Chill-tolerant Gryllus crickets maintain ion balance at low temperatures

Journal of Insect Physiology 77 (2015) 15–25

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Chill-tolerant Gryllus crickets maintain ion balance at low temperatures Litza E. Coello Alvarado, Heath A. MacMillan 1, Brent J. Sinclair ⇑ Department of Biology, University of Western Ontario, London, ON, Canada

a r t i c l e

i n f o

Article history: Received 17 February 2015 Received in revised form 30 March 2015 Accepted 31 March 2015 Available online 3 April 2015 Keywords: Orthoptera Chilling injury Ion homeostasis Plasticity CTmin Chill coma Acclimation Cold tolerance

a b s t r a c t Insect cold tolerance is both phenotypically-plastic and evolutionarily labile, but the mechanisms underlying this variation are uncertain. Chill-susceptible insects lose ion and water homeostasis in the cold, which contributes to the development of injuries and eventually death. We thus hypothesized that more cold-tolerant insects will better maintain ion and water balance at low temperatures. We used rapid cold-hardening (RCH) and cold acclimation to improve cold tolerance of male Gryllus pennsylvanicus, and also compared this species to its cold-tolerant relative (Gryllus veletis). Cold acclimation and RCH decreased the critical thermal minimum (CTmin) and chill coma recovery time (CCR) in G. pennsylvanicus, but while cold acclimation improved survival of 0 °C, RCH did not; G. veletis was consistently more cold-tolerant (and had lower CCR and CTmin) than G. pennsylvanicus. During cold exposure, hemolymph water and Na+ migrated to the gut of warm-acclimated G. pennsylvanicus, which increased hemolymph [K+] and decreased muscle K+ equilibrium potentials. By contrast, cold-acclimated G. pennsylvanicus suffered a smaller loss of ion and water homeostasis during cold exposure, and this redistribution did not occur at all in cold-exposed G. veletis. The loss of ion and water balance was similar between RCH and warm-acclimated G. pennsylvanicus, suggesting that different mechanisms underlie decreased CCR and CTmin compared to increased survival at 0 °C. We conclude that increased tolerance of chilling is associated with improved maintenance of ion and water homeostasis in the cold, and that this is consistent for both phenotypic plasticity and evolved cold tolerance. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction At low temperatures, most insects enter chill coma (characterized by a loss of muscle function and lack of response to external stimuli) at a species-specific temperature, the critical thermal minimum (CTmin; MacMillan and Sinclair, 2011a; Mellanby, 1939). Insects in chill coma can recover with minimal evidence of permanent injury; recovery is typically measured as chill coma recovery time (CCR; e.g. David et al. 1998; MacMillan et al., 2012a). However, in many insects, prolonged exposure or exposure to temperatures below lethal limits can result in the accumulation of chilling injuries, and eventually death, even in the absence of ice formation (Koštál et al., 2006; MacMillan and Sinclair, 2011b). Mortality from chilling injuries, CCR, and CTmin are phenotypically plastic and can evolve, leading to variation within and among species.

⇑ Corresponding author at: Department of Biology, University of Western Ontario, London, ON N6A 5B7, Canada. Tel.: +1 519 661 2111x83138. E-mail address: [email protected] (B.J. Sinclair). 1 Present address: Zoophysiology, Department of Bioscience, Aarhus University, 8000 Aarhus C, Denmark. http://dx.doi.org/10.1016/j.jinsphys.2015.03.015 0022-1910/Ó 2015 Elsevier Ltd. All rights reserved.

Understanding the mechanisms that underlie variation in thermal tolerance is essential in comparing and predicting species’ thermal tolerances (Gaston et al., 2009; Somero, 2010). The CTmin, CCR, and susceptibility to chilling injury all vary among insect species and populations, with insects from colder environments generally having a lower CTmin, faster CCR, and lower incidence of injury or death following a cold stress (David et al., 2003; Hallas et al., 2002; Hoffmann et al., 2001). In addition, all three traits are phenotypically plastic; rearing temperature, hardening treatments of minutes to hours (termed rapid cold-hardening, RCH), acclimation over days or weeks, and different rates of cooling can all affect susceptibility to injury, as well as the CTmin and CCR (e.g. Colinet and Hoffmann, 2012; Findsen et al., 2013; Gibert and Huey, 2001; Gilchrist and Huey, 2001; Hoffmann et al., 2003; Koštál et al., 2004, 2006; Rajamohan and Sinclair, 2008; Ransberry et al., 2011; Sinclair and Roberts, 2005). Insect mortality in the cold can be caused by a range of phenomena, including apoptosis, protein misfolding, membrane phase transitions, and loss of ion balance (Teets and Denlinger, 2013). A wide variety of insects, including cockroaches (Koštál et al., 2006), true bugs (Koštál et al., 2004), locusts (Findsen et al., 2013), crickets (MacMillan and Sinclair 2011b), and Drosophila

