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Climate variability differentially impacts thermal fitness traits in three coprophagic beetle species

  • Casper Nyamukondiwa ,

    Roles Conceptualization, Formal analysis, Funding acquisition, Project administration, Resources, Software, Supervision, Validation, Writing – original draft, Writing – review & editing

    nyamukondiwac@biust.ac.bw

    Affiliation Department of Biological Sciences and Biotechnology, Botswana International University of Science and Technology (BIUST), Palapye, Botswana

  • Frank Chidawanyika,

    Roles Formal analysis, Validation, Writing – review & editing

    Current address: Department of Zoology and Entomology, University of the Free State, Bloemfontein, South Africa

    Affiliations Agricultural Research Council, Plant Protection Research Institute, Weeds Division, Hilton, South Africa, School of Lifesciences, University of KwaZulu-Natal, Pietermaritzburg, South Africa

  • Honest Machekano,

    Roles Data curation, Formal analysis, Investigation, Visualization, Writing – review & editing

    Affiliation Department of Biological Sciences and Biotechnology, Botswana International University of Science and Technology (BIUST), Palapye, Botswana

  • Reyard Mutamiswa,

    Roles Data curation, Formal analysis, Investigation, Writing – review & editing

    Current address: Department of Zoology and Entomology, University of the Free State, Bloemfontein, South Africa

    Affiliation Department of Biological Sciences and Biotechnology, Botswana International University of Science and Technology (BIUST), Palapye, Botswana

  • Bryony Sands,

    Roles Data curation, Methodology, Validation, Writing – review & editing

    Affiliation School of Biological Sciences, University of Bristol, United Kingdom

  • Neludo Mgidiswa,

    Roles Data curation, Investigation, Writing – review & editing

    Affiliation Department of Biological Sciences and Biotechnology, Botswana International University of Science and Technology (BIUST), Palapye, Botswana

  • Richard Wall

    Roles Resources, Supervision, Writing – review & editing

    Affiliation School of Biological Sciences, University of Bristol, United Kingdom

Abstract

While the impacts of extreme and rising mean temperatures are well documented, increased thermal variability associated with climate change may also threaten ectotherm fitness and survival, but remains poorly explored. Using three wild collected coprophagic species Copris elphenor, Metacatharsius opacus and Scarabaeus zambezianus, we explored the effects of thermal amplitude around the mean on thermal tolerance. Using standardized protocols, we measured traits of high- (critical thermal maxima [CTmax] and heat knockdown time [HKDT]) and -low temperature tolerance (critical thermal minima [CTmin], chill coma recovery time [CCRT] and supercooling points [SCPs]) following variable temperature pulses (δ0, δ3, δ6 and δ9°C) around the mean (27°C). Our results show that increased temperature variability may offset basal and plastic responses to temperature and differs across species and metrics tested. Furthermore, we also show differential effects of body mass, body water content (BWC) and body lipid content (BLC) on traits of thermal tolerance. For example, body mass significantly influenced C. elphenor and S. zambezianus CTmax and S. zambezianus HKDT but not CTmin and CCRT. BWC significantly affected M. opacus and C. elphenor CTmax and in only M. opacus HKDT, CTmin and CCRT. Similarly, BLC only had a significant effect for M opacus CTmin. These results suggest differential and species dependent effects of climate variability of thermal fitness traits. It is therefore likely that the ecological services provided by these species may be constrained in the face of climate change. This implies that, to develop more realistic predictions for the effects of climate change on insect biodiversity and ecosystem function, thermal variability is a significant determinant.

Introduction

The frequency and magnitude of extreme thermal events, including deviations from the mean environmental temperatures, is increasing due to climate change [1, 2]. These changes in both seasonal and diurnal temperature fluctuations can pose significant physiological challenges for many species [3,4], with ecological consequences that may have direct implications for biodiversity conservation [58]. For ectothermic arthropods, the ability to withstand both acute and chronic thermal variability is even more critical, as body temperature is largely dependent on ambient temperature [911]. Deviations from optimal ranges not only affect physiological processes (e.g. metabolic rates), but also key activities such as locomotion [12,13], mating, and reproductive success [14,15] and ultimately survival [16,17]. As such, the ability of organisms to operate within varying magnitudes of temperature fluctuation may be a critical trait in the face of climate change.

Phenotypic plasticity is an adaptive response associated with thermal history, that can reduce potential vulnerability to thermal extremes [8,18] both within and across generations [19,20]. For example, rapid hardening, which is a process in which an acute exposure to a sub-lethal temperature can improve subsequent performance and survival in otherwise lethal thermal conditions [2124]. Acclimation through a chronic prior exposure to conditions similar to a future stressful environment, can improve survival and performance [25,26]. This acclimation can be realized in periods ranging from a few days to even months [11,27,28]. In many cases, such phenotypic plasticity leads only to transient physiological and biochemical responses [2931], nevertheless, it still optimizes key life history traits during otherwise deleterious environmental conditions. Under longer timescales plasticity can lead to local genetic adaptation through changes in allele frequency [3233].

Whilst phenotypic plasticity in response to thermal variability is pervasive in nature, several factors mediate its occurrence among different insect species. Diet quality and starvation [3436,16], ontogeny [37,38], body size [39,40] and age [41,16] are all known to influence biochemical processes and subsequent thermal performance or survival. Indeed, strong links have been reported between body size and physiological traits such as body water and lipid content [9,42,40] with concomitant impact on climatic stress resistance [4345,36,46]. Apart from insect physiological attributes, there is increasing evidence of how the pattern of thermal variability can also influence both constitutive (basal) and acquired (plastic) thermal responses. For example, rapid thermal variability, typical of that occurring under climate change scenarios, may exacerbate the impact of warming [4753].

The effects of thermal history pose challenges for studies of the effects of thermal variability, because most use laboratory insect populations, typically reared under constant temperatures [5456]. Furthermore, most studies focus on changes in mean temperatures excluding the magnitude of thermal fluctuations [57]. Thus, more robust studies on thermal variability around the mean rather than variations of the mean itself warrant investigation, in order to better understand the likely impacts of climate change on insect fitness (see [7]). As a result, here we use ecologically relevant standardised protocols (see [58,59]) to determine climate stress resistance among field-collected coprophagic beetles.

Coprophagic beetles (Coleoptera: Scarabaeidae) perform important ecosystem services such as secondary seed dispersal and nutrient cycling through degradation and burying of dung [6062]. Furthermore, dung colonizing beetles serve as important biological control agents of intestinal parasites [63] and several pest flies [64,65] through rapid removal of the dung in the field. Due to their high diversity, abundance and important ecosystem functions, dung beetles have become a focal taxon for various ecological studies including impacts of habitat degradation [61,6669]. The functional diversity of dung beetles has also been reported as an important buffer for such ecosystem services in disturbed agro-ecosystems [61]. In addition, dung beetles exhibit different adult body sizes across species and losses of some ecological functions have already been attributed to poor assemblages of large sized beetles [69]. Even though the factors leading to impoverished assemblages of large sized beetles are not well understood, we hypothesize that body size differentially influences vulnerability to environmental variables, as reported in other insect taxa (e.g. [43,44,40]). How changes in mean temperature and variability affect static and plastic thermal functional traits are important for elucidating insect physiological responses [7,70]. Here we use ecologically relevant protocols [58,59] to determine climate stress resistance among field-collected coprophagic beetles. Thus we test the impact of thermal variability on overall fitness, specifically, the ability of two paracoprid (tunneller) species: Copris elphenor Klug, 1855, Metacatharsius opacus Waterhouse, 1981 and one telocoprid (ball-rolling) Scarabaeus zambezianus Péringuey, 1901 to tolerate low and high temperature stress across a gradient of body mass using field collected specimens. The species to conduct the study were collected from field sites in Botswana, and they were the representatives most abundant that were active at the time of sampling.

