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Journal of Insect Physiology 57 (2011) 108–117

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Journal of Insect Physiology
journal homepage: www.elsevier.com/locate/jins

phys

Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)
Frank Chidawanyika, John S. Terblanche *
Department of Conservation Ecology and Entomology, Faculty of AgriSciences, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 September 2010 Received in revised form 30 September 2010 Accepted 30 September 2010 Keywords: Temperature limits Stress resistance Mortality Climate change Lethal temperatures Overwintering Diel ?uctuations

In order to preserve key activities or improve survival, insects facing variable and unfavourable thermal environments may employ physiological adjustments on a daily basis. Here, we investigate the survival of laboratory-reared adult Cydia pomonella at high or low temperatures and their responses to pre-treatments at sub-lethal temperatures over short time-scales. We also determined critical thermal limits (CTLs) of activity of C. pomonella and the effect of different rates of cooling or heating on CTLs to complement the survival assays. Temperature and duration of exposure signi?cantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between ?20 8C to ?5 8C and 32 8C to 47 8C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were ?12 8C for 2 h and 44 8C for 2 h. Investigation of rapid thermal responses (i.e. hardening) found limited low temperature responses but more pronounced high temperature responses. For example, C. pomonella pre-treated for 2 h at 5 8C improved survival at ?9 8C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 8C for 1 h markedly improved survival at 43 8C for 2 h from 20% to 90% (p < 0.0001). We also examined cross-tolerance of thermal stressors. Here, low temperature pre-treatments did not improve high temperature survival, while high temperature pre-treatment (37 8C for 1 h) signi?cantly improved low temperature survival (?9 8C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not signi?cantly affected by cooling at rates of 0.06, 0.12 and 0.25 8C min?1 (CTmin range: 0.3–1.3 8C). By contrast, critical thermal maxima (CTmax) were signi?cantly affected by heating at these rates and ranged from 42.5 to 44.9 8C. In sum, these results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. We discuss these results in the context of local agroecosystem microclimate recordings. These responses are signi?cant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects. ? 2010 Elsevier Ltd. All rights reserved.

1. Introduction Temperature plays a key role in the life of insects. Over longer periods, temperature in?uences seasonality and evolutionary responses of insects (Bale, 2002; Lee and Denlinger, 2010; Chown and Nicholson, 2004). However, the ability of diel temperature ?uctuations to affect activity and survival at short-time scales is also of critical importance. Indeed, insect responses to temperature extremes over short periods may be an important driver of population dynamics and, consequently, species’ abundance and geographic distribution over longer timescales (reviewed in Bale, 2002; Chown and Terblanche, 2007; Lee and Denlinger, 2010; Hoffmann, 2010). Insect responses to temperature extremes may

* Corresponding author. Tel.: +27 21 808 9225; fax: +21 21 808 3304. E-mail address: jst@sun.ac.za (J.S. Terblanche). 0022-1910/$ – see front matter ? 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.09.013

also be essential in phenological or distribution modelling of climate change impacts (e.g. Estay et al., 2009; Lima et al., 2009; Hazell et al., 2010a; reviewed in Bale, 2002). In addition, most current control methods used in quarantine and post-harvest pest control involve some form of temperature treatment (Neven and Hansen, 2010). However, insect survival in variable thermal environments has been known to be in?uenced by a host of factors including the rate of temperature change (Powell and Bale, 2006; Terblanche et al., 2007; Mitchell and Hoffmann, 2010), thermal history (or acclimation/acclimatization) (Nyamukondiwa and Terblanche, 2010; Hoffmann et al., 2005; Hazell et al., 2010b), and pre-exposure to sub-lethal environments enabling them to survive otherwise lethal ambient temperatures (Ta) (e.g. Powell and Bale, 2005; Loeschcke and Hoffmann, 2007; Slabber and Chown, 2005). Such plasticity of thermal tolerance may make some quarantine protocols less effective in controlling pests if the protocol itself results in enhanced thermal tolerance (Stotter and

