diameter) with 150-micron nylon mesh were used with sweeps performed at a height of <1 m through potential foraging substrates (e.g. over shrubs along rock edges). Ground-dwelling insects (e.g. ants or beetles) were actively picked up using forceps. Sampling was done throughout the active hours of P. dorsalis between 0800 and 1600 h on multiple days across each site. Upon collection, insects were immediately transferred to an insulated container and placed inside an ice box to freeze-kill them. They were then transported to the laboratory where they were sorted to the taxonomic level of Order, counted and stored in a −20°C freezer until further analysis.

To estimate the nitrogen and carbon content of prey Orders, whole prey items belonging to each Order and from each site were dried in hot-air oven at 60°C for a minimum of 12 h. Prey items were also weighed before and after the drying process to determine their wet weight and dry weight, respectively. The loss in weight was an estimate for moisture content of prey Orders. After the drying and weighing process, specimens from each insect Order were manually crushed to a dry homogenous mixture using mortar and pestle. The homogenous mixture was packed in tin foil and analysed with a CHNS analyser (Elementar Analysensysteme GmbH, Germany) to determine their carbon (C) and nitrogen (N) content (%) (see Table S3 for results).

Across sites, the mean dry weight (MDW) of unit prey belonging to any insect Order were calculated as shown in the following Equation 4:

For any given site,

$$\begin{array}{c}\text{MDW}\text{of}\text{unit}\text{prey}\text{from}\text{Order}i\\ =\frac{\text{Total}\text{dry}\text{weight}(\text{mg})\text{of}\text{all}\text{prey}\text{of}\text{Order}i}{\text{Number}\text{of}\text{prey}\text{collected}\text{of}\text{Order}i},\end{array}$$ (4)

where i is any given site-specific prey Order.

The estimated C and N values (%) along with the calculated MDW of unit prey for each insect Order were further used to determine the elemental composition of prey belonging to that Order (on dry weight basis) as shown in the following Equation 5:

$$\begin{array}{c}\left(\text{C}\text{or}\text{N}\right)\text{mg}\text{of}\text{prey}\text{Order}i\\ =\frac{(\text{C}\text{or}\text{N})\%\text{of}\text{prey}\text{Order}i\times \text{MDW}\text{of}\text{unit}\text{prey}\text{of}\text{Order}i}{100}\end{array}$$ (5)

where i is any given site-specific prey Order.

The total elemental content of lizard’s diet across seasons were then estimated by the summation of C or N amounts in different site-specific prey Orders multiplied with the absolute prey numbers found in the diet of the lizard (Alldredge et al., 2002; Sterner & Elser, 2003; Sullivan et al., 2014) as shown in the following Equation 6:

$$(\text{C}\text{or}\text{N})\text{mg}\text{of}\text{diet}\text{for}\text{lizard}n=\sum _i(((\text{C}\text{or}\text{N})\text{mg}\text{of}\text{prey}\text{Order}i)\times (\text{Absolute}\text{prey}\text{number}\text{of}\text{Order}i\text{identified}\text{in}\text{diet}\text{of}n)),$$ (6)

where i is any given site-specific prey Order, and n is any given lizard.

The final estimated C and N content of whole diet of lizards was used to calculate the dietary CN ratio. We also estimated the relative abundance of insect Orders for the early breeding season alone as an additional check to understand if lizard diet composition could be representative of natural prey abundance, in case prey abundance was skewed in some way or the other (see Section S1 for methods).

2.7. Elemental content of faeces

We estimated the carbon and nitrogen content of faecal samples similarly as for prey Orders described above. Faecal samples were dried individually in a hot-air oven at 60°C for a minimum of 12 h and the dry weight was measured. Dry faecal samples were manually crushed to a dry homogenous mixture, packed in tin foils and analysed using a CHNS analyser to determine their carbon (C) and nitrogen (N) content (%). The dry weight of faecal samples and their estimated carbon and nitrogen content (%) was used to determine the faecal total elemental content (on dry weight basis) as shown in the equation:

$$(\text{C}\text{or}\text{N})\text{mg}\text{of}\text{faecal}\text{sample}n=\frac{(\text{C}\text{or}\text{N})\%\text{of}\text{fecal}\text{sample}n\times \text{Dry}\text{weight}\text{of}\text{fecal}\text{sample}n}{100},$$ (7)

where n is any given lizard.