Materials and methods

Sampling site and study animal preparation

A permit was granted from the Ministry of Environment, Wildlife and tourism Botswana [number EWT 8/36/4 XXXIII (18)] to collect experimental organisms. Dung beetles were sampled directly from the field in Khumaga Village (S20.46801; E24. 51491; 918 m.a.s.l), Central District, Botswana–a semi-arid region with Kalahari sand soils. Collections were made at the onset of the rainy season (January and February 2017), the peak period of adult emergence, to increase the likelihood of collecting beetles sharing a similar thermal history. Khumaga is characterized by abundant wildlife and domestic animals which supports high coprophagic species diversity. The area has typical average annual rainfall of 34.4 mm, most of which is recorded in January/February, and average temperatures of 23.6°C (Botswana Meteorological Services). The minimum and maximum temperatures for the two sampling months was 18.5 and 46°C respectively. Dung beetles were collected using pitfall traps baited with fresh cow dung. The traps comprised several ~8000 ml plastic buckets that were covered with a wire mesh (~5cm diameter holes) and then buried so that they were flush with the ground. The traps were equipped with a plastic rain guard that was placed 20 cm above the traps to prevent flooding. Every evening, fresh dung was placed on the wire mesh to attract foraging beetles to land and fall into the trap. A UV light bulb was also mounted on each trapping station as a light source to attract the beetles. Beetle trapping was undertaken over 12 h periods (18:00–06:00 h) and the beetles were then collected from the traps each morning. The beetles were immediately sorted according to species, based on morphological features described by Davis et al [71], and stored in insulated Styrofoam containers that had soil and fresh dung for feeding ad libtum during transportation to the laboratory. During the sampling period, the prevailing temperature was recorded using iButtons (model DS1923, Maxim, Sunnyvale, CA, USA). The iButtons were set within freshly deposited dung and at ground level where they were lightly covered with soil to determine potential differences in dung and ground temperature.

Thermal variability treatments

In the laboratory, insects were acclimated in climate chambers (HPP 260, Memmert GmbH + Co.KG, Germany) at 65 ± 10% relative humidity (RH) under 14L:10D at three different fluctuating thermal regimes (δ3, δ6 and δ9°C) around 27°C (Fig 1) for five days. Control beetles were placed in a chamber at constant 27°C (δ0), 65 ± 10% RH and 14L:10D (Fig 1). Temperature and RH in each cage was verified using iButton data loggers.

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Fig 1. Schematic representation of acclimation treatments to variable temperatures at different amplitudes oscillating around an ambient mean temperature of 27°C.

The control (δ0°C) was kept at benign 27°C.

https://doi.org/10.1371/journal.pone.0198610.g001

Effects of thermal variability on high temperature plasticity

Critical thermal maxima (CTmax).

Critical thermal maximum was measured for insects from the three treatments described above using the protocol outlined by Nyamukondiwa and Terblanche [41] and Mudavanhu et al [72]. First, ten mixed sex adult beetles were numbered and individually weighed using an electronic microbalance (RADWAG® Wagi Elektroczne, Model AS220.R2) for each of the three species and temperature treatments. This was done following 12 h starvation to allow for the clearance of gut contents and reduce the possible confounding effects on temperature tolerance [41]. Thereafter, the beetles were placed in a series of insulated double-jacketed chambers (‘organ pipes’) 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 [9]. The beetles were then first given 10 min to equilibrate at 27°C (equivalent to the rearing benign temperature) before increasing the temperature (CTmax) at a rate of 0.25°C min−1. This was repeated twice to give a sample size of n = 20 for each species per each treatment. A thermocouple (type K 36 SWG) connected to a digital thermometer (53/54IIB, Fluke Cooperation, USA) was inserted into the control chamber to monitor chamber temperatures. Individual beetle body temperature was assumed to be in equilibrium with the chamber temperature under the experimental conditions [58,72]. The CTmax was defined as the temperature at which each individual insect lost coordinated muscle function, consequently losing the ability to self-right or respond to mild stimuli (like prodding with a soft thermally inert camel-hair brush) [19]. After recording, individual beetles were returned to the labeled vials fitted with gauzed lids and then placed in khaki envelopes which were placed in Memmert oven (UF 160, Memmert GmbH + Co.KG, Germany) set at 60°C for 48 h. Individual beetle weight was immediately recorded thereafter and subtracted from initial body weight to determine body water content following methods by Weldon et al. [73]. Body lipid content for M. opacus and C. elphenor was investigated and correlated with CTmax, following modifications from Lease & Wolf [42]. Individual adult beetles were weighed in pre-weighed 50ml Eppendorf tubes, then dried in an oven (UF160, Memmert, Germany) at 60°C for 48 h. Following drying, the flies were weighed on a RADWAG microbalance (model AS 220.R2, Poland; precision 0.001mg). Thereafter, 1.5ml of diethyl ether was added to each tube and then gently agitated at 250 rpm for 24h at 37°C using orbital shaker. The diethyl ether was then removed from the tubes, and the flies dried again at 60°C for 24h, before reweighing. The lipid content of each beetle was calculated by subtracting the lipid free dry mass from the initial beetle dry mass [42].

Heat knockdown time (HKDT).

After exposure to the thermal variability treatments described in the previous section, HKDTs for the three beetle species were assayed as outlined by Weldon et al [73]. Ten beetles were weighed individually and placed in numbered 2 ml Eppendorf tubes (M. opacus) and 30ml polypropylene vials (C. elphenor and S. zambezianus) and placed in a climate chamber set at 48 ± 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. This was repeated two times to yield sample sizes of n = 20 for each species and temperature treatment. All observations from the climate chamber were recorded from a video recording on the computer screen. In this study, HKDT was defined as the time (in minutes) at which an individual beetle lost activity due to heat in the climate chamber as observed from the camera.

Effects of thermal variability on low temperature plasticity

Critical thermal minima (CTmin).

For comparative low temperature tolerance of C. elphenor, M. opacus, and S. zambezianus across all treatments, critical thermal minima (CTmin) were measured using standardized protocol as outlined by Nyamukondiwa and Terblanche [41]; Mudavanhu et al [72]. As in the CTmax experiments, ten replicate beetles were weighed individually and placed in numbered ‘organ pipes’ connected to a programmable water bath before decreasing the temperature at a rate of 0.25°C/min until their CTmin were recorded. This was also repeated twice to yield sample sizes of n = 20 per species. In this study, CTmin was regarded as the temperature at which each individual insect lost coordinated muscle function, consequently losing the ability to respond to mild stimuli (e.g. gentle prodding). The BLC of M. opacus and C. elphenor was determined following previous protocol [42], and its relationship with CTmin was calculated.