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Terblanche, 2009; and see discussions in Denlinger and Lee, 2010) or, on the other hand, may be manipulated to enhance temperature-dependent performance and survival and perhaps also bene?t control programmes (Bloem et al., 2006; Chidawanyika and Terblanche, in press). When threatened by temperature extremes, insects employ a range of mechanisms to adjust their body temperature (Tb), or the extremes they can withstand, using either physiological or behavioural mechanisms or some combination of both. For example, an insect experiencing adverse high Ta can lower its Tb ¨ by avoidance of sunny hot spots and vice versa (e.g. Kuhrt et al., 2006; Huey and Pascual, 2009). However, behavioural adjustments only act as the ?rst line of defence against sub-optimal Ta and depend to a large degree on microsite opportunities in their habitat ¨ (Kuhrt et al., 2006). If unfavourable Ta persist, physiological mechanisms may become critical to ensure survival. Examples of such physiological adjustments include alteration of thermal tolerance at daily (e.g. Sinclair et al., 2003; Overgaard and S?renson, 2008) or seasonal (e.g. Khani et al., 2007; Khani and Moharramipour, 2010) time-scales. Temperatures lethal to insects are a function of both the magnitude of the temperature variation and the duration of exposure (Chown and Nicholson, 2004; Angilletta, 2009; Denlinger and Lee, 2010). However, phenotypic plasticity of thermal tolerance means that insects can modify the time–temperature phase space, thereby promoting survival. Induction of such plastic responses can be achieved after pre-exposure to sub-lethal temperatures or perhaps also in anticipation of extremes (discussed in Chown and Terblanche, 2007), enabling insects to survive what would otherwise be lethal conditions. However, insects unable to increase thermal tolerance through rapid plastic responses may have even greater mortality during the subsequent exposure (e.g. Terblanche et al., 2008). Acute pre-exposures altering thermal tolerance have been referred to as ‘hardening’ responses and sometimes also as cold or heat ‘shock’ (Bowler, 2005; Sinclair and Roberts, 2005; Loeschcke and S?renson, 2005). Here, we use ‘hardening’ to refer to the physiological responses which are of primary interest. Rapid coldhardening (RCH) or rapid heat-hardening (RHH) not only helps to improve survival in lethal conditions but can also help organisms to continue performing routine activities, such as mating and feeding, despite adverse conditions (e.g. Fasolo and Krebs, 2004) and can thereby increase ?tness (e.g. Powell and Bale, 2005 and see discussions in Lee and Denlinger, 2010). Mechanisms of injury caused by extreme temperatures in insects vary from cellular to tissue levels and a range of biochemical responses are probably signi?cant to counter potentially deleterious effects. In freeze intolerant insects, low temperature injury is largely regulated by a depression of supercooling (freezing) point of the body, typically involving polyhydric alcohols (polyols) and sugars which act as cryoprotectants. Also of importance to survival in these insects is removal of potential nucleating agents through, for example, cessation of feeding (Bale, 2002). However, some freeze intolerant insects die at temperatures well above their supercooling point and this type of injury is thought to be related to neuromuscular damage at the tissue level, while at the cellular level injury is attributed to membrane phase transitions, thermoelastic stress and damage to essential proteins (Lee and Denlinger, 1991, 2010; Bale, 2002; Chown and Nicholson, 2004). In freeze tolerant insects, low temperature injury may be associated with extracellular ice formation or the re-establishment of ion homeostasis after thawing and, consequently, much attention has been given to ice nucleating agents, antifreeze proteins and cryoprotective ˇ ? sugars and polyols (e.g. Kostal et al., 2007; Duman et al., 2004). High temperatures result in the disruption of membrane function, DNA lesions, changes in cell microenvironment, and protein