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(MacMillan et al., 2015, in press) lose ion and water balance during exposure to the temperatures that cause chilling injury. The current model proposes that, at low temperatures, thermally-sensitive ion pumps lose activity and can no longer balance relatively thermally-insensitive passive leak. This model of ion balance disruption is based, in part, on the fall field cricket (Gryllus pennsylvanicus Burmeister [Orthoptera: Gryllidae]). In G. pennsylvanicus adults maintained at 25 °C, low temperature exposure leads to a net leak of Na+ down its concentration gradient from the hemolymph to the gut lumen. Water from the hemocoel also moves into the gut; and the resulting reduction in estimated hemolymph volume concentrates the K+ remaining in the hemolymph. Insect muscle resting potential is heavily dependent on extracellular [K+] (Djamgoz, 1987; Hoyle, 1954), and thus the rise in [K+] in the hemolymph depolarizes muscle cells (MacMillan and Sinclair, 2011b). With time spent at low temperature, continued depolarization of the potassium equilibrium potential (EK) leads to slower CCR and correlates with the onset of chilling injury and death in this species (MacMillan and Sinclair, 2011b; MacMillan et al., 2012a). Although this loss of ion balance appears to be closely tied to CCR (MacMillan et al., 2012a) and chilling injury (MacMillan and Sinclair, 2011a), it does not appear to be directly responsible for the onset of chill coma (Findsen et al., 2014; MacMillan et al., 2014). If ion and water balance disruption are a primary cause of chilling injury in Gryllus, variation in survival at low temperatures could arise through variation in the ability to maintain ion and water homeostasis in the cold. In Drosophila, decreased hemolymph [Na+] accompanies lower CTmin (both within and among species), which appears to be associated with suppression of Na+–K+-ATPase activity (MacMillan et al., 2015). In locusts, RCH decreases chill coma recovery time, apparently by increasing the rate at which homeostasis is re-established (Findsen et al., 2013). Together, these imply that both among-species variation in cold tolerance and phenotypic plasticity are tied to modulation of mechanisms of ion and water balance. However, it is not clear whether modified ion and water balance accompanies increased cold tolerance in other genera (such as Gryllus), nor is it clear whether RCH and (longer-term) cold acclimation operate through similar ionoregulatory mechanisms within a species. Here, we explore within-species variation in the ability to maintain ion and water balance at low temperatures. We do this by comparing low temperature maintenance of ion and water homeostasis among individuals of the fall field cricket, G. pennsylvanicus that experienced warm-acclimation (WA), rapid cold-hardening (RCH), or cold-acclimation (CA). Because even cold-acclimated G. pennsylvanicus still enter chill coma and are killed by long exposures to cold, we also compare low temperature ion and water homeostasis of G. pennsylvanicus to that of a more cold-hardy congener, the spring field cricket Gryllus veletis (Alexander and Bigelow). Whereas G. pennsylvanicus overwinters as a diapausing egg (which we do not examine here), G. veletis overwinters as a late-instar nymph (Alexander and Bigelow, 1960), and as a consequence we expect it to have evolved considerably greater cold tolerance than G. pennsylvanicus. This comparison allows us to determine whether loss of ion and water balance is specifically associated with the mortality we observe in G. pennsylvanicus, or is simply a property of Gryllus crickets when exposed to low temperatures. Although we are cognizant that a two species comparison does not allow us to draw specific conclusions about the adaptive nature of any variation we see (Garland and Adolph, 1994), it does give some insight into whether low temperature ion and water balance follow similar patterns in cold-adapted and non-adapted species. We hypothesize that cold tolerance is improved (lower CTmin, faster CCR, and increased survival of prolonged cold exposure)

through the maintenance of water and ion homeostasis at low temperatures, regardless of whether it arises from phenotypic plasticity or adaptive evolution. Thus, we predict that improvements in cold tolerance achieved through different forms of plasticity (RCH and acclimation) will be consistently associated with an improved ability to maintain ion and water balance within G. pennsylvanicus, and that the more cold-tolerant species (G. veletis) will similarly better maintain ion and water homeostasis in the cold. 2. Materials and methods Laboratory colonies of G. pennsylvanicus (originally collected on the University of Toronto at Mississauga campus in 2004) and G. veletis (Collected on the University of Lethbridge campus in 2010) were maintained as previously described by Judge et al. (2010) and reared under constant-temperature summer conditions (25 °C, 14L:10D photoperiod, 70% relative humidity). G. pennsylvanicus eggs required chilling a 4 °C for three months due to obligate diapause, but G. veletis eggs (which hatch in the summer, soon after being laid) did not. All experiments were conducted on adult male G. pennsylvanicus approximately seven weeks post final molt, and third and fourth instar male G. veletis (the overwintering stages; Alexander and Bigelow, 1960) at least 180 mg in body mass. We chose males because previous work (MacMillan and Sinclair, 2011b; MacMillan et al., 2012a,b) had been conducted on females, and we wished to expand our understanding of the responses to cold; also, because we were comparing mature (G. pennsylvanicus) and immature (G. veletis) individuals, we wished to reduce the physiological differences caused by significant resources devoted to egg production. 2.1. Acclimation treatments G. pennsylvanicus were divided into three groups: warm-acclimated (WA, standard rearing conditions), rapid cold-hardened (RCH) and cold-acclimated (CA). Crickets were transferred from their rearing bin into individual 177 mL translucent plastic cups (Polar Plastics, Summit Food Distributors Inc., London, ON, Canada) and provided food, water and a piece of egg carton as shelter. The RCH crickets were transferred from their rearing temperature to a cold room at +4 °C for four hours and given one hour to recover at 25 °C before being cold-exposed. Note that although the RCH pre-treatment is consistent with some RCH protocols (e.g. Nunamaker, 1993; Sinclair and Chown, 2003; Ransberry et al., 2011; Armstrong et al., 2012), it differs from those which utilise slow cooling to the pre-treatment temperature (e.g. Overgaard et al., 2005, 2007), and from others that do not include a recovery period (e.g. Lee et al., 1987; Rajamohan and Sinclair, 2008). Note that our RCH pre-treatment did not change all measures of low temperature performance (see results). Three week post-eclosion individuals in the CA treatment were cooled in an incubator (Sanyo MIR 154, Sanyo Scientific, Bensenville, Illinois) from 25 °C to 12 °C in 2.5 °C day1 steps, and kept at 12 °C under a 10L: 14D photoperiod, 70% RH, and ad libitum food and water for the subsequent three weeks (the entire acclimation therefore took four weeks). G. veletis and WA G. pennsylvanicus were kept under standard rearing conditions for the duration of the acclimation period. 2.2. Low temperature responses We measured critical thermal minima (CTmin) using a method modified from Klok and Chown (1997) and described by MacMillan and Sinclair (2011b). In keeping with these previous studies, crickets were not fasted prior to experiments. Briefly, we