Chill Coma Recovery Time (CCRT).

Chill Coma Recovery Time experiments were assayed as outlined by Weldon et al [73]. As in the CTmin experiments, individual weights of C. elphenor, M. opacus, and S. zambezianus were first recorded as described. After weighing, ten beetles (from each species) were individually placed in 30ml polypropylene vials with gauzed screw-cap lids and then loaded into a large zip-lock bag which was submerged into a water bath (Systronix, Scientific, South Africa) filled with 1:1 water: propylene glycol set at 0°C for 1 h. This temperature by time treatment has been previously reported to elicit chill-coma in other insect taxa (see [73,74]). Following 1 h treatment at chill-coma temperature, the vials were immediately removed from the water bath and placed in a climate chamber set at 27±1°C, 65±10% RH for recovery. The chamber was connected to a video recording camera which was linked to a computer for recording observations. This was repeated two times to yield sample sizes of n = 20 per treatment. CCRT was defined as the time (in mins) required for an adult to stand upright on its legs following recovery from chill-coma [75].

Supercooling points (SCPs).

Supercooling points for the three beetle species were assayed following the protocol of Nyamukondiwa et al [76]. Sixteen beetles of each species were individually placed into 30 ml polypropylene vials with gauzed screw-cap lids. Each beetle was placed in contact with the tip of a type-T copper-constantan thermocouple (762–1121, Cambridge, UK), inserted through the gauzed lid of the vial and both the beetle and thermocouple were secured in contact by a cotton wool. Thermocouples were connected to one of two 8-channel Picotech TC-08 (Pico Technology, Cambridge, UK) thermocouple interfaces and temperatures were recorded at 1s intervals using PicoLog software for windows (Pico Technology, Cambridge, UK). In all treatments, experiments started by holding individual insects at 15°C for 10 mins (for insects’ temperature equilibration) before decreasing the temperature at a rate of 0.5°C min-1 until SCPs were recorded. SCP for each individual was determined as the lowest temperature recorded prior to a spike in temperature associated with the latent heat of crystallization [76].

Data analysis

Data was analyzed in STATISTICA 13.0 (Statsoft Inc., Tulsa, Oklahoma) and R version 3.3.0 [77]. SCPs, HKDT and CCRT data did not meet the assumptions of ANOVA and thus were analyzed using generalized linear models (GLZ) assuming a Gaussian distribution and a logit link function [27] in R3.3.0 statistical software. CTLs (CTmax and CTmin) met the linear model assumptions of constant variance and normal errors, so the data were analyzed using one-way factorial ANOVA in STATISTICA 13.0; Tukey-Kramer’s post-hoc tests were used to separate statistically heterogeneous means. The relationship between traits of thermal tolerance and body mass, body water content (BWC) and BLC were examined using linear regression in STATISTICA. Differences in environmental ambient temperature and inside the dung were compared graphically using Origin 8 software (OriginLab Corporation®, Northampton, Massachusetts, USA).

Results

Effects of thermal variability on high temperature plasticity

Thermal variability significantly affected CTmax across all species tested (F3,11 = 14.7, P < 0.001). Higher thermal variability (δ6 and δ9°C) was associated with a significantly lower high temperature tolerance, and this trend was more pronounced in M. opacus relative to C. elphenor and S. zambezianus (Fig 2A). Similarly, there was a significant species (F2,103 = 92.7, P < 0.001) and species x treatment interaction effect (F6,6 = 16.0, P < 0.001) for CTmax. Overall, S. zambezianus had relatively higher CTmax (47.16±0.083°C) compared to M. opacus (45.83±0.104°C) and C. elphenor (45.85±0.058°C) across all treatments (Fig 2A).

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Fig 2.

Effect of temperature fluctuation (acclimation at temperatures pulsating around a benign of 27°C: 0 = δ0°C (unacclimated), 3 = δ3°C, 6 = δ6°C and 9 = δ9°C) on (A) Critical thermal maxima and (B) Heat knock down time for M. opacus, C. elphenor and S. zambesianus. Vertical bars denote ±95% confidence limits. Means with the same letter are not statistically different.

https://doi.org/10.1371/journal.pone.0198610.g002

Furthermore, thermal variability treatments (χ2 = 118.6, d.f = 3, P < 0.001) and species (χ2 = 592.6.78, d.f = 2, P < 0.001) also affected HKDT across all species. The interaction between species and treatment (χ2 = 219.9, d.f = 6, P < 0.001) also influenced HKDT. Scarabaeus zambezianus had the highest HKDT indicating enhanced high temperature tolerance (Fig 2B). However, higher thermal variability had contrasting effects on the different species. While increased magnitude of temperature variability generally was associated with higher HKDT in C. elphenor (51.99±1.830 minutes) and S. zambezianus, (75.50±4.345 minutes) it was lower in M. opacus (12.20±0.372 minutes) (Fig 2B).

The relationship between traits of high temperature tolerance versus species body mass had contrasting results (Table 1). Generally, when all species were pooled together, body mass was not significantly related to high temperature tolerance. However, when species were analysed individually, body mass was significantly related to C. elphenor and S. zambezianus CTmax and S. zambezianus HKDT (Table 1). The effects of BWC on heat tolerance were more apparent both across and within species (Table 2). Due to limitations in the numbers of beetles available, it was not possible to examine the effect of BLC on all species, and this was only considered for M. opacus and C. elphenor. Nevertheless, BLC was not significantly correlated with high temperature tolerance (measured as CTmax) (Table 3).

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Table 1. The relationship between traits of high and low temperature tolerance and body mass.

Analysis was performed using linear regression in STATISTICA. Dung beetle species body mass were correlated with traits of temperature tolerance independently and then all species combined. R represents the coefficient of correlation.

https://doi.org/10.1371/journal.pone.0198610.t001

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Table 2. The relationship between traits of high and low temperature tolerance and body water content (BWC).

Analysis was performed using linear regression in STATISTICA. Dung beetle species BWC were correlated with traits of temperature tolerance independently and then all species combined. R represents the coefficient of correlation.

https://doi.org/10.1371/journal.pone.0198610.t002

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Table 3. The relationship between body lipid content and traits of high (CTmax) and low (CTmin).

Analyses were performed using linear regression in STATISTICA. Species body mass (M. opacus and C. elphenor) were correlated with traits of temperature tolerance independently and then all species combined.

https://doi.org/10.1371/journal.pone.0198610.t003

Effects of thermal variability on low temperature plasticity

Thermal variability had no significant relationships with traits of low temperature tolerance including CTmin (F3,0.65 = 1.25, P > 0.05). However, it had a significant relationship with CCRT (χ2 = 135.7, d.f = 3, P < 0.001) and SCPs (χ2 = 13.6, d.f = 3, P < 0.001). Nevertheless, higher thermal amplitudes significantly compromised CTmin relative to the control (δ0°C), a trend most apparent in S. zambezianus (Fig 3A).

