ABSTRACT Recent theory suggests that global warming may be catastrophic for tropical ectotherms. Although most studies addressing temperature effects in ectotherms utilize constant

temperatures, Jensen's inequality and thermal stress considerations predict that this approach will underestimate warming effects on species experiencing daily temperature fluctuations

in nature. Here, we tested this prediction in a neotropical pseudoscorpion. Nymphs were reared in control and high-temperature treatments under a constant daily temperature regime and

results compared to a companion fluctuating-temperature study. At constant temperature, pseudoscorpions outperformed their fluctuating-temperature counterparts. Individuals were larger,

microhabitats that dampen temperature oscillations may be critical in avoiding extinction as tropical climates warm. SIMILAR CONTENT BEING VIEWED BY OTHERS VARIATION IN TEMPERATURE OF PEAK

TRAIT PERFORMANCE CONSTRAINS ADAPTATION OF ARTHROPOD POPULATIONS TO CLIMATIC WARMING Article Open access 25 January 2024 TEMPERATE SPECIES UNDERFILL THEIR TROPICAL THERMAL POTENTIALS ON LAND

Article Open access 06 November 2023 INCREASED PUPAL TEMPERATURE HAS REVERSIBLE EFFECTS ON THERMAL PERFORMANCE AND IRREVERSIBLE EFFECTS ON IMMUNE SYSTEM AND FECUNDITY IN ADULT LADYBIRDS

Article Open access 12 August 2023 INTRODUCTION Predicting how climate change will impact on biodiversity requires detailed understanding of the fitness consequences of temperature increase

for tropical terrestrial arthropods, the most diverse animals on earth. With metabolic rate in ectotherms increasing exponentially with temperature1,2, recent theory suggests that the

physiological effects of warming may be most severe for terrestrial ectotherms inhabiting tropical regions3,4. Although median surface temperatures in the tropics are projected to increase

by 3.5–4.0°C by the end of the century5, tropical terrestrial ectotherms, such as arthropods and reptiles, are adapted to an already hot environment and therefore have high baseline

temperatures and narrow thermal safety margins that may limit their capacity to tolerate even small increases in temperature3,4,6. Most studies addressing the effects of temperature on

fitness traits in ectotherms have been conducted under constant temperature conditions (reviewed in Refs. 7,8,9,10,11). However, this approach may yield unrealistic results for the many

organisms that experience regular daily temperature fluctuations in nature. Although rarely taken into account in climate change research11,12, diurnal variance in temperature can

potentially influence fitness traits and mathematical theory suggests that temperature fluctuations are likely to be particularly important for tropical ectotherms that function close to

their critical maximum temperatures. Jensen's inequality is a mathematical property that applies broadly to biological processes, involving non-linear relationships, ranging from

metabolism to species interactions13,14. It states that the average result of a nonlinear function, , does not equal the function evaluated at the average value of _x_, , with variance in

_x_ lowering the response variable, if the function is decelerating and raising it, if the function is accelerating13. Because ectothermic metabolic rate is an accelerating function, i.e.,

it increases exponentially with temperature, the average metabolic rate of an ectotherm across a range of diurnal temperatures will always be greater than its metabolic rate at the average

daily temperature14. While there is little seasonal variation in temperature in the tropics, temperatures typically vary by approximately 8°C on a regular daily basis (NOAA National Climate

Data Center web site, http://www.ncdc.noaa.gov/). For example, at the El Claro site on Barro Colorado Island in lowland central Panamá, shaded air temperature ranged, on average, from a

daily minimum of 23.3°C to a daily maximum of 31.5°C during the period 1994 to 200515. In a recent simulated climate warming study, carried out at fluctuating temperatures that closely

mimicked this natural diurnal temperature cycle, the 3.5°C increase that has been predicted for the tropics by the end of the century5 had highly detrimental consequences for the neotropical

pseudoscorpion, _Cordylochernes scorpioides_16. Although elevated temperature significantly reduced survival, size and level of sexual dimorphism, its greatest impact was on reproductive

traits in the two sexes. At high temperature, males produced only 45% as many sperm as control males and females were rendered sterile. Characteristics of its biology make _C. scorpioides_ a

model system for investigating the life history, morphological and reproductive consequences of climate warming in a tropical ectotherm. Sperm transfer in this pseudoscorpion is indirect,

with males transferring discrete packets of sperm to females by means of a stalked spermatophore deposited on the substrate (Fig. 1a). Indirect sperm transfer provides a window on ejaculate

characteristics that is absent in species that transfer sperm directly via copulatory organs. Immediately following spermatophore deposition, matings can be interrupted and sperm packets

retrieved for evaluation of sperm quality and quantity17. Females are live bearing and nourish developing embryos in an external, translucent brood sac (Fig. 1b) that overlies the genital

opening18,19. This ‘external womb’ mode of viviparous reproduction enables non-invasive monitoring of female reproductive status and embryological development20. The strongly negative

effects of high temperature on fitness-related traits in _C. scorpioides_16 may have been at least partially the result of the fluctuating regime that subjected individuals to temperatures

between 34°C and 35°C for several hours each day. Averaging daily temperature eliminates this upper temperature extreme, and, according to Jensen's inequality13, should reduce high

temperature induced metabolic stress in this tropical ectotherm. Here, we tested this daily temperature regime hypothesis by repeating the simulated climate warming study, but substituted

constant mean temperature for the diurnally fluctuating temperature regime. In a split-brood experiment, we randomly assigned newborn offspring from replicate full-sibling _C. scorpioides_

families to control and high temperature treatments. The control and high temperatures were determined by averaging the diurnally fluctuating temperatures used in the previous study16.