denaturation which can restrict enzyme-catalysed reactions (Chown and Nicholson, 2004). Insects under high temperature stress may produce heat-shock proteins (Hsps) (e.g. McMillan et al., 2005) which act as molecular chaperones protecting other cellular proteins and conserving key enzyme function. Heat shock proteins have also been implicated in low temperature tolerance (e.g. Rinehart et al., 2007) although the role of Hsps over short time-scales (i.e. hardening responses) is more contentious (Sinclair and Roberts, 2005; Chown and Nicholson, 2004). Over brief periods of low temperature exposure, membrane phospholipid composition may also be radically altered to enhance low temperature tolerance (e.g. Overgaard et al., 2006; but see MacMillan et al., 2009). Insects exposed to low temperatures over longer periods, e.g. during initiation of overwintering or entering diapause, may increase the synthesis of various polyols or sugars which function as cryoprotectants and can lower the risk of freezing. For example, glycerol or sorbitol concentration can be elevated during overwintering and is associated with a decrease in supercooling point (Minder et al., 1984; Khani et al., 2007). In this study, we investigate how various acute temperature changes affect the activity limits and survival of 1–2-day-old adult codling moth, Cydia pomonella (Lepidoptera Tortricidae), a polyphagous pest of global agricultural importance (Barnes, 1991; Dorn et al., 1999). Most work to date investigating thermal tolerance of C. pomonella has focused on larvae for post-harvest control and fruit disinfestation (e.g. Neven and Reh?eld-Ray, 2006; Neven and Hansen, 2010) or overwintering physiology (Khani et al., 2007; Khani and Moharramipour, 2010). When other lifestages of C. pomonella have been investigated, these have typically employed extreme, fairly ecologically unrealistic thermal conditions which may nevertheless allow comparison among life-stages (e.g. Wang et al., 2004). Here we speci?cally focus on adult thermal biology as this is the life-stage responsible for reproduction and probably most dispersal in natural and agricultural conditions (Barnes, 1991; Timm et al., 2010). In addition, it is the life-stage used for the sterile insect technique (SIT) control programme (Botto and Glaz, 2010; Vreysen et al., 2010). The SIT programme for C. pomonella usually includes cooling moths for a brief period for ease of handling and to avoid excess damage during transportation, prior to release in the wild (Carpenter et al., 2010; Simmons et al., 2010). However, it is unclear how such chilling, or indeed, short term temperature ?uctuations more generally, may in?uence adult C. pomonella activity and survival upon release in orchards (but see Bloem et al., 2006). It is therefore important to understand how thermal history might in?uence performance and survival as this could help improve quarantine or post-harvest control procedures, or alternatively, may be used to enhance SIT ef?cacy (Bloem et al., 2006; Simmons et al., 2010; Chidawanyika and Terblanche, in press). The aims of this study were several-fold. First, we determined the range of time–temperature combinations which may be lethal at short time-scales to give insight into population dynamics and ?tness of the species (Gilchrist and Huey, 2001; Loeschcke and Hoffmann, 2007). Second, we examined a range of conditions which might induce rapid cold- or rapid heat-hardening responses and thus investigated short-term, plastic responses of survival at extreme temperatures. Third, we assessed cross-tolerance of temperature by assessing survival at low temperatures and their responses to a brief high temperature pre-treatment and vice versa (i.e. responses of high temperature survival after low temperature pre-treatment). Finally, we measured critical thermal limits to activity at high and low temperatures and assessed their plasticity by varying the rates of temperature change in these dynamic assays. These results are discussed in the context of local agroecosystem microclimate recordings and survival of adult C. pomonella.

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2. Methods 2.1. Insect culture The C. pomonella culture used for our experiments was originally established in 2004 at the Deciduous Fruit Producer’s Trust (DFPT) Stellenbosch rearing facility. Rearing was done on a diet described by Guennelon et al. (1981) on trays of food medium for developing larvae. Pupae were held in darkened cardboard boxes (800 mm3) for adult eclosion in the laboratory under (12:12) (L:D) photoperiod in air-conditioned, insulated rooms at 25 ? 1 8C. On emergence, all adult moths had access to 50% sugar/water solution until they were used in thermal tolerance assays as 24–48-h-old adults. This age class was used as it represents the moths typically used for SIT release. Despite the access to the sugar/water solution, we could not distinguish between individual moths that fed from those that did not. Hence, feeding status was not strictly controlled for in these trials but we maintained high sample sizes to randomize these effects across treatments. 2.2. Lethal temperature assays Programmable water baths (GP200-R4, Grant Instruments, UK) were used to measure thermal tolerance using a direct plunge protocol (as in e.g. Sinclair et al., 2006; Terblanche et al., 2008) to determine both the upper lethal temperature (ULT) and lower lethal temperature (LLT) for a range of times (from 0.5 to 4 h). A mixture of propylene glycol and water (1:1 ratio) was used to enable water baths to operate at sub-zero temperatures without freezing. Live 1–2-day-old adult insects were placed in 60 ml polypropylene vials (n = 10 in each vial ? 5 vials) for each temperature/time treatment until a range of 0–100% mortality was covered. Relative humidity (RH) in the ULT experiments was