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Fig 3.

Effects of temperature fluctuations (acclimation at temperatures pulsating around a benign of 27°C: 0 = δ0°C (unacclimated), 3 = δ3°C, 6 = δ6°C and 9 = δ9°C) on (A) Critical thermal minima, (B) Chill coma recovery time and, (C) Supercooling points for M. opacus, C. elphenor and S. zambesianus. Vertical bars denote ±95% confidence limits. Means with the same letter are not statistically different.

https://doi.org/10.1371/journal.pone.0198610.g003

In contrast, higher thermal fluctuations improved CCRT in both M. opacus and C. elphenor, unlike in S. zambezianus (Fig 3B). Similarly, SCPs were significantly enhanced by increased thermal amplitude in both M. opacus and C. elphenor, a trend not apparent in S. zambezianus (Fig 3C). Furthermore, species appeared to play a significant effect on CTmin (F2,72 = 92.9, P < 0.001) and CCRT (χ2 = 370.5 d.f = 2, P < 0.001) but not SCPs (χ2 = 3.48, d.f = 2, P > 0.05). Scarabaeus zambezianus had the most compromised CTmin compared to the other beetle species M. opacus and C. elphenor, which were not different from each other (Fig 3A). Metacatharsius opacus had a significantly shorter recovery time than the other beetle species following chill coma (Fig 3B).

Overall, body mass had contrasting relationships with low temperature tolerance (Table 1). Within species, body mass was not significantly related to CCRT, unlike when all species were pooled together (Table 1). Similarly, the relationship between BWC and low temperature tolerance was less pronounced, showing only significant correlation between CCRT and BWC when all species were combined, and a significant correlation for M. opacus for both CTmin and CCRT. As in high temperature versus BLC correlations, the relationship between BLC and low temperature tolerance was only undertaken for M. opacus and C. elphenor. Nevertheless, BLC had a significant relationship with low temperature tolerance (measured as CTmin) for both M. opacus and C. elphenor (Table 3).

Microclimate temperature recordings revealed largely similar thermal conditions within the dung and the ambient environment (±2°C) with a few exceptions were dung temperature was higher (Fig 4).

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Fig 4. Differences in microclimate temperature records between ambient and ‘inside-dung’ during a one-week dung pat decomposition period in Khumaga village.

Temperature was recorded using i-Buttons (DS1923, Maxim, Sunnyvale, CA, USA) at 15 minutes sampling interval for 5 days (January 19–23 2017). One i-Button was immersed in the middle of a freshly dropped cow dung pat, while the other one was placed in a shaded environment, ~1 m above the ground.

https://doi.org/10.1371/journal.pone.0198610.g004

Discussion

Here the effects of exposure to thermal variability are reported for natural field-derived populations of coprophagic beetles. Our results show that higher amplitudes generally constrain the thermal fitness traits tested, with a few exceptions, highlighting the complexity of insect responses to temperature means and variability. Notably, CTmax and HKDT results generally showed that increased variability in temperature may compromise high temperature tolerance. This suggests that, under projected increase in thermal variability, the coprophagic species studied here may be at more risk of high temperature mortality, a trend that was apparent in M. opacus and C. elphenor (for CTmax) and S. zambezianus for HKDT (Fig 2A and 2B). These results are consistent with findings by Terblanche et al [7] who demonstrated loss of basal and plastic responses to temperature in response to increased amplitude of temperature fluctuations.

Given that global climate change predictions suggest an increased frequency and variability of stressful temperatures [78,79], understanding the effects of these changes to insect population dynamics is important [80]. Related studies have looked at the effects of thermal amplitude on growth [81], fertility [82], density dependent net reproductive rate [57], development rate [83], stress resistance [84] and vector borne disease transmission [85]. However, none has considered natural populations of coprophagic species, with a view of understanding effects of climate variability on their activity and provision of ecosystem services. In addition, more recent studies have focused on how magnitudes of the variation impact species thermal performance curves [47,49, reviewed in 80]. Our current findings are therefore the first to show that increased amplitudes of thermal variations, typical of those predicted under global climate change, may have significant fitness costs to coprophagic beetles. This is likely to affect their provision of important ecosystem services [86,65].

Some studies suggest temperature variability may independently affect fitness traits, owing to the non-linearity of thermal reaction norms or thermal performance curves (see [56,70]). Our results also show that, while thermal amplitude play a significant role in fitness, species also differ significantly in their response to temperature. Among the three species, S. zambezianus appeared more tolerant to high temperature basally, compared to the other two beetle species. This further supports the idea that arthropod responses to climate variability are species-specific, and the rate of anthropogenic climate change may exceed that which may be compensated through genetic and evolutionary plastic mechanisms among the three different beetle species (e.g. [7]). The findings also suggest that M. opacus may be at more risk of high temperature mortality following higher thermal fluctuations compared to C. elphenor and S. zambezianus. Previous studies have also documented that that while plasticity to high temperature tolerance is an evident and near-ubiquitous feature of insect thermal physiology [9,87], the benefits may not be adequate to provide complete compensation for global climate change [88,87]. This therefore suggests that other mechanisms for compensation, e.g. behavioural modification and evolution may offer more benefit. However, it seems likely that many species may not be able to use behaviour e.g. tracking preferred microenvironments in space under prolonged environmental stress (e.g. [89]). This notion appears true for the dung beetle species tested here since ambient and dung temperature records were similar (Fig 4). It has been suggested that telocoprid dung beetle species (e.g. S. zambezianus studied here) withstand high temperatures by frequently mounting the dung to avoid excessive ground heat (of up to 60°C) during the day [90]. However, since our ground temperature recordings closely matched that of the dung it is unlikely that this ball-rolling behaviour would protect the species that were tested here from excessive heat. Furthermore, our study included two paracoprid species C. elphenor and M. opacus. The microclimate climate data recorded here suggest that physiological mechanisms to mitigate excessive heat are more important for their survival and preservation of key activities. However, the species tested here are nocturnal and perhaps gain significant performance and survival advantage due to cooler temperatures at night than day. It is possible that this nocturnal foraging behavior might be an evolutionary response to evade excessive day temperatures.

The correlation between body mass and high temperature tolerance was trait and species dependant. For example, in S. zambezianus, CTmax was positively correlated with body mass whilst HKDT was negative. The current results therefore contradict previous study by Nyamukondiwa and Terblanche [41] which found no evidence for the body mass constraints on thermal tolerance. However, our study supports studies where body mass was shown to correlate with heat tolerance [40,45,91]. We also found that increased heat tolerance was associated with increased BWC, as previously reported [45], perhaps due to enhanced resistance to desiccation [92]. This may be because insect mortality at high temperatures is closely associated with rapid water loss leading to desiccation [9,92]. Thus, insects with higher BWC may take more time to desiccate, and may have improved survival at high temperature through the link between the two stressors (see [93]). We did not find any correlation between traits of high temperature and BLC for the beetle species tested here, indicating that body lipid content may not play a role in high temperature tolerance.