Nymphs were reared to adults and we assessed the effect of temperature treatment on survivorship, developmental rate and sexually monomorphic and dimorphic morphological traits, as well as

on sperm number and viability and female reproductive function. In addition, to distinguish between long-term, developmental and short-term, adult-exposure temperature effects on male

fertility and female reproductive function, we carried out a second experiment, in which adults were switched between temperature treatments 7 days before sperm assessment in males and

mating in females. Data from the constant and fluctuating temperature studies were then pooled, in order to assess the effect of daily temperature regime on response to increased temperature

in this pseudoscorpion. For nearly all traits, including reproductive traits in both males and females, pseudoscorpions at constant temperature outperformed their fluctuating-temperature

counterparts. _Cordylochernes scorpioides_ individuals experiencing constant temperature developed faster and were significantly larger. Males produced more sperm and a higher percentage of

viable sperm and females were more likely to become gravid and produced a greater number of early-stage embryos. The most striking impact of temperature regime involved short-term, adult

exposure, with constant temperature alleviating high-temperature effects on reproductive traits. If these findings are found to be broadly applicable, the differing effects of constant

versus fluctuating daily temperatures have important implications not only for the design and interpretation of simulated climate warming studies but also for the impact of warming on the

community composition of tropical terrestrial ecosystems. RESULTS CONSTANT TEMPERATURE REGIME LIFE HISTORY AND MORPHOLOGICAL TRAITS The high temperature (H) treatment, which involved a 3.5°C

increase above the current mean temperature of 27.3°C (the control (C) treatment) in a forested region of central Panamá, had a significant impact on survivorship, developmental rate,

morphology and level of sexual dimorphism. In the H treatment, the mean proportion of pseudoscorpions surviving from birth to adult was 0.67 compared to 0.80 in the control (_F_1,35 = 9.19,

_P_ = 0.0046). Temperature treatment did not significantly affect sex ratio (_F_1,35 = 1.62, _P_ = 0.2113), but the average proportion of adult male offspring was lower in the H treatment

(0.42) than in the control (0.47). Developmental time at elevated temperature was significantly reduced in both sexes (main effect of temperature: _F_1,139 = 96.22, _P_ < 0.0001; x_x_– ±

SE: C females = 40.38 days ± 0.70, H females = 34.80 days ± 0.71; C males = 44.41 days ± 0.71, H males = 37.06 days ± 0.75). Females developed significantly more rapidly than males (main

effect of sex: _F_1,139 = 18.19, _P_ < 0.0001) but the interaction between sex and temperature was not significant (_F_1,139 = 1.03, _P_ = 0.3113). More rapid maturation at high

temperature was associated with significantly smaller size for all morphological traits in the two sexes (_P_ < 0.0001). Reduced size and sex-specific effects of elevated temperature on

morphology were most apparent in sexually dimorphic traits of the pedipalps, appendages that are used in prey capture and male combat21. Chela hand depth, the most sexually dimorphic trait

(main effect of sex: _F_1,130 = 122.64, _P_ < 0.0001), displayed a much larger reduction in males than in females (sex x treatment interaction: _F_1,130 = 21.58, _P_ < 0.0001).

Consequently, degree of sexual dimorphism, i.e., mean male size relative to mean female size, decreased from 127% in the control to 113% in the high temperature treatment (Fig. 2).

Differences between the sexes in response to elevated temperature was less apparent in cephalothorax length, a sexually monomorphic trait, (main effect of sex: _F_1,139 = 0.85, _P_ = 0.3588;

sex x temperature interaction: _F_1,139 = 7.93, _P_ = 0.0056). Within each sex, scaling between chela hand depth and cephalothorax length was unaffected by temperature (males: _t_202 =

1.22, _P_ = 0.2237; females: _t_201 = 1.00, _P_ = 0.3166). Across the two temperature treatments, however, the allometric coefficient (±SE) was significantly higher in males than in females

(males: β = 1.430 ± 0.045; females: β = 0.841 ± 0.044; _t_404 = 9.367, _P_ < 0.0001). These results indicate that differences between males and females in allometry can explain the

reduced level of sexual dimorphism at high temperature. MALE FERTILITY TRAITS Temperature during development from birth to adult and temperature experienced as an adult were treated as fixed

factors in these analyses, each with C and H treatment levels. We controlled for potential age and size effects on fertility traits in males by including male chela hand depth and male age

as covariates, but neither significantly affected sperm number or sperm viability (_P_ > 0.05). Continuous exposure to high temperature during development dramatically reduced sperm

number (_F_1,79 = 56.00, _P_ < 0.0001). Males that developed to sexual maturity at high temperature and remained at high temperature as adults (H males) produced only 32% as many sperm as

control males (C males) (x_x_– ± SE: C males = 1,447 ± 112, H males = 457 ± 126). The short-term, 7-day exposure of C and H adult males to the alternative temperature treatment (C → H and H

→ C males) did not significantly affect sperm number (_F_1,79 = 0.26, _P_ = 0.6137) but the developmental x adult treatment interaction effect was significant (_F_1,79 = 4.46, _P_ =

0.0378). For sperm viability, the main effects of developmental temperature and adult temperature were not significant (_F_1,73 = 0.17, _P_ = 0.6842 and _F_1,73 = 1.51, _P_ = 0.2226,

respectively). However, there was a significant interaction between developmental and adult temperature treatments, with a mean sperm viability (±SE) of 0.91 ± 0.02 in H males compared to