controlled and maintained at >80% RH using strips of ?lter paper moistened with drops of distilled water suspended from the perforated lids of the vials to avoid desiccation-related mortality. However, care was taken to ensure that there was no free water in the vials during experiments to avoid accidentally drowning the insects. Temperatures in water baths were veri?ed using NIST certi?ed thermometers before each treatment (as in e.g. Stotter and Terblanche, 2009). Vials containing the assayed C. pomonella were placed in a 25 ? 1 8C climate chamber for 24 h whereupon survival was recorded. For the purposes of this study, survival was de?ned as coordinated muscle response to stimuli such as gentle prodding, or normal behaviours such as feeding, ?ying or mating. 2.3. Rapid thermal responses Rapid cold-hardening and rapid heat-hardening experiments were performed using established protocols (e.g. Terblanche et al., 2008; Stotter and Terblanche, 2009; Sinclair and Chown, 2003). In most cases, we used a discriminating temperature at which 25% survival was estimated in the LLT and ULT experiments. The magnitude of pre-treatment temperature and duration of exposure was varied using different temperatures, plunge, gap and ramping rate treatments to allow for potential Hsp responses (for rationale, see e.g. Sinclair and Chown, 2003; Stotter and Terblanche, 2009) (Fig. 1). In all cases, control groups were also included for each day assays were performed in order to eliminate any bias related to handling stress or cohort effects. To investigate the mechanism used by C. pomonella to improve survival after low or high pretreatment, we also undertook cross-tolerance experiments (e.g. MacMillan et al., 2009). In brief, moths were pre-treated at nonlethal low temperatures and then mortality was assayed at lethal high temperatures, or vice versa, before their survival was scored after 24 h recovery at 25 8C.

[()TD$FIG]

Fig. 1. Schematic diagram of experimental protocols for plunge (A), gap (B) and combination of cooling (ramping) and plunge (C) treatments that were followed to elicit hardening responses in adult Cydia pomonella. For a full description see Section 2.3.