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placed crickets individually into covered 200 ml glass beakers jacketed in an insulated acrylic enclosure and cooled by an ethylene glycol:water mixture (1:1 v:v) circulated from a refrigerated bath (Model 1157P, VWR, International, Mississauga, ON, Canada). The temperature inside each well was monitored with a type-T thermocouple connected to a computer via a Picotech TC-08 thermocouple interface and PicoLog software (Pico Technology, Cambridge, UK). The beakers were cooled from the rearing, recovery or acclimation treatment temperature at 0.25 °C min1 while crickets were observed continuously. We defined the CTmin as the first temperature at which the insect was no longer able to move in response to stimulation with a plastic probe. Chill coma recovery time (CCR) was measured using a method similar to that described by MacMillan et al. (2012a). We placed crickets individually into 14 mL plastic tubes and cooled them (as above) at 0.25 °C min1 from their rearing, recovery or acclimation temperature to 0 °C where they were held for 12 h. When removed from the chamber, we laid crickets on their dorsum in a petri dish at room temperature (22–24 °C) and recorded the time taken for individuals to voluntarily right themselves without stimulation. To assess survival following chronic cold exposure, we placed crickets individually into 14 mL plastic tubes, and cooled them at 0.25 °C min1 in a Tenney ETCU Series Chamber (Thermal Product Solutions, White Deer, PA, USA) from their rearing, recovery or acclimation temperature to 0 °C, where they were held for up to 120 h. We removed a subset of crickets from the chamber every 12 h, transferred them to individual cups containing food and water, and allowed them to recover for 24 h at 25 °C before survival assessment (after Koštál et al., 2006; MacMillan and Sinclair, 2011b). We considered crickets alive if they walked in a coordinated fashion and were able to perform a jump. Animals that were injured (walked in an uncoordinated fashion and were unable to jump), or dead (failed to respond to physical stimulus) were pooled for analyses. 2.3. Ion and water balance To assess the impact of cold exposure on ion and water homeostasis, we cooled crickets at 0.25 °C min1 from their rearing, recovery or acclimation temperature and held them at 0 °C (in the Tenney chamber described above). Every 12–24 h thereafter we removed a subset of individuals to a cold room at +4 °C for dissection of their gut and muscle tissue, and to extract hemolymph. Time 0 dissections were carried out at room temperature (24 °C). Tissue collection and ion content analysis followed previouslydescribed methods (MacMillan and Sinclair, 2011a). Briefly, we collected (and weighed, to estimate volume) hemolymph using a micropipette from an incision made at the coxal joint of a hind leg while applying gentle pressure to the abdomen. Muscle tissue was collected (and combined) from the femora of both hind legs using fine forceps to gently squeeze and separate the tissue from the hind leg exoskeleton. A dorsal incision was made from the tip of the abdomen to the back of the head, and the gut was clamped at each end with fine forceps and removed. We blotted all tissues gently on a kimwipe to remove residual hemolymph before storing them at 20 °C in sealed pre-weighed 200 lL tubes. Tissue water content was determined gravimetrically from the mass before and after being dried at 70 °C for 48 h. The total volume of hemolymph collected was used as an approximation of hemolymph volume (after MacMillan and Sinclair, 2011a; MacMillan et al., 2012a). Afterwards, a 200 lL aliquot of concentrated nitric acid was added to the dried samples, which were digested for 24 h at room temperature and then stored at 20 °C until used in analyses. Total tissue Na+ and K+ content were determined using atomic absorption spectrometry (AAS; Model iCE

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3300, Thermo Scientific, Waltham MA, USA) and comparisons to standard curves, as described previously (MacMillan and Sinclair, 2011a). We calculated the total content of ions in the hemolymph and gut, because we could estimate the total volume of hemolymph, and we removed the entire gut. This allowed us to examine the bulk movement of ions from hemolymph to gut. By contrast, we were unable to remove all of the muscle, so we calculated only the concentration of ions in muscle. By comparing concentration of ions in the hemolymph and muscle, we could calculate equilibrium potentials. Muscle equilibrium potentials were calculated using the Nernst equation (Eq. (1)):