0.96 ± 0.02 in C males (_F_1,73 = 5.21, _P_ = 0.0253). FEMALE REPRODUCTION Fecundity was evaluated by counting early-stage embryos and female reproductive success was quantified based on the

number of nymphs born to each female (see Ref. 19 for details). Female reproductive success is a function of both the female's fecundity and the fertility of her mate. We therefore

assessed the effect of temperature experienced throughout development and adulthood on reproduction in females, by pairing virgin females and males in all four possible treatment

combinations: C♀xC♂ (_n_ = 16); C♀xH♂ (_n_ = 16); H♀xC♂ (_n_ = 15) and H♀xH♂ (_n_ = 13). The proportion of females that produced a brood sac containing early-stage embryos varied

significantly across the four female x male mating combinations (Fisher's exact test _P_ = 0.0105), with only five of 28 H females (H♀xC♂ and H♀xH♂) becoming gravid (Fig. 3). Females

reared and maintained as adults at high temperature produced significantly fewer early-stage embryos than their control temperature counterparts (x_x_– ± SE: 3.29 ± 4.32 versus 27.91 ± 3.97;

main effect of female temperature treatment, _F_1,55 = 16.39, _P_ = 0.0002). In addition, females mated to males reared at high temperature produced significantly fewer embryos than females

mated to control males (x_x_– ± SE: 1.06 ± 4.19 versus 30.13 ± 4.11; main effect of male temperature treatment, _F_1,55 = 24.46, _P_ < 0.0001). Male treatment effects were limited to C

females because very few H females produced embryos. There was nonetheless a significant interaction between male and female treatment effects (_F_1,55 = 12.28, _P_ = 0.0009; Fig. 4). The

pattern for number of nymphs born was similar to that for early embryo production (main effect of female treatment: _F_1,55 = 17.41, _P_ = 0.0001; main effect of male treatment: _F_1,55 =

19.95, _P_ < 0.0001; female treatment x male treatment: _F_1,55 = 17.41, _P_ = 0.0001). Switching females between high and control incubators 1 week prior to mating demonstrated a

rescuing effect of short-term adult exposure to the control temperature for females that had developed at high temperature, with 18 of 29 producing embryos, compared to three of 15 H females

maintained at high temperature as adults (Fisher Exact Test, P = 0.0114). By contrast, 18 of the 30 C females switched to the H environment became gravid, compared to 11 of 16 C females

maintained as adults at the control temperature, indicating that females may be resistant to short-term exposure to high temperature as adults (Fisher Exact Test, P = 0.7500). For numbers of

embryos produced and nymphs born, developmental and adult temperature regimes were again analyzed as fixed factors, each with C and H treatment levels. Both the temperature at which females

developed (_F_1,67 = 10.14, _P_ = 0.0022) and the short-term temperature exposure at the adult stage (_F_1,67 = 8.04, _P_ = 0.0060) had statistically significant effects on the average

number of early-stage embryos produced. The interaction between developmental and adult treatments was not significant (_F_1,67 = 0.46, _P_ = 0.4979). This pattern was also evident for the

number of nymphs born (main effect of developmental treatment: _F_1,67 = 14.76, _P_ = 0.0003; main effect of adult treatment: _F_1,67 = 13.08, _P_ = 0.0006; developmental treatment x adult

treatment: _F_1,67 = 1.76, _P_ = 0.1888). COMPARISON OF CONSTANT VERSUS DIURNALLY FLUCTUATING TEMPERATURE REGIME EFFECTS For these analyses, data from this constant diurnal temperature study

and the previous diurnally-fluctuating temperature investigation16 were pooled and temperature regime incorporated as an additional fixed factor in the models. Temperature regime did not

significantly affect the impact of the projected 3.5°C increase in temperature on survival from birth to the adult stage or the proportion of male offspring produced (P > 0.05; data not

shown). However, there were significant regime effects on developmental time, morphological traits, male fertility traits and female reproductive function. The effect of regime on

development time was large and highly significant (_F_1,265 = 66.75, _P_ < 0.0001), with exposure to constant temperatures reducing developmental period for all possible regime x

temperature x sex treatment comparisons (Fig. 5). For both the sexually dimorphic trait, chela hand depth and the sexually monomorphic trait, cephalothorax length, there was a significant

main effect of regime, with larger size under the constant temperature regime (chela hand depth: _F_1,256 = 6.06, _P_ = 0.0145; cephalothorax length: _F_1,265 = 4.41, _P_ = 0.0366) and a

significant interaction between regime and temperature (chela hand depth: _F_1,256 = 12.71, _P_ = 0.0004; cephalothorax length: _F_1,265 = 6.00, _P_ = 0.0150). Sperm number was also

significantly higher under the constant temperature regime (_F_1,173 = 5.96, _P_ = 0.0156). Exposure to fluctuating temperatures decreased sperm number for all regime x temperature

combinations, with the effect of regime most apparent in C males switched to high temperature as adults (Fig. 6). A similar pattern was evident for sperm viability (_F_1,154 = 11.97, _P_ =

0.0007; Fig. 7). Overall, females were much more likely to become gravid under the constant temperature regime than under the fluctuating regime (56% versus 25%) and this difference was

highly significant (Fisher Exact Test, P = 0.0001). While the proportion of females becoming gravid was higher for all female treatment categories under constant temperature, the effect of

regime was again most apparent in the control to high temperature switch treatment. Sixty percent of the 30 constant regime C → H females became gravid, compared to 0% of the 21 fluctuating

regime C → H females (Fisher Exact Test, P < 0.0001). The effect of regime on number of early stage embryos produced by females was also statistically significant (_F_1,122 = 9.66, _P_ =

