Limited thermal plasticity may constrain ecosystem function in a basally heat tolerant tropical telecoprid dung beetle, Allogymnopleurus thalassinus (Klug, 1855)

Tropical organisms are more vulnerable to climate change and associated heat stress as they live close to their upper thermal limits (UTLs). UTLs do not only vary little across tropical species according to the basal versus plasticity ‘trade-off’ theory but may also be further constrained by low genetic variation. We tested this hypothesis, and its effects on ecosystem function using a diurnally active dung rolling beetle (telecoprid), Allogymnopleurus thalassinus (Klug, 1855) that inhabits arid environments. Specifically, (i) we tested basal heat tolerance (critical thermal maxima [CTmax] and heat knockdown time [HKDT]), and (ii) ecological functioning (dung removal) efficiency following dynamic chronic acclimation temperatures of variable high (VT-H) (28–45 °C) and variable low (VT-L) (28–16 °C). Results showed that A. thalassinus had extremely high basal heat tolerance (> 50 °C CTmax and high HKDT). Effects of acclimation were significant for heat tolerance, significantly increasing and reducing CTmax values for variable temperature high and variable temperature low respectively. Similarly, effects of acclimation on HKDT were significant, with variable temperature high significantly increasing HKDT, while variable temperature low reduced HKDT. Effects of acclimation on ecological traits showed that beetles acclimated to variable high temperatures were ecologically more efficient in their ecosystem function (dung removal) compared to those acclimated at variable low temperatures. Allogymnopleurus thalassinus nevertheless, had low acclimation response ratios, signifying limited scope for complete plasticity for UTLs tested here. This result supports the ‘trade-off’ theory, and that observed limited plasticity may unlikely buffer A. thalassinus against effects of climate change, and by extension, albeit with caveats to other tropical ecological service providing insect species. This work provides insights on the survival mechanisms of tropical species against heat and provides a framework for the conservation of these natural capital species that inhabit arid environments under rapidly changing environmental climate.

, Gunderson and Stillman 28 and van Heerwaarden and Kellermann 40 , we hypothesise that (i) A. thalassinus may have high basal heat tolerance as a native tropical day active forager and that (ii) if present, this high basal heat tolerance is likely traded-off with phenotypic plasticity (see also 28,67,68 ). Here we used ecologically relevant physiological-(CT max and heat knockdown time [HKDT]) and ecological-traits as proxies for performance under climate change 46,69 using dynamic protocols 64 . The results will provide a profound understanding of species-specific physiological acclimation capacity and associated ecological implications for this ecologically important species. This information is essential to predict the fate of ecosystem function under climate change 70 and provide a framework for the conservation of such species in order to preserve their benefits to the environment and society in the future.

Results
Basal critical thermal maxima and heat knockdown time. The CT max of A. thalassinus was higher than 50 °C for all treatments and controls ( Fig. 1A; Table 1). The CT max of non-acclimated A. thalassinus beetles was 51.32 ± 0.68 °C and 51.73 ± 1.81 °C, at ramping rate of 0.25° and 0.5 °C/min respectively. These CT max values indicate extreme heat tolerance and are comparable with other related diurnal beetle species in the same tribe (Table 1). Similarly, basal HKDT was long, recording 130.79 ± 41.17 and 55.05 ± 2.12 min at 53 and 55 °C respectively. These HKDT values are also longer that related diurnal species in the same tribe ( Table 1).
Effect of acclimation on heat tolerance. CT max was significantly affected by both acclimation treatments (p < 0.001) and ramping rate (p < 0.001) although combined interactions of acclimation and ramping rate were not statistically significant (p = 0.924) ( Table 2). Variable high temperature acclimation (VT-H) significantly improved CT max compared to control (p < 0.001) at all the two ramping rates while variable low temperature (VT-L) on the other hand significantly reduced CT max (p = 0.0274) compared to the controls (Fig. 1A). Across all acclimation treatments, the 0.5 °C/min ramping rate resulted in significantly higher CT max values than the 0.25 °C/min one (Fig. 1A). HKDT was significantly affected by both acclimation treatments and knockdown temperature (p < 0.05), with combined interactions of both acclimation treatments and knockdown temperature also being statistically significant (p < 0.05) ( Table 3). The HKDT for control adult beetles was 130.79 ± 41.17 and 55.05 ± 2.11 min for knockdown temperature of 53 and 55 °C respectively. HKDT was consistently significantly higher across all treatments for the 53 °C than 55 °C heat knockdown temperature (Table 3). Variable temperature high and low acclimation treatments improved HKDT for the 55 °C heat knockdown temperature but not 53 °C. Therefore, there were no treatment effects for HKDT at the 53 °C heat knockdown temperature. Interactions between acclimation treatment × heat knockdown temperature showed that, at 53 °C the HKDT for both acclimation treatments (VT-H & VT-L) were not statistically different from the control group, while at 55 °C, both variable temperature high and low acclimation treatments significantly increased HKDT (Fig. 1B).