The faecal CN ratio was calculated based on the estimated faecal total C and N content.

2.8. Ethical approval

All protocols employed in capturing, collection of samples and later release were approved by the Animal Ethics Committee of the Indian Institute of Science (CAF/Ethics/867/2021). Collection permits were not required since P. dorsalis is not protected under the Schedules of the Indian Wildlife (Protection) Act, 1972.

2.9. Statistical analyses

We used generalized linear mixed-effects models (GLMM) with a Gamma distribution to determine the seasonal effect on stress-induced CORT and circulating glucose levels for both sexes (using the R package lme4, Bates et al., 2015). We also used a GLMM with a Gamma distribution to determine the seasonal effect on dietary CN estimates for both sexes. In both models, season and sex were used as an interaction effect. To determine the differences between sexes in faecal CN, we ran a similar GLMM analysis as described above, but season was not included in the model since faecal samples were only collected during the early breeding season. Also, since the same value for dietary CN or faecal CN could result from different combinations of carbon and nitrogen estimates from those samples, we also ran separate models for dietary and faecal total carbon and total nitrogen estimates. All models for dietary total C or N and faecal total C or N were corrected for body size differences by dividing by SVL of lizards. We used ‘time of capture’ and ‘site’ as random effects in all models to account for any potential circadian effect or site-specific differences. Post-hoc pairwise comparisons were done when relevant using emmeans (Lenth et al., 2019) with Tukey’s HSD corrections. In addition, we ran Pearson’s product–moment correlation tests to determine the correlations between stress-induced CORT, glucose levels, dietary CN and faecal CN estimates. Finally, to determine the differences in dietary composition across seasons and sexes, we ran the one-way analysis of similarity (ANOSIM) using the Bray-Curtis distance metric. All global tests of ANOSIM were run at 9999 permutations. ANOSIM was performed using the Vegan package in R software (Dixon, 2003). All statistical analyses were done using R, version 4.4.0. (for detailed model outputs, see Tables S5–S7).

3. Results

3.1. Variation in stress-induced corticosterone and glucose levels

We found a significant interaction between season and sex on stress-induced CORT in lizards (χ² = 30.614, p < 0.01). For males, stress-induced CORT levels in the pre-breeding season were significantly higher than levels in the early breeding season (Z = 4.437, p < 0.001) and late breeding season (Z = 5.583, p < 0.001). CORT levels did not differ significantly between early and late breeding seasons for males (Z = 1.795, p = 0.2; Figure 2). In contrast, for females, CORT levels in the pre-breeding season did not differ from the early breeding season (Z = −0.704, p = 0.8). But, CORT levels in the late breeding season were significantly lower than both the pre-breeding (Z = 6.72, p < 0.001) and early breeding seasons (Z = 8.767, p < 0.001; Figure 2).

Figure 2.

Variation in stress-induced corticosterone levels across body size (snout-vent length) in females (green) and males (orange) of Psammophilus dorsalis during the pre-breeding, early breeding and late breeding seasons.

As expected from a sexually dimorphic species, sex and SVL were strongly correlated (r_pb = 0.72, p < 0.01). For both males and females, we found that SVL was negatively correlated with stress-induced CORT (Pearson’s product–moment correlation—males: r = −0.43, p < 0.001; females: r = −0.60, p < 0.001) such that larger lizards showed lower CORT levels. ‘Time of capture’ and ‘site’ was found to have a negligible effect on CORT levels (GLMM random effect: SD < 0.5) (for detailed results, see Tables S5–S7).

Circulating glucose levels of lizards ranged widely across seasons for both sexes and did not correlate with stress-induced CORT levels (t = 1.540, df = 228, p = 0.1). There was no significant interaction effect of season and sex on circulating glucose (χ² = 0.535, p = 0.8) (see Figure S1). Also, glucose values did not vary across seasons (χ² = 0.947, p = 0.9) or sexes (χ² = 6.582, p = 0.1). ‘Time of day’ and ‘site’ had a negligible effect on glucose levels (GLMM random effect: SD < 0.5).