Thermal variability had more dramatic effects on CCRT compared to CTmin and SCPs. The results therefore highlight differences in sensitivity to thermal variability among related traits of thermal tolerance. For S. zambezianus CTmin, higher thermal amplitudes also compromised low temperature tolerance (Fig 3A). It is also interesting to note that S. zambezianus had the highest lower activity temperature limit (CTmin), which perhaps relates to its superior high temperature tolerance compared to the other species (see Fig 2A and 2B), since this may have come at a cost of fitness at low temperature (CTmin) [94]. Indeed, similar fitness costs have been reported in other insect taxa, [9597]. Similar modest thermal fluctuations have been observed to affect many insect life history traits including mating behaviour and fertility (see [9]). Similarly, Marshall and Sinclair [98] showed that at stressful low temperatures, D. melanogaster trades off immediate survival for future reproductive output. Thus, thermal variability reported here may also have significant life history trade-offs for the beetle species tested. Coupled with individual reduced fitness, thermal variability under changing climates may therefore have far reaching population level consequences [47]. Nevertheless, exact trade-offs resulting from the magnitude of the thermal variability in M. opacus, C. elphenor and S. zambezianus warrants future investigation.

Previous studies have indicated that ectotherms are already experiencing sub-lethal low temperatures, e.g. chill coma and CTmin in natural environments [47], and consistently compromise fitness traits under increased magnitude of temperature changes [9,70]. While the direct and indirect effects of increased thermal variability have been appreciated by evolutionary ecologists [56,70], mechanistic models for surviving variable stressful environments have been limited. These mechanisms however remain significant in explaining how organisms likely cope with imminent and increased thermal variability associated with climate change. Without compensatory physiological or behavioural modifications to fluctuating thermal regimes, current findings thus suggest fitness losses for the beetle species tested here. Measurement of traits of low temperature vis CTmin, CCRT and chill coma also reveal significant patterns of species local low temperature adaptation and compensatory responses under thermal variability (see [9]). However, Donat and Alexander [99] suggest cold events may be less likely as opposed to extreme high temperature events under climate change. Thus survival of species under variable high temperature may be more imminent as opposed to low temperatures. Nevertheless, while low temperature variability may not be that critical here, it has shifted key life history traits, seasonal timing, and population phenologies of many insect species [100].

Body mass generally had little impact on low temperature tolerance traits (Table 1) even when traits for all species were pooled together, in particular for CCRT. This result corroborates with Nyamukondiwa and Terblanche [41] who showed no effects of body mass on insect CTLs to activity. Similarly, the relationship between high temperature tolerance and BWC did not follow a specific pattern. For example, BWC appeared to be positively correlated with CTmin and CCRT for M. opacus, and when species were pooled together for CCRT. This suggests taxa and trait dependent effects of varying thermal amplitudes to low temperature tolerance. Our results also showed significant correlations between traits of low temperature and BLC. Indeed, [101] elaborated on the role of metabolites, including carbohydrates (i.e. trehalose), free amino acids, lipids, osmoprotectants and polyols are upregulated during low temperature conditions. Our results therefore suggest lipid accumulation may be an essential mechanism sustaining low temperature survival by insects faced with increased magnitude of low temperature fluctuations.

Age and body condition e.g. nutrition are among some of the factors which may affect physiological and biochemical processes in insects, including traits of thermal tolerance measured here [9,34,38]. Thus, one confounding effect in our study here is that, age and nutritional history of the test organisms was not strictly controlled for. Nevertheless, our assessments of body mass, BWC and BLC and their correlation with thermal tolerance traits may be indicative of body condition which can correlate with age. In conclusion our results show that coprophagic insects M. opacus, C. elphenor and S. zambezianus may suffer fitness losses (for both high and low temperature traits) in response to increased temperature variability with climate change. Tolerance levels varied significantly across variability (δ) levels, and likely depend on the trait in question and the species. Field studies have reported that pesticide residues in the dung of treated animals may have deleterious consequences on different developmental stages of dung beetles [102,103]. Thus loss of fitness with thermal variability reported here, coupled with reports on the effects of pesticides (e.g. [103]), represent a significant burden to conservation of dung beetle ecological services.

Acknowledgments

We appreciate logistical support from the Ministry of Environment, Wildlife and Tourism Botswana and Michael Flyman for the research permits, Botswana International University of Science and Technology (BIUST), and University of KwaZulu-Natal. We are also grateful to the Insect Physiological Ecology research group (BIUST) for assistance in running experiments and several anonymous reviewers for their comments on an earlier version of this manuscript.