Acclimation response ratio (ARR) of CT max . The acclimation response ratio (ARR) of CT max at benign ramping rate (0.25 °C/min) for A. thalassinus following variable temperature high and variable temperature low acclimation treatments was 0.0284 °C/°C and − 0.02556 °C/°C respectively, indicating ~ 2.84% compensation capacity and ~ − 2.56% fitness cost respectively. However, when the CT max ramping rate was increased to 0.5 °C/min, the CT max ARR for variable temperature high acclimation treatment increased to ~ 0.06 °C/°C, (i.e. 6% compensation capacity) while it decreased for variable temperature low (− 0.026 °C/°C) (-2.6% fitness cost). Compared to results of ARR values of other insects' taxa at a chosen ramping rate of 0.25 °C/min, the CT max ARR values for A. thalassinus was low except compared to Scarabaeus zambezianus and Copris elephenor (Table5).

Discussion
Our results showed that the native, arid environment inhabiting dung beetle A. thalassinus has high basal heat tolerance as exhibited by CT max values > 50 °C and long HKDT values (> 2 h), consistent with other arid/desert habitat insect species that exhibit these striking basal heat tolerance traits 18,40,43,78,79 . Second, variable temperature high acclimation improved CT max for all ramping rates, while variable temperature low acclimation generally reduced heat tolerance (CT max ) This may show plastic responses to CT max at high temperature acclimation. Similarly, heat knockdown temperature influenced HKDT, suggesting that the magnitude of temperature stress may affect insect fitness in a warming climate (see 40 ). Similar, to CT max , effects of acclimation on HKDT were significant, with variable temperature high and low temperature acclimations both increasing HKDT than controls at 55 °C while no acclimation treatment effects were recorded for 53 °C. The improved CT max at higher ramping rate is in agreement with the notion that thermal stress and injury is highly additive 19 . Thirdly, high temperature acclimation increased net ecological function through significantly higher dung removal and wider ball diameters than low temperature acclimation. Fourth, a qualitative comparison of ARRs to like tropical insect species showed that phenotypic plasticity of heat tolerance in A. thalassinus adults was low, indicating that plasticity is constrained 28,37 . When A. thalassinus ARR was compared to the general mean ARR for most terrestrial arthropods (0.12-0.16) 28 its ARR was ~ 5 times lower, indicating considerably constrained high temperature plasticity compared to other insect species. Thus, this work supports the trade-off theory 28,29,40,67 , and that selection for high basal heat tolerance in A. thalassinus may come at a cost of plasticity. This therefore affirms the notion that although having high basal heat tolerance than temperate species, tropical species may be more vulnerable to www.nature.com/scientificreports/ future global warming because they lack complete plastic responses to heat stress, i.e. phenotypic plasticity is ecologically insufficient ( 29 , but see 36 ). Acclimation effects were significantly positive on ecosystem function only following high temperature acclimation and would likely plateau. We therefore summarise that although plasticity is present in A. thalassinus, it may be insufficient to buffer this species against projected increase in in global warming, moreso in its arid, tropical native habitat of southern Africa. Thus, A. thalassinus' field fitness and efficiency in contributing to ecological functions in the future may also be constrained under increasing heat stress with climate change.