3.2. Diet composition

A total of 166 lizards were gut-flushed across the pre-breeding (N = 51), early breeding (N = 61) and late breeding (N = 54) seasons. We found only eight lizards (three males and five females) with an empty stomach. Identification of prey items from the remaining samples revealed that most of the prey (>99%) belonged to eight insect Orders. Prey items belonging to the Formicidae Family (or ants) of Hymenoptera Order showed 90%–100% frequency of occurrence in diet and had the highest relative abundance of 55% to 85% across seasons and sites. Other notable insect Orders in the diet were other Hymenoptera (non Formicidae, wasps/bees), Lepidoptera (butterflies/moths), Hemiptera (true bugs), Coleoptera (beetles), Isoptera (termites), Diptera (flies), Orthoptera (locusts/grasshoppers) and Neuroptera (antlions), with relative abundances varying between 1% and 35% in diets across different seasons and sites (see Table S1).

We also identified some other rare prey Orders in the diet, such as, Order Araneae (spiders, <3% frequency of occurrence), Order Dermaptera (earwigs, <2%), Order Odonata (dragonflies, <1%), Order Decapoda (crabs, only in 1 sample) and Order Scorpiones (scorpions, only in 1 sample). These Orders were not included for the interpretation of the diet composition because these were rare occurrences in diet. We also found some plant parts, such as leaf fragments or flower petals, small pebbles and shed lizard skin remains in some of the diet samples. These were also not included in the analysis of diet because they contributed to <3% (frequency of occurrence) to the gut-flushed contents.

ANOSIM results revealed that sexes did not differ in their diet composition (R = 0.01, p = 0.2), but composition did vary across seasons (R = 0.07, p < 0.01). Since one prey taxonomic group, Order Hymenoptera: Family Formicidae (or ants), made up ~75% (relative abundance) of this species’ diet, the ANOSIM R statistic was very low (R = 0.07) (i.e. even distribution of higher and lower ranks within and between the groups). Overall, irrespective of sexes, there were seasonal differences in diet composition of P. dorsalis (for further details on diet composition across seasons and sites, see Table S1; Figure S2). This is also supported by the Shannon’s Diversity Index that increases from pre-breeding to early breeding to late breeding season (Figure 3).

Figure 3.

Composition of diet of Psammophilus dorsalis from pre-breeding, early breeding and late breeding seasons. Absolute prey numbers are shown on the x-axis. All major insect prey Orders identified in the diet are shown in the y-axis which includes Order Hymenoptera: Family Formicidae (Hym:For), Order Hymenoptera excluding ants (Hym), Order Coleoptera (Col), Order Lepidoptera (Lep), Order Hemiptera (Hem), Order Isoptera (Iso), Order Diptera (Dip), Order Orthoptera (Ort) and Order Neuroptera (Neu). Each facet grid also has the Shannon’s Diversity Index value (H) at the bottom-right corner.

3.3. Elemental composition of identified prey Orders

Insect prey Orders showed an average moisture content of 58.8 ± 1.6% (mean ± SE) on a wet matter basis across all sites (see Table S2), similar to Razeng and Watson (2015). Nitrogen and carbon estimates of the insect Orders (see Table S3) was comparable to total nitrogen and carbon estimates reported in Reeves et al. (2021) (for detailed results, see Table S3).

3.4. Dietary carbon and nitrogen estimates

Dietary total (by weight) carbon estimates were not significantly affected by an interaction between season and sex (χ² = 2.348, p = 0.3). However, sex had a significant main effect (χ² = 8.557, p = 0.03). When corrected for SVL, females showed a higher total carbon intake than males (Figure 4a,c). Similarly, dietary total nitrogen estimates were not significantly affected by an interaction effect of season and sex (χ² = 2.599, p = 0.3), but sex had a significant main effect (χ² = 8.664, p = 0.03). When corrected for SVL, females showed a higher total nitrogen intake than males (Figure 4b,c). ‘Time of capture’ and ‘site’ had a negligible effect on dietary total carbon and nitrogen estimates (GLMM random effect: SD < 0.5).

Figure 4.