References

  1. 1. Diffenbaugh NS, Pal JS, Trapp RJ, Giorgi F (2005) Fine-scale processes regulate the response of extreme events to global climate change. Proceedings of the National Academy of Sciences of the United States of America 102: 15774–15778. pmid:16236722
  2. 2. Diffenbaugh NS, Field CB (2013) Changes in ecologically critical terrestrial climate conditions. Science 341: 486–492. pmid:23908225
  3. 3. Helmuth B (2009) From cells to coastlines: how can we use physiology to forecast the impacts of climate change? Journal of Experimental Biology 212: 753–760. pmid:19251989
  4. 4. Sunday JM, Bates AE, Kearney MR, Colwel RK, Dulvy NK, Longino JT et al. (2014) Thermal-safety margins and the necessity of thermoregulatory behavior across latitude and elevation. Proceedings of the National Academy of Sciences of the United States of America 111: 5610–5615. pmid:24616528
  5. 5. Sinclair BJ, Addo-Bediako A, Chown SL (2003) Climatic variability and the evolution of insect freeze tolerance. Biological reviews of the Cambridge Philosophical Society 78: 181–195. pmid:12803420
  6. 6. Chown SL, Terblanche JS (2006) Physiological Diversity in Insects: Ecological and Evolutionary Contexts. Advances in Insect Physiology 33: 50–152. pmid:19212462
  7. 7. Terblanche JS, Nyamukondiwa C, Kleynhans E (2010) Thermal variability alters climatic stress resistance and plastic responses in a globally invasive pest, the Mediterranean fruit fly (Ceratitis capitata). Entomologia Experimentalis et Applicata 137: 304–315.
  8. 8. Gunderson AR, Stillman JH (2015) Plasticity in thermal tolerance has limited potential to buffer ectotherms from global warming. Proceedings of the Royal Society of London B 282: 1808. pmid:25994676
  9. 9. Chown SL, Nicolson SW (2004) Insect physiological ecology; mechanisms and patterns. Oxford University Press. New York.
  10. 10. Speight MR, Hunter MD, Watt AD (2008) Ecology of Insects: Concepts and Applications. Wiley-Blackwell, West Sussex, UK Chichester, U.K. 626 pp.
  11. 11. Fischer K, Dierks A, Franke K, Geister TL, Liska M, Winter S et al. (2010) Environmental effects on temperature stress resistance in the tropical butterfly Bicyclus anynana. PLOS ONE 5: e15284. pmid:21187968
  12. 12. Clusella-Trullas S, Terblanche JS, Chown SL (2010) Phenotypic Plasticity of locomotion performance in the seed harvester Messor capensis (Formicidae) Physiological and Biochemical Zoology 83: 519–530. pmid:20367320
  13. 13. Lachenicht MW, Clusella-Trullas S, Boardman L, Le Roux C, Terblanche JS (2010) Effects of acclimation temperature on thermal tolerance, locomotion performance and respiratory metabolism in Acheta domesticus L. (Orthoptera: Gryllidae). Journal of Insect Physiology 56: 822–830. pmid:20197070
  14. 14. Piyaphongkul J, Pritchard J, Bale J (2012) Heat stress impedes development and lowers fecundity of the brown planthopper Nilaparvata lugens (Stål). PLoS ONE 7: e47413. pmid:23071803
  15. 15. Dick CA, Rank NE, McCarthy M, McWeeney S, Hollis D, Dahlhoff E P (2013) Effects of temperature variation on male behavior and mating success in a Montane beetle. Physiological and Biochemical Zoology 86: 432–40. pmid:23799837
  16. 16. Chidawanyika F, Nyamukondiwa C, Strathie L, Fischer K (2017) Effects of thermal regimes, starvation and age on heat tolerance of the Parthenium beetle Zygogramma bicolorata (Coleoptera: Chrysomelidae) following dynamic and static protocols. PLos One 12(1): e0169371. pmid:28052099
  17. 17. Mutamiswa R, Chidawanyika F, Nyamukondiwa C (2017) Comparative assessment of the thermal tolerance of spotted stemborer, Chilo Partellus (Lepidoptera: Crambidae) and Its larval parasitoid, Cotesia Sesamiae (Hymenoptera: Braconidae). Insect Science. 00: 1–14.
  18. 18. Valladares F, Matesanz S, Guilhaumon F, Araujo MB, Balaguer L, Benito-Garzon M et al. (2014) The effects of phenotypic plasticity and local adaptation on forecasts of species range shifts under climate change. Ecology Letters 17: 1351–1364. pmid:25205436
  19. 19. Nyamukondiwa C, Terblanche JS (2010) Within-generation variation of critical thermal limits in adult Mediterranean and Natal fruit flies Ceratitis capitata and Ceratitis rosa: thermal history affects short-term responses to temperature. Physiological Entomology 35: 255–264.
  20. 20. Zhou Z-S, Rasmann S, Li M, Guo J-Y, Chen H-S, Wan F-H (2013) Cold temperatures increase cold hardiness in the next generation Ophraella communa beetles. PLoS ONE 8: e74760. pmid:24098666
  21. 21. Sejerkilde M, Jesper G, Sørensen JG, Loeschcke V (2003) Effects of cold- and heat hardening on thermal resistance in Drosophila melanogaster. Journal of Insect Physiology 49: 719–726. pmid:12880651
  22. 22. Kellett M, Hoffmann AA, Mckechnie SW (2005) Hardening capacity in the Drosophila melanogaster species group is constrained by basal thermotolerance. Functional Ecology 19: 853–858.
  23. 23. Stotter RL, Terblanche JS (2009) Low temperature tolerance of false codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa. Journal of Thermal Biology 34: 320–325
  24. 24. Chidawanyika F, Terblanche JS (2011a) Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae). Journal of Insect Physiology 57: 108–117. pmid:20933517
  25. 25. Huey RB, Berrigan D (1996) Testing evolutionary hypotheses of acclimation. In Johnston IA and Bennett AE (eds.), Phenotypic and evolutionary adaptation to temperature, pp. 205–237. U. Cambridge Press, Cambridge, U.K.
  26. 26. Geister TL, Fischer K (2007) Testing the beneficial acclimation hypothesis: temperature effects on mating success in a butterfly. Behavioural Ecology 18: 658–664.
  27. 27. Chidawanyika F, Terblanche JS (2011b) Costs and benefits of thermal acclimation for codling moth, Cydia pomonella (Lepidoptera: Tortricidae): implications for pest control and the sterile insect release programme. Evolutionary Applications 4: 534–544. pmid:25568003
  28. 28. Kristensen TN, Hoffmann AA, Overgaard J, Sorensen JG, Hallas R, Loeschcke V (2008) Costs and benefits of cold acclimation in field-released Drosophila. Proceedings of the National Academy of Sciences of the United States of America 105: 216–221. pmid:18162547
  29. 29. Boardman L, Sørensen JG, Terblanche JS (2013) Physiological responses to fluctuating thermal and hydration regimes in the chill susceptible insect, Thaumatotibia leucotreta. Journal of Insect Physiology 59: 781–794. pmid:23684741
  30. 30. Boardman L, Sorensen JG, Terblanche JS (2015) Physiological and molecular mechanisms associated with cross tolerance between hypoxia and low temperature in Thaumatotibia leucotreta. Journal of Insect Physiology 82: 75–84. pmid:26376454
  31. 31. Sgrò CM, Terblanche JS, Hoffmann AA (2016) What Can Plasticity Contribute to Insect Responses to Climate Change? Annual review of Entomology 61: 433–451. pmid:26667379
  32. 32. Angilletta MJ Jr Bennett AF, Guderley H Navas CA, Seebacher F Wilson RS (2006) Coadaptation: A Unifying Principle in Evolutionary Thermal Biology. Physiological and Biochemical Zoology 79: 282–294. pmid:16555188