Critical thermal maximum is a popular ecologically relevant index of heat tolerance measurement 18 that is used by many ecologists to measure the capacity of organisms to survive extreme heat and thus as proxy for estimating climate change risks in many species 40,69,72,75 . Our results showed that regardless of acclimation and ramping rates, the basal CT max for A. thalassinus was high (> 50 °C), indicating that A. thalassinus is thermophilic (survives extreme temperatures of > 41 °C) 17 . This could be attributed to its tropical origins 57,59 and day foraging activities 61,71 in tropical environments where environmental temperatures are normally high during daytime. Similarly, in seed bugs, Käfer et al. 79 showed high correlation between CT max with environmental annual mean temperature and mean maximum temperature of warmest months in Austria. In separate studies, the desert ant, Cataglyphis bombycin had an extremely high heat tolerance (CT max = 53.6 °C) (reviewed in 17,80 ), partly attributable to its high habitat temperature (desert) environment. Similar reports on dung beetles Gymnopleurus Table 1. The basal CT max (°C) and HKDT (minutes) for Allogymnopleurus thalassinus compared to other diurnal telecoprid species in the same tribe occurring in the same hot and arid environment.    72 , using dynamic acclimation protocols for nocturnally active dung beetle Scarabaeus zambezianus. This high basal HKDT (and CT max ) in A. thalassinus could have evolved as an adaptation to daytime activity (foraging during peak heat stress) in stressful arid environments 71,79,81 where it is native 57 . Although diurnal species are highly heat tolerant physiologically, telecoprids were observed to particularly employ behavioural plasticity mechanisms such as utilising microhabitats (see details in 35,36 ) or using the moist dung balls as thermal refuge (heat sinks) to cool their bodies during rolling 82 . Although it is not clear under what environmental temperature this thermal respite behaviour would be initiated, in the midday foraging desert ant, Ocymyrmex robustior, thermal respite behaviour was shown to increase when soil temperatures reach 51 °C, coinciding with A. thalassius CT max . Thus, extreme basal heat tolerance for A. thalassius reported, here may be attributable to its tropical origins 57 and the latitudinal hypothesis 66,79 and may form part of its main survival strategy against warming climates.
Our results showed that variable temperature high acclimation improved CT max , while variable temperature low acclimation generally reduced CT max . This result suggests plastic responses for this species for CT max following dynamic high temperature acclimation. These results are consistent with results from other studies which showed that heat acclimation improves CT max , while on the contrary, low temperature acclimation may not improve heat tolerance 17 . Similar to what was observed in other studies, CT max values increased with increase in ramping rate, while slower ramping rates reduced this trait potentially owing to cumulative stress effects 17,19 . The effects of ramping rates, test temperatures and duration of acclimation are reportedly complex to disentangle 17,46 . Indeed, CT max varies with methodological context e.g., starting temperature and ramping rates 83,84 . Thus, the higher plasticity (CT max ) and survival consequences for higher ramping rates (for CT max ) may be attributable to the effect of reduced timing (and stress thereof) at faster heating rates (0.5 °C/min) relative to slower one (0.25 °C/min). This is in keeping with recent models that assume heat stress and consequent injury is a function of temperature severity and that it is additive 19 .
Acclimation to high temperature often improves low temperature traits and vice versa owing to shared physiological response mechanisms e.g., Hsps 85 . However, phenotypic plasticity may also be maladaptive and tradedoff with other life history traits 86 . In related studies, Kristensen et al. 87 showed that acclimation to low temperature negatively affected heat tolerance in Drosophila melanogaster. This suggests a possible trade-off between heat and cold tolerance, as such, represents an additional constraint for this species when facing changing environments (i.e. acute high and low temperature events) in nature. This also affirms the notion that the relationship between heat and cold shock responses is highly asymmetrical e.g., heat acclimation 'always' improves low temperature survival while the reverse is not always true (see discussions in 17 ). Similarly, in Nezara viridula, CT max showed more plastic responses post heat acclimation than CT min , showing that CTLs may be typically decoupled 88 . In wolf spiders, acclimation also did not modify thermal breadth showing that low thermal plasticity, as reported for A. thalassinus here, may not cushion these species from high temperature stress 89 . Thus, the role of short-to medium-term plasticity in the adaptation to variable climatic environments remains largely contested 30 .
One of the more intriguing aspects of our data is the implications of variable temperature acclimation on A. thalassius functional responses (dung removal efficiency). High temperature acclimated adult beetles made significantly bigger balls and removed a significantly higher proportion of dung compared to both control and low temperature acclimated beetles. In our view, this translates to relatively higher ecological functions at high Table 5. The critical thermal maxima (CT max ) acclimation response ratios (ARR) for A. thalassinus compared to other different Orders and their specific species obtained from literature. The list may not be purely exhaustive but represents a significant number of studies found in literature at the time of publication. Also note that CT max values and consequently ARRs may vary depending on CT max methodological context e.g. starting temperature 17 . All the acclamatory response ratio values were calculated only for CT max at ramping rate of 0.25 °C/min. www.nature.com/scientificreports/ temperature acclimation compared to the control and low temperature acclimated beetles. In a similar study, Mamantov and Sheldon 25 showed that Onthophagus taurus increased ball size and depth of dung burial following high temperature acclimation, signifying that ecological responses were linked to temperature acclimation. During acclimation, higher temperatures increase metabolic enzyme activity 90 that likely plateaus at peak (yet unknown) temperature or duration of exposure.