Variation in total dietary (a) carbon and (b) nitrogen content for females (green) and males (orange) during the pre-breeding, early breeding and late breeding seasons. Carbon and nitrogen estimates were controlled for the snout to vent length of lizards and is represented as weight (mg) of whole diet dry mass. (c) Nutrient state-space showing composition, that is carbon and nitrogen content (mean ± SE) of all female (green) and male (orange) lizard diets across all three seasons. The grey dotted line represents a nutrient rail that connects the CN ratios of intakes.

Across all seasons, the mean dietary intakes for females ranged between 4.4-9.25 mg for carbon and 0.95–2.13 mg for nitrogen. This was greater than the mean intakes for males that ranged between 2.47-6.36 mg for carbon and 0.53–1.41 mg for nitrogen (Figure 4c). The resulting dietary CN ratio across seasons for both sexes was similar, with a mean (±SE) of 4.57 ± 0.07 for females and 4.55 ± 0.06 for males. Overall, the ratio of C to N in males and females (corrected for their differences in SVL) was within the available elemental compositional CN range of the identified prey Orders for all the three sites (Figure 5).

Figure 5.

Elemental composition for all identified insect Orders from lizard diets, represented as percent (%) carbon and nitrogen per unit dry mass. Mean elemental content of diets of females (black solid circle) and males (black solid triangle) across all seasons are shown as %C and %N per unit of body size. (Note that the C and N content for females and males overlap completely.) The grey dotted line represents a nutrient rail that connects CN ratios of intakes with CN ratios of insects.

3.5. Relationship between dietary elemental composition and stress-induced corticosterone and glucose

Contrary to our expectation, dietary total carbon estimates did not correlate with either stress-induced CORT (t = 1.505, p = 0.1) or circulating glucose levels (t = −1.641, p = 0.1) across seasons. Similarly, dietary total nitrogen estimates also did not correlate with stress-induced CORT (t = 1.509, p = 0.1) or circulating glucose levels (t = −1.334, p = 0.2) across seasons. However, the CN ratio of lizard diets was negatively correlated only with circulating glucose levels (r = −0.2, p = 0.01, Figure 6a), but not with stress-induced CORT (t = −0.3, p = 0.8, Figure 6b).

Figure 6.

Relationship between dietary CN estimates and stress-induced (a) glucose levels, and (b) corticosterone levels in Psammophilus dorsalis of both sexes from pre-breeding (green), early breeding (orange) and late breeding (blue) seasons.

3.6. Faecal carbon and nitrogen estimates from early breeding season

Faecal samples collected from the early breeding season revealed that sexes significantly differed in the amounts of faecal carbon (χ² = 4.317, p = 0.04, Figure 7a) and faecal nitrogen (χ² = 6.252, p = 0.01, Figure 7b). Specifically, after controlling for differences in SVL, females excreted lower carbon and nitrogen amounts in their faeces than males (Figure 7). ‘Time of capture’ and ‘site’ had a negligible effect on faecal carbon and nitrogen estimates (GLMM random effect: SD < 0.5).

Figure 7.

Variation in total faecal (a) carbon content, (b) nitrogen content and (c) CN ratio in females (green) and males (orange) of Psammophilus dorsalis. Relationship between stress-induced corticosterone level and total faecal (d) carbon content, (e) nitrogen content and (f) CN ratio, in female (green) and male (orange) of P. dorsalis. Carbon or nitrogen estimates are controlled for snout to vent length of lizards and are represented as weight (mg) of whole faecal dry mass.

We also found that stress-induced CORT was negatively correlated with the amounts of both faecal carbon (r = −0.3, p = 0.03, Figure 7d) and faecal nitrogen (r = −0.3, p = 0.04, Figure 7e). Glucose levels were uncorrelated with both faecal total carbon (t = −0.72, p = 0.5) and faecal total nitrogen (t = −0.84, p = 0.4).

Sex differences in total carbon and nitrogen estimates in faecal matter did not hold (χ² = 0.023, p = 0.9) when we calculated the ratio of carbon to nitrogen (i.e. faecal CN). Faecal CN ratio of lizards also did not correlate with either stress-induced CORT (t = −0.45, p = 0.7, Figure 7f) or circulating glucose levels (t = −0.177, p = 0.9).