  33. 33. Gerken AR, Eller OC, Hahn DA, Morgan TJ (2015) Constraints, independence, and evolution of thermal plasticity: Probing genetic architecture of long- and short-term thermal acclimation. Proceedings of the National Academy of Sciences of the United States of America 112: 4399–4404. pmid:25805817
  34. 34. Shreve SM, Yi SX, Lee RE (2007) Increased dietary cholesterol enhances cold tolerance in Drosophila melanogaster. Cryoletters 28: 33–37. pmid:17369960
  35. 35. Diamond SE, Kingsolver JG (2010) Environmental dependence of thermal reaction norms: host plant quality can reverse the temperature-size rule. American Naturalist 175: 1–10. pmid:19911984
  36. 36. Scharf I, Wexler Y, MacMillan HA, Presman S, Simson E, Rosenstein S (2016) The negative effect of starvation and the positive effect of mild thermal stress on thermal tolerance of the red flour beetle, Tribolium castaneum. Science of Nature 103: 20. pmid:26888763
  37. 37. Marais K, Terblanche JS, Chown SL (2009) Life stage-related differences in hardening and acclimation of thermal tolerance traits in the kelp fly, Paractora dreuxi (Diptera, Helcomyzidae). Journal of Insect Physiology 55: 336–43. pmid:19171152
  38. 38. Bowler K, Terblanche JS (2008) Insect thermal tolerance: what is the role of ontogeny, ageing and senescence? Biological Reviews 83: 339–355. pmid:18979595
  39. 39. Spicer JI, Gaston KJ (1999) Physiological diversity and its ecological implications. Oxford, UK: Blackwell Science.
  40. 40. Klockmann M, Gunter F, Fischer K (2017) Heat resistance throughout ontogeny: body size constrains thermal tolerance. Global Change Biology 23: 686–696. pmid:27371939
  41. 41. Nyamukondiwa C, Terblanche JS (2009) Thermal tolerance in adult Mediterranean and Natal fruit flies (Ceratitis capitata and Ceratitis rosa): effects of age, gender and feeding status. Journal of Thermal Biology 34: 406–414.
  42. 42. Lease HM, Wolf BO (2011) Lipid content of terrestrial arthropods in relation to body size, phylogeny, ontogeny and sex. Physiological Entomology 36: 29–38.
  43. 43. Le Lagadec M, Chown S, Scholtz C (1998) Desiccation resistance and water balance in southern African keratin beetles (Coleoptera, Trogidae): the influence of body size and habitat. Journal of Comparative Physiology B 168: 112–122.
  44. 44. Chown SL, Le Lagadec MD, Scholtz CH (1999) Partitioning variance in a physiological trait: desiccation resistance in keratin beetles (Coleoptera, Trogidae). Functional Ecology 13: 838–844.
  45. 45. Oberg EW, Toro ID, Pelini SL (2012) Characterization of the thermal tolerances of forest ants of New England. Insectes Sociaux 59: 167–174.
  46. 46. Mitchell KA, Boardman L, Clusella-Trullas S, Terblanche JS (2017) Effects of nutrient and water restriction on thermal tolerance: A test of mechanisms and hypotheses. Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology 212: 15–23. pmid:28676379
  47. 47. Deutsch CA, Tewksbury JJ, Huey RB, Sheldon KS, Ghalambor CK, Haak DC et al. (2008) Impacts of climate warming on terrestrial ectotherms across latitude. Proceedings of the National Academy of Science USA 105: 6668–6672.
  48. 48. Paaijmans KP, Heinig RL, Seliga RA, Blanford JI, Blanford S, Murdock CC et al. (2013) Temperature variation makes ectotherms more sensitive to climate change. Global Change Biology 19: 2373–2380. pmid:23630036
  49. 49. Kingsolver JG, Diamond SE, Buckley LB, (2013) Heat stress and the fitness consequences of climate change for terrestrial ectotherms. Functional Ecology 27: 1415–1423.
  50. 50. Estay SA, Lima M, Bozinovic F (2014) The role of temperature variability on insect performance and population dynamics in a warming world. Oikos 123: 131–140.
  51. 51. Dowd WW, King FA, Denny MW (2015) Thermal variation, thermal extremes and the physiological performance of individuals. Journal of Experimental Biology 218: 1956–1967. pmid:26085672
  52. 52. Ma FZ, Lu ZC, Wang R, Wan FH (2014) Heritability and evolutionary potential in thermal tolerance traits in the invasive Mediterranean cryptic species of Bemisia tabaci (Hemiptera: Aleyrodidae). PLoS ONE 9:e103279. pmid:25054554
  53. 53. Shah AA, Gill BA, Encalada AC, Flecker AS, Funk WC, Guayasamin JM et al. (2017) Climate variability predicts thermal limits of aquatic insects across elevation and latitude. Functional Ecology 31: 2118–2127.
  54. 54. Scheiner SM (2002) Selection experiments and the study of phenotypic plasticity. Journal of Evolutionary Biology 15: 889–898.
  55. 55. Schulte PM, Healy TM, Fangue NA (2011) Thermal performance curves, phenotypic plasticity, and the time scales of temperature exposure. Integrative and Comparative Biology 51: 691–702. pmid:21841184
  56. 56. Vasseur DA, DeLong JP, Gilbert B, Greig HS, Harley CDG, McCann KS et al. (2014) Increased temperature variation poses a greater risk to species than climate warming. Proceedings of the Royal Society of London B 281:20132612.
  57. 57. Clavijo-Baquet S, Boher F, Ziegler L, Martel SI, Estay SA, Bozinovic F (2014) Differential responses to thermal variation between fitness metrics. Scientific Reports 4: 5349. pmid:24954717
  58. 58. Terblanche JS, Deere JA, Clusella-Trullas S, Janion C, Chown SL (2007) Critical thermal limits depend on methodological context. Proceedings of the Royal Society of London Series B, Biological Sciences 274: 2935–2943.
  59. 59. Terblanche JS, Hoffmann AA, Mitchell KA, Rako L, le Roux PC, Chown SL (2011) Ecologically relevant measures of tolerance to potentially lethal temperatures. Journal of Experimental Biology 214: 3713–3725. pmid:22031735
  60. 60. Yamada D, Imura O, Shi K, Shibuya T (2007) Effect of tunneler dung beetles on cattle dung decomposition, soil nutrients and herbage growth. Grassland Science 53: 121–129.
  61. 61. Beynon SA, Mann DJ, Slade EM, Lewis OT (2012) Species-rich dung beetle communities buffer ecosystem services in perturbed agro-ecosystems. Journal of Applied Ecology 49: 1365–1372.
  62. 62. Beynon SA, Wainwright WA, Christie M (2015) The application of an ecosystem services framework to estimate the economic value of dung beetles to the U.K. cattle industry. Ecological Entomology 40: 124–135.
  63. 63. Sands B, Wall R (2016) Dung beetles reduce livestock gastrointestinal parasite availability on pasture. Journal of Applied Ecology 54: 1180–1189.
  64. 64. Bergstrom RC, Maki LR, Werner BA (1976). Small dung beetles as biological control agents: laboratory studies of beetle action on trichostrongylid eggs in sheep and cattle feces. Proceedings of the Helminthological Society of Washington 43: 171–174.
  65. 65. Nichols E, Spector S, Louzada J, Larsen T, Amezquita S, Favila ME (2008) Ecological functions and ecosystem services provided by Scarabaeinae dung beetles. Biological Conservation 141: 1461–1474.
  66. 66. Nichols E, Larsen T, Spector S, Davis AL, Escobar F, Favila M et al. (2007) Dung beetle response to tropical forest modification and fragmentation: a quantitative literature review and meta-analysis. Biological Conservation 137: 1–19.
  67. 67. Jacobs CT, Scholtz CH, Escobar F, Davis ALV (2010) How might intensification of farming influence dung beetle diversity (Coleoptera: Scarabaeidae) in Maputo Special Reserve (Mozambique)? Journal of Insect Conservation 14: 389–399.