Our results also showed that low temperature acclimation had negative effects on dung beetle ecosystem function, manifesting as (significantly smaller dung balls, and lower dung mass removal). Acclimation responses to low temperature may be highly species dependant 61 ; as such, we speculate, with caveats that A. thalassius may not be adapted to low but high temperature stress owing to its warm tropical origin and diurnal activity patterns (see details in 61 ). In a similar study, Wu and Sun 24 , showed that a 2.3 °C increase in temperature delayed oviposition maturity and egg hatching by 4.1 and 7.2 days respectively and egg and larval size by 22.1 and 33.4% respectively in Aphodius erractus. This signifies that high temperature acclimation affects beetle life history fitness traits. The current study only tested within-generation adult acclimation responses; thus, future studies should aim to investigate the effects of temperature variability across generations and testing more diverse life history traits. In addition, we could not account for the cost of mounting plasticity and the role of behavioural adaptation (the Borget Effect) 25,28,40,74 . Thus, future work may need to test acclimation across ontogeny and investigate the role of carry-over and/or transgenerational plasticity in A. thalassius. To avoid competition, dung rollers are also known to abandon their dung balls if they cannot migrate far enough from the dung pat source. As such, future experiments may consider increasing experimental arena sizes, considering mesocosm-or field-approaches to better explain the effects of temperature variability on ecological services. Similarly, future studies may also incorporate dung beetles from diverse locations to better understand the role of local adaptation in buffering climate change effects.
Although critical thermal limits (e.g., CT max ) have received considerable attention under climate change, the fate of ecosystem functioning under climate change have been limited (but see 22,61,91 ). Our work thus, provide novel data on how a diurnally active tropical dung beetles species may be adapted to the predicted global warming and how the ecological service delivery of this species may be affected by heat stress under climate change. This is evidence to argue that future models for species survival under climate change should account for potential losses in ecosystem function and/services. Our results showed that (i) A. thalassius has extreme basal heat tolerance that presumably helps the species forage diurnally in heat stressing tropical environments; (ii) acclimation to variable high temperature improves A. thalassius heat tolerance indicating thermal plasticity (albeit limited), while simultaneously improving ecosystem function (dung removal), (iii) low temperature acclimation constrained A. thalassius ecosystem function (dung removal), and (iv) A. thalassius has low plasticity as exhibited by the low ARRs, as such phenotypic plasticity may unlikely cushion physiological fitness and survival of this species and indeed ecosystem functioning in the face of heat stress associated with climate change. Our study thus contributes empirical evidence to literature supporting the phenotypic plasticity versus basal tolerance 'trade-off ' theory and contributes to the growing recognition of the need to make practical decisions for ecosystem management to enable continued provision of ecological functions under a range of future human-mediated environmental conditions in sub-Saharan Africa and similar environments.

Materials and methods
Study animals. Study beetles were collected from Khumaga Village (S20.46801; E24. 51491; 918 m.a.s.l), Central District, Botswana, in February 2020. The summer season represents the peak activity time of most dung beetles. Khumaga village is characterised by small scale pastoralism (mainly cattle and goats) and is at the interface with a protected area (Wildlife Park), Makgadikgadi Pans National Park, that hosts several wild large ruminants, and non-ruminants 92 . This rich animal diversity provides diverse and overlapping dung resources that promote abundant and diverse beetle communities. The beetles were captured using pit fall traps consisting of mini-plastic buckets (~ 2 L) buried flush with the ground and covered with fine wire mesh of 15 mm internal diameter (modified from 71,72,93,94 ). About 350 g of fresh cattle dung was placed on top of the wire mesh as bait. The traps were covered with overhead shading to protect from rain and direct sunlight 94 . Traps were set at 06:00 h every morning and captured beetles were collected from about 1000 h till 1800 h for 6 consecutive days. Collected beetles were placed in insulated cooler boxes with perforated lids containing moist soil and dung for feeding during transportation to the Eco-physiology Laboratory, Department of Biological Sciences and Biotechnology, Botswana International University of Science and Technology in Botswana. In the laboratory, beetles were identified using gross morphology 95 and Voucher specimens were deposited at the Botswana National Museum. Beetles were kept in a climate chamber set at conditions like those at site of collection (28 ± 1 °C, 65 ± 10% RH, 14L:10D photoperiod) (see 72 ) prior to experimentation. All acclimations and/or experiments were done within 7 days (as in 96 ) of specimen collection to minimise confounding effects of laboratory captivity.