Considering only the early breeding season for which both dietary and faecal data were collected, we found that after controlling for body size (SVL), female lizards ingested and excreted a greater proportion of nitrogen and carbon (by percent) than males (see Figure 8).

Figure 8.

Variation in carbon and nitrogen (%) ingested (solid squares) and excreted (solid triangles) for females (green) and males (orange) of Psammophilus dorsalis for the early breeding season. Carbon or nitrogen estimates are controlled for snout to vent length of lizards and are represented as % of diet ingested and faecal excreted. (Note that the ingested and excreted values shown here are from separate group of lizards.)

4. Discussion

Animals face predictable seasonal and sex-specific challenges governed by their life history stage (Schoenle et al., 2021; Tokarz et al., 1998). These challenges are energy intensive and need to be mitigated by behavioural and physiological adaptations that help to maintain allostasis and maximize survival and reproductive success (Bonier et al., 2009; Jimeno & Verhulst, 2023). During the breeding season, animals are expected to prioritize reproductive activities and therefore modulate glucocorticoid responses to avoid their potential negative effects in the long term (Moore & Mason, 2001; Romero-Diaz et al., 2019). Changes in CORT levels could further inflict new physiological demands and can lead to changes in dietary or faecal macronutrient composition (Hawlena & Schmitz, 2010b). In this study, we measured the seasonal and sex-specific variation in the elemental composition of diets and faeces in wild lizards and tested whether elemental intakes and faecal eliminations are correlated with the stress-induced CORT levels of individuals. For the tropical insectivorous lizard P. dorsalis, stress-induced CORT, measured as circulating CORT after handling stress, was significantly lower during the breeding season compared to pre-breeding season. Reduced CORT responsiveness does not necessarily imply low stress (MacDougall-Shackleton et al., 2019; Vitousek et al., 2019), but CORT levels could influence metabolic processes (Jimeno & Verhulst, 2023). We found that differences in CORT responsiveness across individuals and seasons did not correlate with the elemental composition of diet. Interestingly, stress-induced CORT was, however, negatively correlated with carbon and nitrogen estimates of faeces. These results challenge the existing link between glucocorticoid hormone levels and nutrition in the wild, illustrating how animals can maintain elemental intake ratios despite variation in physiological and ecological conditions.

When we tracked natural variation in CORT responsiveness in free ranging lizards across different stages of the breeding season, we found that stress-induced CORT was lowest during the late breeding season for both sexes (Figure 2). This pattern is in line with predictions from other studies in lizards and birds (Klukowski, 2011; Romero, 2002; Selman et al., 2012), as reduced stress-induced CORT during the breeding season could be an adaptive physiological strategy to avoid their negative effects on reproductive success (Romero, 2002; Schoenle et al., 2021). Males of many lizards and birds experience an increase in testosterone production during breeding season, which promotes mating activities, competition or aggressive behaviours (Adkins-Regan, 2013; John-Alder et al., 2009). Similarly, in females of lizards, there is a surge in β-estradiol and progesterone during the breeding phase, which directly facilitates reproductive processes involving ovulation or egg-shell formation (Radder et al., 2001). Elevated CORT levels can potentially disrupt these reproductive functions in males (Romero, 2002) and females (Liu et al., 2020; Saino et al., 2005). We also find that males of P. dorsalis reduce CORT responsiveness for a longer part of the breeding season than females, suggesting that the androgen-facilitated breeding-related activities of males, which involves territory defence, courtship and spermatogenesis, are required for a longer period. For female lizards, CORT responsiveness was constrained only in the late breeding season when they were expected to be developing eggs. Circulating CORT in females of several avian and reptile species have been known to transfer to developing eggs, which when elevated, can lead to decreased overall reproductive output, reduced offspring hatching size, and altered offspring physiology and behaviour (Ensminger et al., 2018; Liu et al., 2020; Saino et al., 2005). Thus, reducing the levels of CORT during critical breeding periods for females would be adaptive.