0.0023). As with sperm number and sperm viability, exposure to constant temperatures resulted in a higher number of embryos for all possible regime by temperature treatment combinations, but

again, the effect of regime was greatest in C→H treatment (Fig. 8). Although females also gave birth to more nymphs at constant temperature than under fluctuating temperature conditions,

the main effect of regime on number of protoymphs born was not statistically significant (_F_1,122 = 1.91, _P_ = 0.1695). DISCUSSION In a recently published study carried out under diurnally

fluctuating temperature regimes designed to simulate current and predicted temperatures in the species' natural environment, the projected 3.5°C increase in tropical temperatures by

the end of the 21st century had extremely detrimental consequences for important fitness-related traits in the harlequin beetle riding pseudoscorpion, _C. scorpioides_16. In the companion

study reported here, the same experimental design performed under constant diurnal temperatures yielded results that were qualitatively but not quantitatively similar to the fluctuating

temperature regime study. In both studies, the projected increase in temperature resulted in a marked reduction in survival, size, level of sexual dimorphism and sperm number and

dramatically decreased female fecundity and reproductive success. However, for most of the assayed fitness-related traits, pseudoscorpions exposed to constant temperatures were superior to

their fluctuating-temperature counterparts. At constant temperature, _C. scorpioides_ individuals were larger and developed faster than pseudoscorpions that experienced diurnally fluctuating

temperatures. Males produced more sperm and a higher percentage of viable sperm and females were more likely to become gravid and produced a greater number of early-stage embryos. The

strong effects of daily temperature regime reported here underscore the importance of taking into account both Jensen's inequality13 and thermal stress-mediated physiological disruption

in investigating the effects of climate warming on tropical ectotherms. Because ectothermic metabolic rate increases exponentially with temperature, the average metabolic rate of an

ectotherm across a range of diurnal temperatures should exceed its metabolic rate at the average daily temperature. This variance-driven increase in metabolic rate is likely to reduce both

the optimum and critical maximum temperatures, as has recently been demonstrated empirically in the mosquito, _Anopheles stephensi_ and theoretically in four tropical insect species11. In

addition, under fluctuating high temperature regimes, daily exposure to temperatures approaching the critical maximum temperature for tropical arthropods is likely to generate physiological

stress, damage associated with the metabolic production of reactive oxygen species22, and/or stress-induced activation or increased expression of transposable elements23,24,25. Heat shock

experienced during larval development has been shown to disrupt adult wing morphology in _Drosophila_26 and genes regulating physiological responses to heat shock appear to be particularly

vulnerable to transposable element-induced mutagenesis resulting from the insertion of _P_ elements into their promoters27. With diurnal temperatures in the tropics typically varying by

approximately 8°C (NOAA National Climate Data Center web site, http://www.ncdc.noaa.gov/), studies based on constant temperature regimes are likely to significantly underestimate the

negative effects of climate warming on the many tropical terrestrial ectotherms that experience daily fluctuations in temperature. In our study, depending on the traits involved, both

variance-driven and stress-related mechanisms appear responsible for the reduced performance of individuals subjected to fluctuating temperatures. Temperature regime did not significantly

affect survival and sex-specific survival to the adult stage, but fluctuating temperatures did result in small but significant reductions in developmental rate and body size, which, taken

together, are consistent with predictions of Jensen's equality. However, the effects of temperature regime were most marked in the experimental treatments involving short-term, adult

exposure, with constant temperature alleviating high-temperature effects on reproductive traits. For example, at constant temperature, the sperm packets of males switched to the elevated

temperature as adults contained an average of 1,646 sperm, compared to an average of only 1,051 for C → H males under the fluctuating temperature regime (Fig. 6). Similarly, at constant

temperature, females switched as adults to high temperature produced an average of 35.73 embryos, while none of their counterparts produced embryos under fluctuating temperature conditions

(Fig. 8). These results suggest that physiological stress resulting from an increase in daily maximum temperature rather than increase in mean daily temperature is the most critical factor

undermining reproductive success in this pseudoscorpion and that _C. scorpioides_ could at least partially circumvent the threat posed by climate warming through adult exploitation of

microhabitats that dampen daily fluctuations in temperature. At a broader level, species that reproduce and develop in exposed environments, such as phytophagous beetles, bugs and

butterflies, might be at much greater risk from climate warming than species, such as ants, bees and termites with subterranean nests, whose microhabitat use and/or social systems buffer

diurnal temperature fluctuations. It is important to acknowledge that several factors, in addition to microhabitat-based dampening of diurnal temperature fluctuations could alleviate the

negative impacts of climate warming on tropical ectotherms. From an abiotic perspective, climate models predict that diurnal temperature range will decrease due to greenhouse warming, with

daily maximum temperatures rising less rapidly than daily minimum temperatures5. However, this prediction has received equivocal empirical support, with diurnal range declining through the

mid 1980s28 but then followed by an increase between 1987 and 201029. Tropical ectotherms could respond to elevated temperature by range shifts to higher elevations or higher latitudes,

although latitudinal range shifts in the tropics are complicated by shallow latitudinal temperature gradients30. However, shifting range boundaries upslope is a plausible option for _C.