EX ¼

RT ½Xo ln zF ½Xi

ð1Þ

where E is the equilibrium potential for ion X, R is the gas constant, T is the absolute temperature, z is the charge of the ion, F is the Faraday constant, [X]o is the external (hemolymph) and [X]i is the internal (muscle) concentration of the ion, respectively. 2.4. Data analysis We compared CTmin among treatments and species using a oneway ANOVA followed by a Tukey’s post hoc test in Sigma Plot (v12.2, San Jose, CA, USA). Chill coma recovery time was compared among species and treatments using Log Rank survival curve analyses followed by a Holm-Sidak multiple comparison post hoc tests in SPSS (v21.0. Armonk, NY: USA). We compared survival data among treatments and species in SPSS using a logistic regression with logit link and binomial error distribution. To account for changes in both ion and water content during cold exposure, we express ion data as both concentration and total tissue content. Tissue water and ion content was significantly correlated with tissue dry mass and body mass (P < 0.001), therefore analyses of water and ion content of the tissues (with the exception of hemolymph) were conducted on the residuals of a regression of ion content on dry mass. Estimated hemolymph volume was square-root-transformed prior to analysis to improve normality. We used generalized linear models built in R (version 2.1.3; R Development Core Team, 2010) to determine the effects of time, acclimation treatment, and species on water content, ion content and concentration in hemolymph, muscle gut tissue and equilibrium potentials between time point 0–12 h and across time points 12–120 h. We took this approach because there were clear differences in the direction and rate of change of most response variables between the first 12 h and the prolonged cold exposure (see also MacMillan and Sinclair, 2011a). A model simplification approach was used, beginning with the saturated model including time, group and their interaction (Crawley, 2005). Model terms were retained on the basis of Akaike’s Information Criterion until minimal adequate model was reached (Crawley, 2005). Tukey’s HSD post hoc tests were used to identify statistically-significant differences among the different treatment groups. Because G. veletis and G. pennsylvanicus are different species, if there was a significant difference across time or groups between WA G. pennsylvanicus and G. veletis, we evaluated the effects of time and treatment on G. veletis separately. 3. Results 3.1. Low temperature performance All G. pennsylvanicus crickets exposed to 0 °C for 12 h were in chill coma when removed to room temperature. Both RCH and

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3.2. Hemolymph volume and gut water content Estimated hemolymph volume decreased significantly with increasing time at 0 °C in all G. pennsylvanicus treatment groups (F1,120 = 98.64, P < 0.001; Fig. 2). The rate of decrease in hemolymph volume over the entire duration of cold exposure was slower in CA individuals than in WA and RCH individuals, such that estimated hemolymph volume at 120 h was significantly higher in CA individuals than in their RCH or WA counterparts (F2,120 = 13.71, P < 0.001, Fig. 2A). Male G. veletis are smaller than male G. pennsylvanicus (means of 271 ± 12.3 and 300 ± 8.9 mg, respectively), and therefore contain less total hemolymph (8.42 ± 1.42 vs. 16.41 ± 1.99 ll). The estimated hemolymph volume of G. veletis did not significantly change during exposure to 0 °C (F1,28 = 0.48, P = 0.496; Fig. 2B). Gut water content significantly increased during the first 12 h of cold exposure in all G. pennsylvanicus treatment groups (F1,31 = 13.26, P < 0.001), but then did not change significantly during the subsequent 108 h of cold exposure (F1,118 = 0.06, P = 0.811; Fig. 2C). Gut water content of G. pennsylvanicus did not differ among treatment groups during the first 12 h of cold exposure (F2,31 = 0.46, P = 0.638). However, in the subsequent 108 h at 0 °C, CA individuals had significantly less water in their gut at each point than did RCH and WA individuals, which did not differ (F2,118 = 7.14, P = 0.005; Fig. 2C). Gut water content in G. veletis did not change during the first 12 h of cold exposure (F1,10 = 0.41, P = 0.538) but increased significantly during the subsequent 108 h (F1,20 = 12.45, P < 0.005; Fig. 2D). Gut water content not differ significantly between WA G. pennsylvanicus and G. veletis after 120 h exposure to 0 °C (F1,56 = 0.01, P = 0.913). 3.3. Hemolymph and gut ion content Total hemolymph Na+ content significantly declined with increased time spent at 0 °C in all G. pennsylvanicus treatment groups (F1,100 = 49.20, P < 0.001; Fig. 3). However, the rate of decrease of hemolymph Na+ content during the last 108 h of cold exposure was slower in CA individuals compared to WA and RCH individuals (F3,88 = 31.27, P < 0.001; Fig. 3). The decrease in hemolymph Na+ content we observed in cold-exposed WA and RCH individuals was accompanied by an increase in gut Na+ content in first 12 h of exposure to 0 °C (F1,26 = 9.23, P < 0.001). Gut Na+ content did not change significantly during the subsequent 108 h of cold exposure (F1,105 = 3.66, P = 0.059; Fig. 3C). Conversely, gut Na+ content in CA G. pennsylvanicus did not significantly change