Similar to patterns seen in passerine birds (Hau et al., 2010), we found a negative correlation between the size of lizards (SVL) and stress-induced CORT in both sexes (Figure 2), with smaller sized lizards having higher CORT responses than larger lizards (DuRant et al., 2010; Ott et al., 2000). This negative correlation reflects the developmental role of glucocorticoids, as smaller sized individuals have higher mass-specific metabolic rates (Hau et al., 2010). This pattern is also supported by a recent meta-analysis that finds CORT levels to be tightly linked with metabolic rates (Jimeno & Verhulst, 2023). Contrary to our initial expectations, stress-induced CORT did not seem to correlate with glucose levels in circulation. Other studies in birds and reptiles report a wide range of relationships between glucose and CORT, ranging from a positive correlation to no correlation (see Flower et al., 2015; Millanes et al., 2024; Neuman-Lee et al., 2019). For P. dorsalis in the wild, variation in blood glucose levels between individuals could be indicative of variation in recent foraging input and the short residence time of glucose in circulation (Rinehart & Hawlena, 2020), which is also supported by the fact that lizards with higher CN ratios in their diet had lower circulating glucose levels (Figure 6a). The relationship between CORT and glucose levels is also dependent on the timing of sampling. The elevated CORT levels in response to a 30 min acute handling stress may not have resulted in increased expenditures that would be reflected in the glucose levels at that time (Walsberg, 2003).

Insectivorous lizards are thought to be non-selective in their diets, eating whatever is available or accessible (Rodríguez et al., 2008). Variation in diets have been commonly attributed to ecological variation in prey abundance, depending on what prey are available (Rodríguez et al., 2008) or morphological constraints of the forager, bounded by what can fit within the jaws (Lopez-Darias et al., 2015). Morphological constraints from sexual dimorphism of head and body size are likely why males and females of many species show differences in their diet (Lopez-Darias et al., 2015; Tucker et al., 1995; Verwaijen et al., 2002). Alternatively, seasonal or spatial variation in diet could be due to predation pressure leading to avoidance of risky foraging patches (Des Roches et al., 2022; Lehtiniemi, 2005), or inter- or intra-specific competition leading to resource partitioning and dietary niche separation (Linnebjerg et al., 2013; Tucker et al., 1995). Given these, we expected seasonal and sex differences in diets of P. dorsalis. Diet analyses reinforce existing evidence that P. dorsalis is insectivorous and predominantly myrmecophagous, with no sex-specific differences in dietary composition (see also Balakrishna et al., 2016). Despite the dominance of ants in the diet, we found seasonal differences in diet. Diversity of diet composition was highest in the late breeding season compared to the pre-breeding season (Figure 3). Many reasons could explain this variation. One could be active selection or preference for a diverse diet during the late breeding season, which could increase micronutrient ingestion that would facilitate the maintenance of carotenoid-based sexual coloration (seen in P. dorsalis) or the development of eggs (Dierenfeld et al., 2002; García-de Blas et al., 2015). Diet composition could also vary due to heterogeneity in food resource abundance across habitats (Morey et al., 2007; Rodríguez et al., 2008), which is quite likely in our study since sampling included multiple seasons and sites.

Although the link between stress (mediated by corticosterone) and nutrition is, in principle, clear, the direction of change in macronutrient or elemental stoichiometry of diets in stressed animals shows mixed results across studies (Rinehart & Hawlena, 2020). Most studies that test the effects of stress responses on macronutrient intake expose prey to increased predation risk, which results in heightened metabolic expenditure that can lead to a change in diet preference towards higher carbohydrates (Hawlena & Schmitz, 2010a; Rinehart & Hawlena, 2020). In our study, we measured the natural seasonal variation in corticosterone responsiveness, mediated for breeding activities, and explored their correlation with dietary and faecal elemental compositions. Breeding activities are energy intensive for both sexes and these metabolic challenges require increased energetic investment (Del Giudice et al., 2018; Jimeno & Verhulst, 2023), which would result in a similar shift to carbon-rich diets for both sexes. Alternatively, males and females can differ in diet because of sex-specific differences in macronutrient requirements (Reddiex et al., 2013). For example, female crickets require protein-rich food sources for egg production, but males prefer carbohydrate-rich food sources for energetically demanding breeding behaviours such as courtship (Maklakov et al., 2008). In our study, we found that female lizards ingested greater amounts of both nitrogen and carbon than males across all seasons, after we controlled for differences in body size between the sexes. Females could be selecting for greater nitrogen (or proteins) for its direct use in egg production (Casagrande et al., 2018; Maklakov et al., 2008), which is likely given that females lay multiple large clutches (2–3 clutches with 8–12 eggs) during the breeding season that typically lasts for a short period, that is 4–5 months (Deodhar & Isvaran, 2017). Females are also smaller than males and so higher carbon consumption by females may be needed to meet the higher metabolic needs for the smaller sized vertebrate (Nagy, 2005). Contrary to our expectation, elemental composition of diets in P. dorsalis did not differ across the seasons or correlate with CORT responsiveness. The best explanation for our results would be the fact that most of the species’ diet is made up of ants (~75% relative abundance). Thus, the bulk of the nutritional intake in terms of carbon and nitrogen is dominated by one prey type. From our estimate from one season of sampling, ants were also the most abundant insect group in the wild and thus were the most available prey Order for these ambush predators (see Table S4).