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2.4. Effects of ramping rates on critical thermal limits We assessed CTLs under a range of heating and cooling rates to determine if RCH and RHH responses may be elicited by different rates (e.g. Powell and Bale, 2006; Overgaard et al., 2006), and to assess ecologically relevant CTLs. In these experiments, moths were individually placed in insulated double-jacketed series of chambers (‘organ pipes’) connected to a programmable water bath and subjected to different constant rates of heating or cooling (0.06, 0.12 and 0.25 8C min?1) starting from 25 8C to determine their critical thermal maximum (CTmax) and critical thermal minimum (CTmin), respectively (see e.g. Nyamukondiwa and Terblanche, 2010; Mitchell and Hoffmann, 2010). A mixture of water and glycol solution (1:1 ratio) was used in the water bath to enable sub-zero operation. Upon placement in the ‘organ pipes’, moths were given 10 min to equilibrate at 25 8C before temperature ramping started and their CTmax or CTmin was measured. A copper-constantan (Type T, 36 SWG) thermocouple connected to a digital thermometer (Fluke 53/54II, Fluke Cooperation South Africa; accuracy 0.01 8C) was inserted into the control chamber to detect and verify organ pipe chamber temperatures. One concern that might be raised with using different cooling or heating rates for CTL assessment is that thermal inertia might in?uence the results obtained thereby resulting in a difference between body and chamber temperature, especially at faster rates for larger individuals. However, it is well established that at such small body sizes and at these relatively slow rates of temperature change, there is little difference between organism body temperature and air temperature (for theoretical discussion, see e.g. Stevenson, 1985; Campell and Norman, 1998). This has also been veri?ed in a ?y of similar size to the moth in our study by recording body and chamber temperatures during experimental ramping (Terblanche et al., 2007). Here we therefore assumed the same was true and that body temperature of the moths was in equilibrium with the chamber temperature. The CTmin and CTmax were de?ned as the temperature at which a moth lost coordinated muscle function or experienced onset of muscle spasms, respectively (e.g. Nyamukondiwa and Terblanche, 2010). Moths were never removed from the organ pipes to assess behaviour. Because age can have a major effect on thermal tolerance of insects (Bowler and Terblanche, 2008; Nyamukondiwa and Terblanche, 2009) it was strictly controlled in all assays. For CTL assays, 1–2-day-old moths which had access to sugar and water solution (1:1 ratio) were used in all experiments. However, gender was not taken into consideration as preliminary assays showed no effect on C. pomonella CTLs. Each individual moth was treated as a replicate and twenty individuals were used per ramping rate for all CTmin and CTmax experiments. Individuals used in CTmin assays were not re-used for CTmax assays and were discarded. 2.5. Statistical analyses Temperature–time interaction effects on survival (number of moths alive/total moths exposed as the dependent variable) for both lower and upper lethal limits were analysed using non-linear models (proc probit) in SAS 9.1 (SAS Institute Inc., Cary, USA). Tests for signi?cance of temperature, duration of exposure and their interactions were undertaken using generalized linear models (Wald x2 test) with a single degree-of-freedom approach, corrected for overdispersion and assuming a bimodal distribution. A wafer-estimation approach in Statistica 9.0 (Statsoft, Oklahoma, USA) was used to generate surface plots of time and temperature effects on survival (as in e.g. Stotter and Terblanche, 2009). Generalized linear models (GLZ), performed in SAS 9.1 (proc genmod) were used to assess the effects of hardening pretreatments on the survival of C. pomonella. A bimodal distribution

was assumed for survival data with a logit link function and correction for overdispersion. In addition, all comparisons of pretreatments were made relative to the corresponding replicated control groups. Analyses of high and low pre-treatments were also performed separately to avoid biased outcomes of statistical tests due to pooling of test groups. Similar GLZ analyses were also used to test the signi?cance of hardening pre-treatments on the crosstolerance of C. pomonella. Statistically homogenous groups were identi?ed using overlap in 95% con?dence limits. The effects of ramping rates on critical thermal limits were compared using One-Way ANOVAs in Statistica 9.0 (Statsoft, Tulsa, OK, USA). In this instance, the categorical predictor was the ramping rate (0.06, 0.12 or 0.25 8C min?1) and the dependent variable was either CTmin or CTmax. Key assumptions of ANOVA were checked and were met for homogeneity of variance and normality of data distributions. Tukey’s HSD post hoc tests were used to identify statistically homogenous groups at p = 0.05. 3. Results 3.1. Lethal temperature assays The temperature and time period C. pomonella were exposed to signi?cantly affected their survival at either high or low temperatures (Table 1). An increase in severity of exposure at low or high temperatures resulted in increased mortality (Figs. 2 and 3). Similarly, an increase in the duration of exposure at any given temperature resulted in a reduction in C. pomonella survival (Figs. 2 and 3). The interaction of temperature and the duration of exposure was highly signi?cant resulting in shorter periods of time required to in?ict 100% mortality at extremely severe low or high temperatures, suggesting limited plasticity of survival in these trials (Table 1; Figs. 2 and 3). 3.2. Effect of ramping rates on critical thermal limits Critical thermal minima were not signi?cantly affected by variation in cooling rates (F2,57 = 2.322, p = 0.107) (Fig. 4a). By contrast, CTmax was signi?cantly affected by variation in heating rates (F2,57 = 19.28, p < 0.0001). However, mean CTmax for moths exposed to heating rates of 0.12 and 0.25 8C min?1 were statistically homogenous while 0.06 min?1 yielded higher CTmax (Fig. 4b). 3.3. Rapid thermal responses Low temperature pre-treatment of ?7, 0 or 5 8C for 1 h signi?cantly reduced survival of C. pomonella relative to the
Table 1 Summary of probit non-linear analyses results of the effects of temperature and duration of exposure on the survival of Cydia pomonella. Tests of signi?cance were done using generalized linear models (GLZ) (Type III) analyses assuming a logit link function and correcting for overdispersion. Five replicates of ten individuals were used for both low (n = 1700 moths, n = 170 replicates) and high (n = 2000 moths, n = 200 replicates) temperature treatments. Lethal temperatures ranged from ?20 to ?5 8C and 32 to 47 8C for 0.5–4 h treatments (see Figs. 2 and 3). Parameter Lower lethal temperature Intercept Time Temperature Time ? temperature Upper lethal temperature Intercept Time Temperature Time ? temperature d.f. 1 1 1 1 Estimate ? S.E. 23.0510 ? 2.8417 3.8732 ? 0.5852 1.9595 ? 0.2363 0.0485 ? 0.0485 35.6723 ? 2.9514 3.4180 ? 0.7700 0.8428 ? 0.0690 0.0834 ? 0.0179