4

A: Chill-coma onset

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CA of G. pennsylvanicus lowered the CTmin (F3,48 = 54.62, P < 0.001) and shortened CCR following a 12 h exposure at 0 °C (Log-rank statistic = 79.16, df = 3 P < 0.001) in comparison to WA individuals (Fig. 1); CTmin and CCR did not differ between RCH and CA G. pennsylvanicus. All G. veletis individuals remained active during cold exposure, and were observed contracting their abdomen and moving their limbs while at 0 °C, so their recovery time (Fig. 1) represents the amount of time before a (cold) cricket righted itself when placed on its dorsum. The CTmin of G. veletis was lower than that of G. pennsylvanicus, regardless of the treatment applied to G. pennsylvanicus (F3,48 = 54.62, P < 0.001; Fig. 1). We observed >70% mortality in WA and RCH G. pennsylvanicus after 120 h exposure to 0 °C (Fig. 1C); there was no difference in survival between these groups (Wald v2: 18.88, df = 2, P = 0.340). By contrast, 75% of CA G. pennsylvanicus were uninjured after 120 h at 0 °C (Wald v2: 54.93, df = 1, P < 0.001; Fig. 1C). Several G. veletis individuals still exhibited abdominal contractions after a 120 h exposure at 0 °C (implying they were not in chill coma), and we did not observe mortality or signs of chilling injury in G. veletis exposed to 0 °C for up to 120 h (Fig. 1).

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Exposure to 0 °C (h) Fig. 1. Differences in cold tolerance between Gryllus species and induced by rapid cold hardening and cold acclimation in G. pennsylvanicus. Critical thermal minimum (A), chill coma recovery time after 12 h at 0 °C (B), and mortality after up to 120 h at 0 °C (C) of warm-acclimated (WA), cold-acclimated (CA), and rapid cold-hardened (RCH) G. pennsylvanicus, and G. veletis. Note that G. veletis was not in chill coma, and the ‘recovery’ time is therefore just the amount of time that elapsed before the (cold) cricket righted itself. All values are mean ± S.E.M. Different letters denote a significant difference in CTmin or CCR among treatments (Tukey’s HSD, P < 0.05). N = 10–14 crickets per species/treatment. See methods for details of thermal acclimation and RCH treatments.

during the first 12 h of cold exposure (F1,26 = 9.23, P = 0.217; Fig. 3). Hemolymph Na+ content in G. veletis did not change with increasing time exposed to 0 °C, (F1,20 = 1.05, P = 0.319, Fig. 3B). However, G. veletis gut Na+ content significantly increased (by approximately 213%) during the first 12 h of cold exposure (F1,19 = 10.36, P < 0.005; Fig. 3C) before remaining unchanged throughout the subsequent 108 h of cold exposure (F1,56 = 0.67, P = 0.418). The total content of potassium in the hemolymph varied considerably among individuals (even after correction for body size,

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Exposure to 0 °C (h) Fig. 2. Exposure to 0 °C causes a loss of hemolymph volume that is attenuated by cold acclimation or adaptation. Estimated hemolymph volume (A, B) and residual water content in gut (C, D) of warm-acclimated (WA), cold-acclimated (CA), and rapid cold-hardened (RCH) Gryllus pennsylvanicus, and G. veletis exposed to 0 °C for up to 120 h. All values are mean ± SEM. Lines denote a significant effect of cold exposure over time. Different letters at the end of each line denote a significant effect of treatment during cold exposure (Tukey’s HSD, P < 0.05). N = 6 per point/species/treatment. Error bars that are not visible are obscured by the symbols. See methods for details of thermal acclimation and RCH treatments.

resulting especially in high variance at the 20 h timepoint for WA G. pennsylvanicus), and did not significantly differ among G. pennsylvanicus treatment groups (F4,121 = 0.06, P = 0.994; Fig. 3D) or between G. veletis and WA G. pennsylvanicus (F2,49 = 2.60, P = 0.085; Fig. 3E), nor did hemolymph K+ content change with increasing time spent at 0 °C in any of the treatments or either species (F4,121 = 0.06, P = 0.898; Fig. 3); this lack of change was not modified by the removal of the 20 h timepoint for WA G. pennsylvanicus (not shown). Gut K+ content did not change over time in G. veletis (F1,20 = 0.12, P = 0.737; Fig. 3F). Gut K+ content increased significantly (but marginally) during cold exposure in G. pennsylvanicus WA and RCH individuals, but declined with cold exposure in CA individuals (F4,126 = 7.83, P = 0.005; Fig. 3F).

3.4. Hemolymph and muscle ion concentration Hemolymph [Na+] remained unchanged throughout cold exposure in all G. pennsylvanicus treatment groups (F2,88 = 0.15, P = 0.863; Fig. 4A). The hemolymph [Na+] of G. veletis significantly decreased during the first 12 h of cold exposure (t11 = 11.86, P < 0.001), but returned to control levels during the remainder of the cold exposure (F1,18 = 6.06, P < 0.05; Fig. 4B). Hemolymph [K+] did not significantly change in G. pennsylvanicus treatment groups during the first 12 h of cold exposure (F1,24 = 0.19, P = 0.670), but significantly increased in all G. pennsylvanicus groups during the last 108 h of cold exposure (F3,84 = 35.06, P < 0.001; Fig. 4C). This increase in hemolymph [K+] was similar in WA and RCH individuals (c. 40–50 mM; F3,84 = 35.06, P = 0.220), but the magnitude of hemolymph [K+] increase during cold exposure was reduced in CA individuals compared to the other groups (<20 mM;

F2,24 = 4.35, P < 0.005). Hemolymph [K+] did not change with cold exposure in G. veletis (F1,18 = 0.41, P = 0.528; Fig. 4D). Exposure to 0 °C did not significantly affect [Na+] in the muscle tissue of any G. pennsylvanicus treatment groups or G. veletis (F1,116 = 1.15, P = 0.286; Fig. 5). Muscle [K+] increased slightly throughout the last 108 h of cold exposure (albeit by less than 13%) for all G. pennsylvanicus treatment groups and by about 4% in G. veletis (F1,116 = 10.34, P < 0.01; Fig. 5).