Animals can meet their energetic needs during physiologically challenging periods either by actively ingesting carbon-rich food resources in the wild or by selectively retaining nutrients post ingestion (Simpson & Raubenheimer, 2012). Hence, it is possible that P. dorsalis lizards ingest similar amounts of macronutrients but are retaining different amounts based on their physiological nutritional requirements. This is somewhat evident from the fact that in the early breeding season, dietary intake levels of lizards were similar regardless of stress-induced CORT levels, but the stress-induced CORT was negatively correlated with both faecal carbon and nitrogen estimates. Within the early breeding season, when stress-induced CORT levels varied a lot between individuals, we find that lizards with higher CORT levels eliminated lower amounts of carbon and nitrogen. The above finding implies that lizards with higher stress-induced CORT were retaining more of these elements, via macronutrients (Hawlena & Schmitz, 2010a; Reeves et al., 2021). The lizards in our study were in the middle of their breeding phase when we measured their faecal elemental levels. Therefore, they should be experiencing a metabolic challenge for the nutrients required for developing eggs (in females) and seminal fluid (in males), as well as for energetic investments in territory defence (both sexes) (Deodhar & Isvaran, 2017; Maklakov et al., 2008; Reddiex et al., 2013). Selective retention of nutritional elements seems to be a possible mechanism to meet macronutrient demands. Since all the above reproductive activities require additional nutrients beyond growth and maintenance, it is difficult to determine what specific outcome is utilizing the increased retention. What is clear is that additional studies that measure both ingested and egested elemental and macronutrient ratios in wild free ranging animals are needed to better understand nutrient regulation.

Despite the wealth of information about dietary variation in animals, there is a lack of studies that link seasonal or sex-specific physiological differences with dietary intake and faecal elimination differences in the wild, especially from a nutritional point of view. Our study addresses this gap in literature by examining the interplay between physiological challenges, mediated by glucocorticoids, and nutritional ecology in a wild lizard species. We found that stress-induced CORT levels in lizards and the insect composition in lizard diets varied across seasons, and yet the overall elemental ratio of diets were not different across seasons and were not associated with the physiological state of the animals. In fact, the elemental intake in P. dorsalis remained fairly consistent at 4.56 CN ratio for both sexes, despite variation in physiological and ecological conditions. It is worth noting, however, that arthropods are made up of both digestible and undigestible components (Wilder et al., 2019). Hence, our estimates of dietary carbon and nitrogen of lizards, which include both digestible and indigestible components, represents the potential upper limits of dietary elemental composition. Our results suggest no link between natural variation in CORT responsiveness and elemental intake in wild lizards, which challenges the predictions of the general stress paradigm (Hawlena & Schmitz, 2010b). However, our results on faecal elemental compositions posits the possibility that lizards are modulating macronutrient needs post-ingestion with selective retention of elements. Also, our study makes it clear that more studies of elemental or macronutrient intake on free-ranging vertebrates are needed to better understand their optimal diet choice and the constraints that mediate foraging decisions.

Data availability Statement

All data presented in this manuscript are available from the Dryad Digital Repository https://doi.org/10.5061/dryad.k98sf7mgb (Banerjee & Thaker, 2025).