  68. 68. Almeida S, Louzada J, Sperber C, Barlow J (2011) Subtle land-use change and tropical biodiversity: dung beetle communities in cerrado grasslands and exotic pastures. Biotropica 43: 704–710.
  69. 69. Braga RF, Korasaki V, Andresen E, Louzada J (2013) Dung beetle community and functions along a habitat-disturbance gradient in the Amazon: a rapid assessment of ecological functions associated to biodiversity. PLoS One 8:e57786. pmid:23460906
  70. 70. Bozinovic F, Medina NR, Alruiz JM, Cavieres G, Sabat P (2016) Thermal tolerance and survival responses to scenarios of experimental climatic change: changing thermal variability reduces the heat and cold tolerance in a fly. Journal of Comparative Physiology 186: 581–587. pmid:27003422
  71. 71. Davis ALV, Frolov AV, Scholtz CH (2008) The African Dung Beetle Genera. Protea Book House. Pretoria, South Africa.
  72. 72. Mudavanhu P, Addison P, Conlong DE (2014) Impact of mass rearing and gamma irradiation on thermal tolerance of Eldana saccharina. Entomologia Experimentalis et Applicata 153: 55–63.
  73. 73. Weldon CW, Terblanche JS, Chown SL (2011) Time-course for attainment and reversal of acclimation to constant temperature in two Ceratitis species. Journal of Thermal Biology 36: 479–485.
  74. 74. Sinclair BJ, Coello Alvarado LE, Ferguson LV (2015) An invitation to measure insect cold tolerance: Methods, approaches, and workflow. Journal of Thermal Biology 53: 180–197. pmid:26590471
  75. 75. Milton CC, Partridge L (2008) Brief carbon dioxide exposure blocks heat hardening but not cold acclimation in Drosophila melanogaster. Journal of Insect Physiology 54: 32–40. pmid:17884085
  76. 76. Nyamukondiwa C, Weldon CW, Chown SL, le Roux PC, Terblanche JS (2013) Thermal biology, population fluctuations and implications of temperature extremes for the management of two globally significant insect pests. Journal of Insect Physiology 59: 1199–1211. pmid:24080125
  77. 77. R Development Core Team (2016) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.
  78. 78. Easterling DR, Meehl GA, Parmesan C, Changnon SA, Karl TR, Mearns LO (2000) Climate extremes: observations, modeling, and impacts. Science 289: 2068–2074. pmid:11000103
  79. 79. Meehl GA, Tebaldi C (2004) More intense, more requent, and longer lasting heat waves in the 21st century. Science 305: 994–997. pmid:15310900
  80. 80. Thompson RM, Beardall J, Beringer J, Grace M, Sardina P (2013) Means and extremes: building variability into community-level climate change experiments. Ecology Letters 16: 799–806. pmid:23438320
  81. 81. Estay SA, Clavijo-Baquet S, Lima M, Bozinovic F (2011) Beyond average: an experimental test of temperature variability on the population dynamics of Tribolium confusum. Population Ecology 53: 53–58.
  82. 82. Ragland GJ, Kingsolver JG (2008) The effect of fluctuating temperatures on ectotherm life history traits: comparisons among geographic populations of Wyeomyia smithii. Evolutionary Ecology Research 10: 29.
  83. 83. Saunders LM, Tompkins DM, Hudson PJ (2002) Stochasticity accelerates nematode egg development. Journal of Parasitology 88: 1271–1272. pmid:12537129
  84. 84. Folguera G, Bastı´as DA, Caers J, Rojas JM, Piulachs M-D, Belle´s X et al. (2011) An experimental test of the role of environmental temperature variability on ectotherm molecular, physiological and life-history traits: implications for global warming. Comparative Biochemistry and Physiology A 159: 242–246.
  85. 85. Paaijmans KP, Blanford S, Bell AS, Blanford JI, Read AF, Thomas MB (2010) Influence of climate on malaria transmission depends on daily temperature variation. Proceedings of the National Academy of Science USA 107: 15135–15139.
  86. 86. Losey JE, Vaughan M (2006) The economic value of ecological services provided by insects. Bioscience 56: 311–323.
  87. 87. Gunderson AR, Dillon ME, Stillman JH (2017) Estimating the benefits of plasticity in ectotherm heat tolerance under natural thermal variability. Functional Ecology 31: 529–1539.
  88. 88. Mitchell KA, Hoffmann AA (2017) Thermal ramping rate influences evolutionary potential and species differences for upper thermal limits in Drosophila. Functional Ecology 24: 694–700.
  89. 89. Chevin LM, Lande R, Mace GM (2010) Adaptation, plasticity and extinction in a changing environment. Towards a predictive theory. PLoS Biology 8: e1000357. pmid:20463950
  90. 90. Smolka J, Baird E, Bryne MJ, Jundi B, Warrant EJ, Dacke M (2012) Dung beetles use their dung ball ball as mobile thermal refuge. Current Biology 22: 863–864.
  91. 91. Brans KI, Jansen M, Vanoverbeke J, Tuzun N, Stoks R, De Meester L (2017) The heat is on: Genetic adaptation to urbanization mediated by thermal tolerance and body size. Global Change Biology. pmid:28614592
  92. 92. Weldon CW, Boardman L, Marlin D, Terblanche JS (2016) Physiological mechanisms of dehydration tolerance contribute to the invasion potential of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) relative to its less widely distributed congeners. Frontiers in Zoology 13: 15. pmid:27034703
  93. 93. Bubliy OA, Kristensen TN, Kellermann V, Loeschcke V (2012) Plastic responses to four environmental stresses and cross resistance in a laboratory population of Drosophila melanogaster. Functional Ecology 26: 245–253.
  94. 94. Nyamukondiwa C, Terblanche JS, Marshall KE, Sinclair BJ (2011) Basal cold but not heat tolerance constraints plasticity among Drosophila species (Diptera: Drosophilidae). Journal of Evolutionary Biology 24: 1927–1938. pmid:21658189
  95. 95. Hoffmann AA, Hewa-Kapuge S (2000) Acclimation for heat resistance in Trichogramma nr. brassicae: can it occur without costs? Functional Ecology 14: 55–60.
  96. 96. Coulson SC, Bale JS. (1992) Effect of rapid cold hardening on reproduction and survival of offspring in the housefly Musca domestica. Journal of Insect Physiology 38: 421–424.
  97. 97. Basson CH, Nyamukondiwa C, Terblanche JS (2011). Fitness costs of rapid cold-hardening in Ceratitis capitata. Evolution 66: 296–304. pmid:22220884
  98. 98. Marshall KE, Sinclair BJ (2010) Repeated stress exposure results in a survival-reproduction trade-off in Drosophila melanogaster. Proceedings of the Royal Society of London B 277: 963–969.
  99. 99. Donat MG, Alexander LV (2012) The shifting probability distribution of global daytime and night-time temperatures. Geophysics Research Letters 39:
  100. 100. Parmesan C (2006) Ecological and evolutionary responses to recent climate change. Annual Review of Ecology, Evolution and Systematics 37: 637–669.
  101. 101. Sinclair BJ (2015) Linking energetics and overwintering in temperate insects. Journal of Thermal Biology 54: 5–11. pmid:26615721
  102. 102. Kruger K, Lukhele OM, Scholtz CH (1999) Survival and reproduction of Euoniticellus intermedius (Coleoptera: Scarabaeidae) in dung following application of cypermethrin and flumethrin pour-ons to cattle. Bulletin of Entomological Research 89: 543–548.
  103. 103. Mann CM, Barnes S, Offer B, Wall R (2015) Lethal and sub-lethal effects of faecal deltamethrin residues on dung-feeding insects. Medical and Veterinary Entomology 29: 189–195. pmid:25594879