scorpioides_ and other saproxylic species in mountainous regions of Central and South America. In the province of Chiriqui in western Panamá, for example, the elevational range of this

pseudoscorpion spans at least 1500 m31. Nonetheless, warming-driven upward shifts in range are likely to result in the loss of considerable biodiversity, since tropical lowlands have no

source pool of species that have adapted to withstand higher temperatures30. Finally, populations of tropical ectotherms could respond through acclimation32, reduced metabolic sensitivity33

or adaptation34 to elevated temperatures, although recent studies suggest that tropical ectotherms have limited heritable genetic variation for heat resistance35,36. The natural history of

_C. scorpioides_ illustrates the complexity of species interactions in tropical ecosystems and their vulnerability to climate warming. _Cordylochernes scorpioides_ is distributed throughout

rain forests in Central and South America and inhabits decaying trees in the families Moraceae and Apocynaceae, particularly _Ficus_ species. The pseudoscorpion disperses between these rich

but patchily-distributed and ephemeral habitats by hitchhiking under the elytra of the giant harlequin beetle, _Acrocinus longimanus_21. Dying and newly dead trees bring in _A. longimanus_

adults for mating, oviposition and larval development. The wood-boring activity of the beetle larvae then creates a microhabitat of exfoliating bark and frass that is exploited by a diverse

community of invertebrate species that colonize the decaying tree31,37. While the significance of fig trees as keystone resources for folivorous and frugivorous vertebrates and invertebrates

has been extensively documented (reviewed in Ref. 38), the post-mortem importance of _Ficus_ in providing a habitat for saproxylic invertebrates that play an essential role in nutrient

recycling is less well appreciated. _Ficus_ species are unique in possessing an enclosed inflorescence, the syconium and are pollinated through reciprocally obligate relationships with

agaonid fig wasps that have an adult life span of only 1–2 days39. Although evaporative cooling in developing syconia moderates temperatures that would otherwise be lethal to fig wasp

larvae40, recent research suggests that an increase of 3°C in daytime temperature would reduce adult lifespan in four species of fig wasps by 33%–60%41. If fig wasps are more vulnerable to

increased temperature than longer-lived tropical species, the fate of the harlequin beetle riding pseudoscorpion and the myriad of other invertebrates and vertebrates that depend on fig

trees may ultimately hinge on the ability of fig wasps to live long enough to disperse and pollinate _Ficus_ in a rapidly warming tropical climate. METHODS With the exception of constant

temperature, the experimental methods employed in this study were identical to those reported for the diurnally fluctuating temperature study16 and will therefore be described only briefly

here. STUDY POPULATION AND DESIGN OF EXPERIMENT _Cordylochernes scorpioides_ were drawn from a subsequent generation of the same large laboratory population used in the diurnally fluctuating

temperature regime investigation16. To provide full-sibling families for this study, one virgin female from each of 36 families was mated to a randomly selected, unrelated male. Thirty of

these families were derived from the same matrilines represented in the diurnally fluctuating temperature study16. We employed a split-brood design and randomly assigned 24 first-stage

nymphs (protonymphs) from each female, 12 to a 27.3°C control treatment (C) and 12 to a 30.8°C high temperature treatment (H). These temperatures were determined by averaging the diurnally

fluctuating temperatures used in the previous study16. Identical Percival I-36NL incubators were used for the H and C temperature treatments, the incubators were located in the same room and

relative humidity was maintained at 85% in both temperature treatments. Nymphs were reared in individual vials to ensure virginity, as described elsewhere42. Developmental time was

determined by daily visual inspection of vials for the presence of an adult during the period between 27 days and 70 days after birth. We emptied and inspected vials not yielding an adult

after 70 days. If no nymphs or adults were found, individuals in these vials were scored as having died during development. The two sexes are morphologically indistinguishable as nymphs and

gender was therefore assessed only for individuals that survived to the adult stage (_n_ = 620). The effect of high temperature on male and female survivorship was assessed, using a paired

comparison by full-sibling family, of the proportion of survivors that were male in the two temperature treatments. MORPHOMETRICS To evaluate high temperature effects on male and female size

and degree of sexual dimorphism, adult morphometric data were obtained by holding individuals flat under a glass slide with the right pedipalp fully extended and photographing them at high

magnification (approximately 30×)16. ImageJ 1.43 (National Institutes of Health, USA) was then used to measure five pedipalp and cephalothorax trait for a large sample of these images (_n_ =

488). These traits, i.e., movable finger length, chela hand length, chela hand depth, tibia depth and cephalothorax length, are fixed in size at the final molt to the adult stage. We used

principal components analysis to extract composite measures of size (PC1) and shape (PC2) from these five traits. As in the previous study16, hand depth in males (_r_ = 0.99) and

cephalothorax length in females (_r_ = 0.92) were found to be the traits most closely correlated with PC1 score. Because of the strength of these correlations, female cephalothorax length

and male hand depth were used as size covariates in analyses of treatment effects on female and male reproductive function (see below). MALE FERTILITY TRAITS We evaluated male fertility

traits for a subset of males (_n_ = 139), reared and maintained at either the high or the control temperature, by staging matings between experimental males and non-experimental, virgin

females, collecting sperm packets and quantifying sperm number and sperm viability, as described elsewhere17. Briefly, each male was placed in an arena with a female and the mating was

observed under a stereomicroscope. Immediately after the spermatophore was deposited, the mating was interrupted and the sperm packet collected. Sperm packets were ruptured in phosphate

buffered saline and stained with SYBR 14 and propidium iodide (Invitrogen Live/Dead Sperm Viability Kit). Each 11 μL stained sample was pipetted onto a hemocytometer and viewed under an