3.5. Equilibrium potentials We used the concentrations of Na+ and K+ in the hemolymph and muscle to calculate the transmembrane equilibrium potentials (ENa and EK, respectively) for the muscle. ENa did not differ among G. pennsylvanicus treatments nor between WA G. pennsylvanicus and G. veletis during the 120 h exposure at 0 °C (F1,121 = 0.10, P = 0.957; Fig. 6). Across these comparisons, crickets with better low temperature performance (lower CCR and CTmin, increased survival at 0 °C) had consistently less polarized ENa compared to their less cold-hardy counterparts (Fig. 6A and B). Muscle EK was depolarized in all G. pennsylvanicus treatment groups during exposure to 0 °C for 120 h (F1,87 = 30.02, P < 0.001, Fig. 6C). The muscle EK of G. veletis also depolarized during the first 12 h of cold exposure, but reached a plateau around 54 mV and did not change during the last 108 h of cold exposure (F1,48 = 11.87, P = 0.574; Fig. 6D), nor was depolarization as extensive in G. veletis as it was in any of the other G. pennsylvanicus treatment groups (F1,48 = 12.94, P < 0.001).

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4. Discussion When exposed to 0 °C, water and sodium migrated from the hemolymph to the gut of warm-acclimated male G. pennsylvanicus, increasing hemolymph [K+], and depolarizing muscle EK, as previously reported for comparably-treated females of this species (MacMillan and Sinclair, 2011a; MacMillan et al., 2012a,b), and for other chill-susceptible insects, including Hemiptera, Blattaria, Diptera and other Orthoptera (Koštál et al., 2004, 2006; Findsen et al., 2013; MacMillan et al., 2015). Long-term cold acclimation – which decreased CCR and CTmin and improved survival following exposure to 0 °C – reduced the rate of this migration of water and ions, but did not prevent it altogether. Consistent with findings in locusts (Findsen et al., 2013), the rapid cold-hardening treatment also decreased CCR and CTmin; however, RCH did not markedly change tolerance to 0 °C. In contrast to our observations, Niehaus et al. (2012) were not able to induce a consistent acclimation response (under fluctuating thermal regimes) in G. pennsylvanicus.

The discrepancy with our results (we saw robust acclimation and RCH responses in chill coma onset and recovery) could be because the fluctuating acclimation regime used by Niehaus et al. (2012) did not provide sufficient cues to induce acclimation, because the traits they measured were not affected by cold acclimation, or because their population of G. pennsylvanicus has lost the capacity for acclimation. In most cases, the dynamics of ion and water homeostasis of RCH G. pennsylvanicus in the cold were similar to those of WA crickets of this species. By contrast, G. veletis nymphs were very chill-tolerant, with a low CTmin and no mortality at 0 °C (indeed, they probably did not enter chill coma). As predicted, G. veletis was also largely able to maintain ion and water balance during cold exposure. Cold-acclimated G. pennsylvanicus, which survived better during a chronic exposure to 0 °C, had significantly less water in their gut during the last 108 h at 0 °C compared to their warm-acclimated and RCH counterparts. The slower rate of hemolymph volume decrease, gut Na+ content increase, and lower overall

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Exposure to 0 °C (h) Fig. 4. Changes in hemolymph Na+ and K+ concentration during cold exposure. Concentration of Na+ (A and B) and K+ (C and D) in the hemolymph of warm-acclimated (WA), cold-acclimated (CA), and rapid cold-hardened (RCH) Gryllus pennsylvanicus, and G. veletis exposed to 0 °C for up to 120 h. All values are mean ± SEM. Panels B and D show G. veletis with WA G. pennsylvanicus for comparative purposes. Lines denote a significant effect of increasing time exposure to 0 °C, and different letters at the end of each line denote a significant effect of treatment during that same time period. N = 6 crickets per point. See methods for details of thermal acclimation and RCH treatments.

volumes of water in the gut of cold-exposed CA crickets suggest that cold acclimation results in changes that either reduce the permeability of the gut to Na+ and/or water (preventing water migration in the cold), or enhance the ability to remove Na+ and water from the gut back into the hemocoel at low temperatures. In G. veletis, we did not detect any change in estimated hemolymph volume with cold exposure, but we did detect a small but significant linear increase in relative gut water content over time. This increase is difficult to explain, given the lack of change in estimated hemolymph volume. It is possible that the gut contents changed over time: G. veletis were not in chill coma during cold exposure, and may have continued digesting during the five-day treatment. However, neither gut dry mass nor ion content changed systematically in the cold. It is also possible that this additional water is derived from changes in tissue hydration (which would be consistent with the changes in muscle [K+] observed in G. veletis). Further work, perhaps with isotope-labeled water, would be necessary to unravel the origin of this small increase in gut water during cold exposure. All of our observations are consistent with the current model for a loss of ion homeostasis that accompanies chilling injury (MacMillan and Sinclair, 2011b), and supports our hypothesis that improved cold tolerance is associated with an improved ability to maintain ion and water balance in the cold. We note that there is some disruption of ion and water homeostasis with no accompanying mortality in G. veletis, suggesting that this loss