Thermal variability acclimation treatments. Beetles were acclimated using a combination dynamic (fluctuating temperature) protocol in climate chambers (HPP 260, Memmert GmbH + Co.KG, Germany) at 65 ± 10% relative humidity (RH) under 14L:10D photoperiod. This dynamic acclimation protocol is ecologically sound and may give more reliable estimates of species responses to variability typical of environmental climate change 97,98 . For variable high temperature acclimation (VT-H), temperature was ramped up at 0.5 °C/min from a benign (optimum) temperature of 28-45 °C, allowed to remain at 45 °C for a duration of 2 h before being ramped down back to 28 °C, remain constant for 2 h (at 28 °C) before ramping up again in continuous repeated dailycycles (Fig. 3). Similarly, for variable low temperature acclimation (VT-L), temperature was ramped down at 0.5 °C/min from a benign of 28 °C (ambient) to 16 °C, and then held at 16 °C (2 h) before being ramped up back to 28 °C, held there for 2 h before ramping back down in repeated daily cycles (see Fig. 3). Acclimation at Scientific Reports | (2021) 11:22192 | https://doi.org/10.1038/s41598-021-01478-x www.nature.com/scientificreports/ both temperature extremes may improve both high and low temperature tolerance 85,99 owing to potential overlap in heat and cold stress resistance mechanisms. Control beetles were maintained at a constant 28 °C. Relative humidity and photoperiod were maintained at 65 ± 10% (RH) and 14D:10L respectively for all treatments (see also 72 ).

Physiological assays.
To investigate the effects of plasticity on physiological fitness, we measured physiological functional traits vis CT max and HKDT in adults following standardised protocols from 72 . Critical thermal maximum is a good indicator for an organism's ability to survive extreme events, and as such a good measure of resistance mechanism under extreme heat exposures 100 . A series of insulated double-jacketed chambers ('organ pipes') was connected to a programmable water bath (Lauda Eco Gold, Lauda DR.R. Wobser GMBH and Co. KG, Germany) filled with 1:1 water:propylene glycol to allow for sub-zero temperatures at the same time regulating the flow of liquid around the chambers. Ten mixed sex adult beetles were counted and individually placed randomly into the organ pipes. In the organ pipes, beetles were allowed to first equilibrate for 10 min at 28 °C (benign temperature), before ramping waterbath temperature up at a benign rate of 0.25 °C/min (see 69 ) until the CT max for each beetle was recorded. Thermal ramping rates may affect adaptive capacity for UTLs 64,69 . Thus, the process was repeated using a faster ramping rate of 0.5 °C/min with a fresh set of beetles. A thermocouple (type K 36 SWG) connected to a digital thermometer (53/54IIB, Fluke Cooperation, USA) was inserted into a control chamber to measure beetle temperature. The body temperature of each individual beetle was assumed to be in equilibrium with the organ pipe temperature as in similar work (see 72 ). Each beetle was discarded after recording and for each ramping rate, the process was repeated three times with fresh beetles each time to yield sample size of n = 30 (30 replications) for each treatment. In this study, CT max was defined as the temperature at which each individual beetle lost coordinated muscle function, consequently losing the ability to respond to mild stimuli like prodding with thermally inert camel-hair brush (e.g. 72,101 ). For HKDT, ten mixed sex beetles were individually placed in numbered 30 ml polypropylene vials and placed in a climate chamber set at 53 ± 0.5 °C (65 ± 10% RH) connected to a camera (HD Covert Network Camera, DS-2CD6412FWD-20, Hikvision Digital Technology Co., Ltd, China) linked to a computer from where observations were recorded (in minutes). The process was repeated at 55 ± 0.5 °C (65 ± 10% RH) and each experiment was run three times with fresh beetles each time to yield a sample size of n = 30 (30 replications) for each acclimation treatment and each HKDT temperature. The HKTs were selected following both preliminary assays and previous studies 71 . Beetles were discarded after each recording. HKT was defined as the time (in minutes) at which each individual beetle lost coordinated activity due to acute heat stress (see 72,102 ). All treatments and replicates were all randomised across the different experimental blocks. www.nature.com/scientificreports/ Ecological functions. To investigate ecological effects of acclimation, we assessed the effects of treatments on two essential ecosystem functions, dung ball size and dung removal. Ball sizes were measured for each of the three treatments following modifications of methods by 103,104 . Fifty mixed sex beetles from each treatment were provided with 500 g of manually homogenised fresh cattle dung 54 in plastic containers of 4.09 L total volume, with effective soil depth of 6 cm. The experiment was replicated 3 times for each of the treatments (VT-H, VT-L and controls). Experimental containers were placed in a climate chamber (HPP 260, Memmert GmbH + Co.KG, Germany) set at 28 °C and 65 ± 10% RH under 14L:10D. After 24 h, 50 completely formed balls (see 103 ) were randomly picked from each container and ball sizes (diameter) were recorded. Ball diameter was measured using an electronic digital Vanier calliper (E-base Measuring Tools Co., model: SV-03-150, size 6 in./150 mm, Pert Industries, Johannesburg, South Africa). Since most balls were more spheroidal in shape, both the longest and shortest diameters of each ball were measured. The final diameter of each ball was thus calculated as the average of the longest and the shortest diameters.