Olympus BX51 fluorescence microscope. We estimated total number of sperm by multiplying sperm counted in a 0.9-μL volume of the sample by a factor of 12.2 (11 μL/0.9 μL). Sperm viability was

estimated as the proportion of live sperm in the sample. In addition, 20 C males and 15 H males were switched between treatment incubators seven days before mating, in order to distinguish

between long-term, developmental and short-term, adult exposure effects of temperature on male reproductive traits. Sperm number and sperm viability were then measured for these switch

treatment males, as indicated above. FEMALE REPRODUCTIVE FUNCTION Virgin females were paired with males in the four possible treatment combinations (C♀xC♂; C♀xH♂; H♀xC♂ and H♀xH♂). Pairs

were placed in a mating arena and given 40 min to mate. Typically, males produce a spermatophore within 7 min of encountering a female and previous research has established that 98% of

virgin females are sexually receptive20,43. _Cordylochernes scorpioides_ mated females exhibit one of the three possible outcomes: (i) failure to become gravid; (ii) production of a brood

sac containing embryos followed by spontaneous abortion of the entire brood, or (iii) brood production, in which embryos are carried to term and nymphs are born simultaneously. Beginning on

the fifth day after mating, each female was monitored until she gave birth, spontaneously aborted her brood of embryos or failed to become gravid within 30 d. Females that became gravid were

gently removed from their vials as soon as individual embryos became clearly discernable and digital images were taken of their brood sacs for early-stage embryo counting, as described

elsewhere19. We then returned females their vials and monitored them until they gave birth or spontaneously aborted the brood. Females remain in a silken nest constructed on the vial wall

throughout gestation and embryonic development and brood status can therefore be monitored without further disturbance to the female20. Nymphs were removed from the nest and counted within

24 h of birth. In order to differentiate between long-term, developmental and short-term, adult-exposure temperature effects on the ability of females to reproduce, we carried out a second

experiment, in which, seven days prior to mating, 30 females from the control treatment and 29 females from the high temperature treatment were switched between temperature treatment

incubators. We controlled for male effects in this “switch” experiment by mating all females to C males. Matings were carried out and female fecundity and reproductive success were

evaluated, as described above. STATISTICAL ANALYSES Temperature effects on survival, developmental time, morphology and male and female reproductive traits under the constant diurnal

temperature regime were analyzed, using a general linear mixed model (GLMM), as performed in PROC GLIMMIX in SAS, v9.344. We avoided pseudoreplication and controlled for genetic and maternal

effects by including full-sibling family identity in the models as a random effect. Because morphological traits were normally or approximately normally distributed, GLMMs for analyzing

these variables incorporated a Gaussian (Normal) distribution, an identity link function, a Laplace maximum likelihood approximation and the SAS containment method for determining degrees of

freedom44,45. Embryo and protonymph count data were square root transformed and analyzed as above. To accommodate non-normality and overdispersion in the developmental time and sperm number

data, GLMMs for analyzing these variables incorporated a log link function, a Gauss-Hermite Quadrature maximum likelihood approximation and a generalized Poisson mixed model for

overdispersed count data (see Ref. 44, pp. 3123–3124). Sperm viability was evaluated as the proportion of live sperm in an ejaculate and was not normally distributed. We therefore analyzed

this response variable, using the GLIMMIX logit link function to fit a binomial response variable44. To assess the effects of experimental diurnal temperature regime on _C.

scorpioides_' response to the 3.5°C projected temperature increase in the tropics, data from this constant daily temperature study and the previous fluctuating temperature study16 were

pooled and analyzed, as above, with diurnal temperature regime incorporated as an additional fixed factor in the models. For all hypothesis testing, we used two-tailed P values. REFERENCES *

Gillooly, J. F., Brown, J. H., West, G. B., Savage, V. M. & Charnov, E. L. Effects of size and temperature on metabolic rate. Science 293, 2248–2251 (2001). Article  CAS  ADS  Google

Scholar  * Clark, A. Temperature and the metabolic theory of ecology. Funct. Ecol. 20, 405–412 (2006). Article  Google Scholar  * Deutsch, C. A. et al. Impacts of climate warming on

terrestrial ectotherms across latitude. Proc. Natl. Acad. Sci. USA 105, 6668–6672 (2008). Article  CAS  ADS  Google Scholar  * Dillon, M. E., Wang, G. & Huey, R. B. Global metabolic

impacts of recent climate warming. Nature 467, 704–706 (2010). Article  CAS  ADS  Google Scholar  * IPCC. Climate Change 2007. In: The Physical Basis. Contribution of Working Group I to the

Fourth Assessment Report of the Intergovernmental Panel on Climate Change (eds Solomon, S. et al.) Cambridge University Press, Cambridge, UK (2007). * Addo-Bediako, A., Chown, S. L. &

Gaston, K. J. Thermal tolerance, climatic variability and latitude. Proc. R. Soc. Lond. B 267, 739–745 (2000). Article  CAS  Google Scholar  * Terblanche, J. S., Nyamukondiwa, C. &

Kleynhans, E. Thermal variability alters climatic stress resistance and plastic responses in a globally invasive pest, the Mediterranean fruit fly (_Ceratitis capitata_). Entomol. Exp. Appl.