of ion balance is not the only cause of low-temperature mortality, or that a specific threshold loss is necessary to cause injury. Thus, it is unclear whether mortality is a consequence of the loss of ion homeostasis or if loss of ion homeostasis is a symptom of tissue damage from other sources. It is probable that improved cold tolerance is accompanied by more complex processes, such as increased tolerance to EK perturbation, protection of cellular macromolecules from cold-induced disruption, or retardation of apoptotic signalling (see also Yi et al., 2007; Teets and Denlinger, 2013), in addition to a simple modification of ion homeostasis at the organismal scale such as we investigate here. Hemolymph [Na+] remained unchanged throughout the cold exposure in all G. pennsylvanicus treatment groups. This trend contrasts with the small, but significant decline observed in hemolymph [Na+] throughout a 120 h exposure to 0 °C in females of the same species (MacMillan and Sinclair, 2011b). The lack of change in hemolymph [Na+] despite a decrease in hemolymph Na+ content suggests that Na+ and water move into the gut of male G. pennsylvanicus in approximately equal proportions. Similarly, hemolymph [Na+] remains largely unchanged during cold exposure in D. melanogaster (MacMillan et al., 2015). At the cellular level, cold-acclimated G. pennsylvanicus had consistently lower muscle ENa before cold exposure than their less cold-hardy counterparts. G. veletis also had a lower muscle ENa than WA G. pennsylvanicus, suggesting that this may be a common pattern in more cold-hardy

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Exposure to 0 °C (h) Fig. 6. Sodium and potassium equilibrium potentials in cold-exposed crickets. Muscle equilibrium potentials for sodium (A and B) and potassium (C and D) in warmacclimated (WA), cold-acclimated (CA), or rapid cold-hardened (RCH) Gryllus pennsylvanicus, and G. veletis exposed to 0 °C for up to 120 h. All values are mean ± SEM. Lines denote a significant effect of cold exposure over time. Different letters at the end of each line denote a significant effect of treatment during that same time period (Tukey’s HSD, P < 0.05). N = 6 crickets per point. See methods for details of thermal acclimation and RCH treatments.

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species, and is also apparent in Drosophila (MacMillan et al., in press). The increase in hemolymph [K+] we observed in cold-exposed male G. pennsylvanicus is consistent with observations for female G. pennsylvanicus (MacMillan and Sinclair, 2011b). We observed high variance in hemolymph [K+] at the 120 h time point in WA and RCH G. pennsylvanicus. We hypothesize that this high variance reflects increasing chilling injury (and incipient mortality) after this cold exposure, and therefore inter-individual variation in chilling injuries accrued at that point. Because hemolymph K+ content does not change during cold exposure, decreased hemolymph volume, rather than increased quantity of extracellular K+ appears to drive an increase in hemolymph [K+] in both males and females in this species. The consequent depolarization of muscle EK (from c. 70 to c. 30 mV) in G. pennsylvanicus is probably sufficient to dissipate muscle membrane potential, as membrane potential closely follows EK in non-lepidopteran insects (Hoyle, 1953; Wood, 1957). Muscle EK of cold-acclimated individuals also depolarized during cold exposure, but the depolarization was slower than in the other groups, likely delaying the onset of muscle EK depolarization during a 120 h exposure to 0 °C. Overgaard and colleagues (Findsen et al., 2014; MacMillan et al., 2014) suggest that EK depolarization is not the primary cause of chill coma onset. This is supported by our observation that cold-acclimated individuals were in chill coma after as little as 12 h at 0 °C, despite maintaining muscle EK. Rapid cold-hardening improves cold tolerance after a short pretreatment, and has been reported in a range of insects and other arthropods to improve chilling tolerance, decrease CTmin and CCR, aid in the maintenance of reproductive behaviors after cold exposure and even increase freeze tolerance (Lee and Denlinger, 2010; Teets and Denlinger, 2013). However, there is a range of thresholds for inducing RCH (e.g. Sinclair and Chown, 2006), some species do not show an RCH response at all (e.g. Nyamukondiwa et al., 2011), and approaches to inducing RCH vary, depending on whether a slow cooling rate is used (e.g. Overgaard et al., 2005, 2007), and whether a recovery period is included in the RCH treatment (e.g. Nunamaker, 1993; Armstrong et al., 2012). In a previous study on beetles (Sinclair and Chown, 2006), RCH pre-treatments at 0 °C only elicited an RCH response if combined with a recovery period; therefore, we included a recovery period in our study to maximize the probability of eliciting an RCH response. Because the mechanisms underlying RCH are not yet fully understood (Teets and Denlinger, 2013), these different RCH treatments may be inducing different physiological processes, and may not be strictly equivalent. We found that an RCH treatment decreased CCR and CTmin, but had relatively little impact on survival of exposure to 0 °C in G. pennsylvanicus. In contrast to the effects of cold acclimation, our RCH treatment did not appear to modify the hemolymph volume or cation concentration dynamics during cold exposure. The similar pattern of muscle depolarization in warm-acclimated and RCH crickets suggests that RCH did not induce the physiological changes that CA induces to make G. pennsylvanicus individuals more resistant to a decrease in muscle EK. RCH had different impacts on three different measures of low temperature performance, which is consistent with the current model of chill coma recovery (MacMillan et al., 2012a), which posits that CCR is determined by the rate of recovery of ion concentrations, and further supports a separation of the mechanisms underlying CCR, CTmin, and chilling injury (Findsen et al., 2014; Sinclair and Roberts, 2005; MacMillan et al., 2014). Findsen et al. (2013) showed that an RCH pre-treatment increased the rate of ion balance recovery in locusts, which is consistent with our observation of faster CCR by RCH crickets. However, if improved tolerance to prolonged cold exposure requires substantial structural changes (e.g. remodeling of the cytoskeleton to reduce the impact of water imbalance, or to the gut epithelium to change the rate of