Dung removal experiments were conducted following modified protocols 54,104,105 . Following acclimation treatments, 50 mixed sex beetles were exposed to 200 g homogenised dung pats (RADWAG1 Wagi Elektroczne, Model AS220. R2, Poland) in plastic containers of 27 × 17.8 × 8.5 cm (4.09 L volume) with effective soil depth of 6 cm. A thin film of clean multipurpose wiping paper was placed beneath each dung pat to avoid soil sticking to the dung. The experiment was replicated 3 times for each of the treatments. Experimental containers were placed in a climate chamber (HPP 260, Memmert GmbH + Co.KG, Germany) set at 28 °C and 65 ± 10% RH under 14L:10D photoperiod. After 24 h, residual dung that was not balled or buried was weighed and recorded. Water loss was accounted for by using a parallel control experiment with only 200 g dung pats but no beetles 93 . Data analysis. All the statistical analyses were performed in R version R4.0.2 106 . We built models based on how treatments (acclimation and ramping rates) affected traits of CT max (°C) , HKDT (minutes), ball sizes (mm) and dung removal (%). A total of 180 observations were used to assess the effects of the 3 acclimation treatments (Control, VT-H and VT-L) and ramping rate (0.25 and 0.50 °C/min) on CT max .
A sample of 180 observations was also considered for HKDT model at three different acclimation treatments (Control, VT-H and VT-L) with 2 different heat knockdown temperature levels (53 °C and 55 °C) within each treatment respectively. A balanced 90 observations for each of the temperature levels was used treating the data at 53 °C and 55 °C separately for analysis. A preliminary two-way ANOVA on both CT max and HKDT models were run and showed that the residuals were not normally distributed for HKDT. Thus, a rank based non-parametric approach was adopted to assess the effects of acclimation on HKDT under the different acclimation treatments, and heat knockdown temperatures. We used the aligned rank transformation (ART) for nonparametric factorial analyses using only ANOVA procedures 107 . A multifactor contracts procedure by 108 was implemented to distinguish significant differences on the different factors and levels for the ART method. In order to determine how ball size and dung removal were influenced by the different acclimation treatment levels, one-way analysis of variance (ANOVA) was used. The models were both appropriate as both Shapiro Wilk's test for normality and Levene's test for equal variance assumptions on residuals were satisfied. Shapiro Wilk's p values were (0.85, 0.34) whilst Levene's test p values were (0.17, 0.93) for the ball diameter and dung model, respectively.
The responses of A. thalassinus to acclimation, termed acclimation response ratio (ARR) was calculated for CT max using the formula: where ΔCT max = recorded change in (CT max ) (°C), and ΔAcclimation = Difference between holding and acclimation temperature (°C) following methods by 44,76,77 . This was compared to ARRs from literature to interpret how the magnitude of plasticity of thermal tolerance may likely buffer A. thalassinus under changing climates. Acclimation Response Ratio of 1 shows a positive 1 °C shift in CT max for each 1 °C acclimation temperature investment suggesting positive plasticity while an ARR of close to zero indicates lack of plasticity and ARR = 0.5 indicates little effects of acclimation on CT max plasticity 100 .