137, 304–315 (2010). Article  Google Scholar  * Estay, S. A., Clavijo-Baquet, S. M. & Bozinovic, F. Beyond average: an experimental test of temperature variability on the population

dynamics of _Tribolium confusum_. Popul. Ecol. 53, 53–58 (2011). Article  Google Scholar  * Fischer, K., Kölzow, N., Höltje, H. & Karl, I. Assay conditions in laboratory experiments: is

the use of constant rather than fluctuating temperatures justified when investigating temperature-induced plasticity? Oecologia 166, 23–33 (2011). Article  ADS  Google Scholar  * Chen,

C.-Y., Chiu, M.-C. & Kuo, M.-H. Effect of warming with temperature oscillations on a low-latitude aphid, _Aphis craccivora_. Bull. Entomol. Res. 103, 406–413 (2013). Article  CAS  Google

Scholar  * Paaijmans, K. P. et al. Temperature variation makes ectotherms more sensitive to climate change. Glob. Change Biol. 19, 2373–2380 (2013). Article  ADS  Google Scholar  *

Blanford, J. I. et al. Implications of temperature variation for malaria parasite development across Africa. Sci. Rep. 3, 1300 (2013). Article  CAS  Google Scholar  * Jensen, J. L. Sur les

fonctions convexes et les inégalités entre les valeurs moyennes. Acta Math. 30, 175–193 (1906). Article  MathSciNet  Google Scholar  * Ruel, J. J. & Ayres, M. P. Jensen's inequality

predicts effects of environmental variation. Trends Ecol Evol 14, 361–366 (1999). Article  CAS  Google Scholar  * Paton, S. Meteorological and hydrological summary for Barro Colorado

Island. Smithsonian Tropical Research Institute (1994–2005 * Zeh, J. A. et al. Degrees of disruption: projected temperature increase has catastrophic consequences for reproduction in a

tropical ectotherm. Glob. Change Biol. 18, 1833–1842 (2012). Article  ADS  Google Scholar  * Bonilla, M. M., Zeh, D. W., White, A. M. & Zeh, J. A. Discriminating males and unpredictable

females: males bias sperm allocation in favor of virgin females. Ethology 117, 740–748 (2011). Article  Google Scholar  * Weygoldt, P. The Biology of Pseudoscorpions. Harvard University

Press, Cambridge (1969). * Koop, J., Zeh, D. W., Bonilla, M. M. & Zeh, J. A. Reproductive compensation favours male-killing _Wolbachia_ in a live-bearing host. Proc. R. Soc. Lond. B 276,

4021–4028 (2009). Article  CAS  Google Scholar  * Newcomer, S. D., Zeh, J. A. & Zeh, D. W. Genetic benefits enhance the reproductive success of polyandrous females. Proc. Natl. Acad.

Sci. USA 96, 10236–10241 (1999). Article  CAS  ADS  Google Scholar  * Zeh, D. W., Zeh, J. A. & Bermingham, E. Polyandrous, sperm-storing females: carriers of male genotypes through

episodes of adverse selection. Proc. R. Soc. Lond. B 264, 119–125 (1997). Article  ADS  Google Scholar  * Yang, L. H., Huang, H. & Wang, J. J. Antioxidant responses of citrus red mite,

_Panonychus citri_ (McGregor) (Acari: Tetranychidae), exposed to thermal stress. J. Insect Physiol. 56, 1871–1876 (2010). Article  CAS  Google Scholar  * Ratner, V. A., Zabanov, S. A.,

Kolesnikova, O. V. & Vasilyeva, L. A. Induction of the mobile genetic element Dm-412 transpositions in the _Drosophila_ genome by heat-shock treatment. Proc. Natl. Acad. Sci. USA 89,

5650–5654 (1992). Article  CAS  ADS  Google Scholar  * de la Vega, E. et al. Stress-induced gene expression profiling in the black tiger shrimp _Penaeus monodon_. Physiol. Genomics 31,

126–138 (2007). Article  CAS  Google Scholar  * Desalvo, M. K. et al. Differential gene expression during thermal stress and bleaching in the Caribbean coral _Montastraea faveolata_. Mol.

Ecol. 17, 3952–3971 (2008). Article  CAS  Google Scholar  * Williams, K. D., Helin, A. B., Posluszny, J., Roberts, S. P. & Feder, M. E. Effect of heat shock, pretreatment and _hsp70_

copy number on wing development in _Drosophila melanogaster_. Mol. Ecol. 12, 1165–1177 (2003). Article  CAS  Google Scholar  * Walker, J.-C., Chen, B. & Feder, M. E. Heat-shock

promoters: targets for evolution by _P_ transposable elements in _Drosophila_. PLoS Genet 2, e165 (2006). Article  Google Scholar  * Vose, R. S., Easterling, D. R. & Gleason, B. Maximum

and minimum temperature trends for the globe: An update through 2004. Geophys. Res. Lett. 32, L23822 (2005). Article  ADS  Google Scholar  * Rohde, R. et al. A new estimate of the average

earth surface land temperature spanning 1753 to 2011. Geoinfor Geostat: An overview. 1, 1 (2012). Google Scholar  * Colwell, R. K., Brehm, G., Cardelús, C. L., Gilman, A. C. & Longino,

J. T. Global warming, elevational range shifts and lowland biotic attrition in the wet tropics. Science 322, 258–261 (2008). Article  CAS  ADS  Google Scholar  * Zeh, J. A., Zeh, D. W. &

Bonilla, M. M. Phylogeography of the harlequin beetle-riding pseudoscorpion and the rise of the Isthmus of Panamá. Mol. Ecol. 12, 2759–2769 (2003). Article  CAS  Google Scholar  * Mitchell,