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ion leak), those may occur over a time frame that is incompatible with RCH, which could explain the lack of impact of RCH on survival of 120 h at 0 °C. We do note that some small structural changes, such as membrane remodeling, can occur very quickly, and are thought to be directly related to improved tolerance of acute cold shock after RCH (Teets and Denlinger, 2013), but that chilling injury is not thought to be mediated by membrane phase transitions (Lee, 2010). By contrast, stimulation of primary ion transport could be driven via signaling pathways (Teets et al., 2013; Terhzaz et al., 2015), and rapid post-translational modification and recruitment of existing proteins (e.g. McMullen and Storey, 2008; McMullen et al., 2010; Hou et al., 2014), and lead to immediate shifts in the re-establishment of ion homeostasis during CCR as a result of RCH. By all measures, G. veletis is more cold-tolerant than G. pennsylvanicus. Consistent with our expectation that increased cold tolerance is accompanied by maintenance of ion and water balance, G. veletis maintained estimated hemolymph volume, Na+ and K+ content, K+ concentration, and both muscle EK and ENa during cold exposure. However, there were small but steady increases in gut water content and hemolymph [Na+] in G. veletis during cold exposure. Cold-acclimated D. melanogaster maintain low hemolymph [Na+], a modification that is likely to reduce disruption of water balance during cold exposure by reducing the impact of Na+ on hemolymph osmolality (MacMillan et al., 2015). Given that G. veletis were not in chill coma, we may have been observing an ongoing acclimation response. The depolarization of muscle EK during the first 12 h of cold exposure in both G. veletis (which was not in chill coma) and G. pennsylvanicus (which was in chill coma) supports the suggestion that changed potassium concentrations are not the sole underlying cause of paralysis in chill coma (Findsen et al., 2014; MacMillan et al., 2014). Although we demonstrate that enhanced maintenance of ion and water homeostasis accompanies improved cold tolerance both within and among species, our study does not reveal the mechanisms that underlie the improved capacity for maintenance of ion homeostasis in cold-tolerant individuals or species. Improved cold tolerance could arise from changes in the rates or thermal sensitivity of ion transport, or the permeability of epithelia (particularly gut epithelia) to ions and water (or some combination of the two) (MacMillan and Sinclair, 2011b). In Drosophila, increased cold tolerance via acclimation or across species is accompanied by a decreased hemolymph [Na+], suggesting a shared mechanism for evolved and plastic responses to cold in that group (MacMillan et al., 2015). There is a prevailing hypothesis that basal tolerance constrains plasticity, such that more cold tolerant species have reduced capacity for phenotypic plasticity (Stillman, 2003), and this appears to hold true for Drosophila at low temperatures (e.g. Nyamukondiwa et al., 2011; but see Overgaard et al., 2011 for a different picture at high temperatures). Understanding the mechanisms underlying plasticity in other species groups such as Gryllus spp. will determine whether these constraints are consistent across species, or whether this mechanistic basis for the plasticity-basal cold tolerance trade-off is unique to Drosophila.

5. Conclusions Our data partially support our hypothesis: increased cold tolerance is indeed associated with maintenance of ion and water balance at low temperatures. However, although RCH significantly decreased both chill coma onset and recovery time in G. pennsylvanicus, ion and water balance dynamics in RCH crickets remained unchanged from warm-acclimated individuals, as did survival at low temperatures. Our data are consistent with the growing body of literature that suggests that different measures of cold tolerance

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can yield very different conclusions (Ransberry et al., 2011; Sinclair and Roberts, 2005; Andersen et al., 2015). However, our data do suggest that changes in water and ion balance dynamics contributing to increased survival of extended chilling. The model for the processes underlying chill coma onset and recovery (MacMillan et al., 2012a) has received empirical tests recently (e.g. Findsen et al., 2014; MacMillan et al., 2014), and although the link between ion balance and CCR remains robust, it increasingly appears that other processes determine CTmin. We observed a strong concordance between the changes in ion and water balance that accompanied improvement in cold tolerance with cold-acclimation in G. pennsylvanicus and the differences between G. pennsylvanicus and the more cold-hardy G. veletis. This suggests that maintenance of ion homeostasis at low temperatures underlies both within- and among-species variation in chilling tolerance, as it does in Drosophila (MacMillan et al., 2015).

Acknowledgements Thanks to Lisa Wu and Francis Dang for technical assistance and Lauren Des Marteaux and two anonymous referees for constructive comments on earlier drafts of the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) via a Discovery grant to BJS and a Canada Graduate Scholarship to HAM.

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