K. A. & Hoffmann, A. A. Thermal ramping rate influences evolutionary potential and species differences for upper thermal limits in _Drosophila_. Funct. Ecol. 24, 694–700 (2010). Article

  Google Scholar  * Williams, C. M. et al. Thermal variability increases the impact of autumnal warming and drives metabolic depression in an overwintering butterfly. PLoS ONE 7, e34470

(2012). Article  CAS  ADS  Google Scholar  * Williams, B. R., Van Heerwaarden, B., Dowling, D. K. & Sgrò, C. M. A multivariate test of evolutionary constraints for thermal tolerance in

_Drosophila melanogaster_. J. Evol. Biol. 25, 1415–1426 (2012). Article  CAS  Google Scholar  * Overgaard, J., Kristensen, T. N., Mitchell, K. A. & Hoffman, A. A. Thermal tolerance in

widespread and tropical _Drosophila_ species: does phenotypic plasticity increase with latitude? Am. Nat. 178, S80–S96 (2011). Article  Google Scholar  * Grigg, J. W. & Buckley, L. B.

Conservatism of lizard thermal tolerances and body temperatures across evolutionary history and geography. Biol. Lett. 9, 20121056 (2013). Article  Google Scholar  * Zeh, J. A. & Zeh, D.

W. Tropical liaisons on a beetle's back. Nat. Hist. 103, 36–43 (1994). Google Scholar  * Herre, E. A., Jandér, K. C. & Machado, C. A. Evolutionary ecology of figs and their

associates: recent progress and outstanding puzzles. Annu. Rev. Ecol. Evol. Syst. 39, 439–458 (2008). Article  Google Scholar  * Cook, J. M. & Rasplus, J.-Y. 2003 Mutualists with

attitude: coevolving fig wasps and figs. Trends Ecol. Evol. 18, 241–248 (2003). Article  Google Scholar  * Patino, S., Herre, E. A. & Tyree, M. T. Physiological determinants of _Ficus_

fruit temperature and implications for survival of pollinator wasp species: comparative physiology through an energy budget approach. Oecologia 100, 13–20 (1994). Article  CAS  ADS  Google

Scholar  * Jevanandam, N., Goh, A. G. R. & Corlett, R. T. Climate warming and the potential extinction of fig wasps, the obligate pollinators of figs. Biol. Lett. 9, 20130041 (2013).

Article  Google Scholar  * Zeh, D. W., Zeh, J. A. & Bonilla, M. M. Wolbachia, sex ratio bias and apparent male killing in the harlequin beetle riding pseudoscorpion. Heredity 95, 41–49

(2005). CAS  Google Scholar  * Zeh, J. A., Newcomer, S. D. & Zeh, D. W. Polyandrous females discriminate against previous mates. Proc. Natl. Acad. Sci. USA 95, 13732–13736 (1998).

Article  CAS  ADS  Google Scholar  * SAS Institute Inc. SAS/STAT® 9.3 User's Guide. Cary, NC: SAS Institute Inc. (2011). * Ferron, J. M., Bell, B. A., Hess, M. R., Rendina-Gobioff, G.

& Hibbard, S. T. Making treatment effect inferences from multiple-baseline data: the utility of multilevel modeling approaches. Behav. Res. Meth. 41, 372–384 (2009). Article  Google

Scholar  * Zeh, J. A., Bonilla, M. M., Adrian, A. J., Mesfin, S. & Zeh, D. W. From father to son: transgenerational effect of tetracycline on sperm viability. Sci. Rep. 2, 375 (2012).

Article  ADS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank La Autoridad Nacional del Ambiente (A.N.A.M.) for permission to collect pseudoscorpions in Panamá, the Smithsonian

Tropical Research Institute for extensive logistical support. This research was supported by grants from the National Geographic Society and the National Science Foundation (DEB-0721226) to

J.A.Z. and D.W.Z. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Biology and Program in Ecology, Evolution and Conservation Biology, University of Nevada, Reno, NV, 89557, Reno

Jeanne A. Zeh, Melvin M. Bonilla, Eleanor J. Su, Michael V. Padua, Rachel V. Anderson & David W. Zeh Authors * Jeanne A. Zeh View author publications You can also search for this author

inPubMed Google Scholar * Melvin M. Bonilla View author publications You can also search for this author inPubMed Google Scholar * Eleanor J. Su View author publications You can also search

for this author inPubMed Google Scholar * Michael V. Padua View author publications You can also search for this author inPubMed Google Scholar * Rachel V. Anderson View author publications

You can also search for this author inPubMed Google Scholar * David W. Zeh View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.A.Z. designed

the study, J.A.Z., M.M.B., E.J.S., M.V.P. and R.V.A. performed the experiments, D.W.Z. analyzed the data and J.A.Z. and D.W.Z. wrote the manuscript. ETHICS DECLARATIONS COMPETING INTERESTS

The authors declare no competing financial interests. RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a

copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Zeh, J., Bonilla, M., Su, E. _et al._ Constant

diurnal temperature regime alters the impact of simulated climate warming on a tropical pseudoscorpion. _Sci Rep_ 4, 3706 (2014). https://doi.org/10.1038/srep03706 Download citation *

Received: 15 July 2013 * Accepted: 13 November 2013 * Published: 15 January 2014 * DOI: https://doi.org/10.1038/srep03706 SHARE THIS ARTICLE Anyone you share the following link with will be

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