x2
65.80 43.80 68.75 49.01

p-Value

<0.0001 <0.0001 <0.0001 <0.0001

1 1 1 1

146.08 19.71 149.34 21.80

<0.0001 <0.0001 <0.0001 <0.0001

[()TD$FIG]

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[()TD$FIG]

Fig. 2. Mean survival (?95% con?dence limits (CL)) of C. pomonella under different low temperatures during different durations of exposure (A). Three-dimensional surface plot of the relationship between survival of C. pomonella, low temperature and time ?tted using the wafer estimation method in Statistica (B). Data points for the ?gures represent averages of 5 replicates of n = 10 individual moths per treatment.

Fig. 3. Mean survival (?95% CL) of Cydia pomonella under different high temperatures during different durations of exposure (A). Three-dimensional surface plot (Wafer ?t method) of the relationship between C. pomonella high temperature survival and time (B). Data points for the ?gures represent averages of 5 replicates of n = 10 individual moths per treatment.

control group (Table 2; Fig. 5a). Adult moths that were pre-treated at 5 8C for 1 or 2 h and given 1 h (gap treatment) at 25 8C signi?cantly increased survival at ?9 8C for 2 h (Table 2; Fig. 5a). Survival of adult moths pre-treated at 5 8C for 2 h and exposed to ?10 8C for 2 h using direct plunge protocols was not signi?cantly different from controls (Table 2; Fig. 5a). A slow cooling protocol, at a rate of 0.01 8C min?1 from 25 8C to 5 8C and then holding moths at this temperature for 1 h signi?cantly improved survival when assayed at ?10 8C for 2 h (Table 2.) However, cooling rates of 0.1 and 0.5 8C min?1 did not signi?cantly improve C. pomonella survival at ?10 8C for 2 h (Table 2). Adult C. pomonella pre-treated at 35 8C for 1 h did not show signi?cant improvements in survival after a direct exposure to 43 8C for 2 h (Table 3; Fig. 5b). However, there was a dramatic improvement in survival in moths exposed to 37 8C for 1 h followed by a 1 h gap at 25 8C and then assayed for survival at 43 8C for 2 h (Table 3; Fig. 5b). One or 2 h pre-treatment at 32 8C, with or without a gap period at 25 8C, did not improve survival at 45 8C for 2 h (Fig. 5b). Cross tolerance experiments showed that adult C. pomonella can improve survival at low temperatures after high temperature

pre-treatment, but not vice versa. A pre-treatment at 37 8C for 1 h improved C. pomonella survival of 2 h at ?9 8C (Table 4, Fig. 6a). However, a 2 h exposure at 5 8C did not improve survival at 45 8C (2 h) (Table 4, Fig. 6b). 4. Discussion 4.1. Lethal temperatures The adult life-stage in C. pomonella is probably the stage responsible for most dispersal. In addition, it contributes directly to changes in population size through reproduction, and is also the life-stage used for SIT (see Introduction). Yet most work examining thermal tolerance of C. pomonella has not explicitly focused on adult thermal biology. It is clear, however, that adult thermal tolerance of C. pomonella at daily scales is critical to SIT success (through e.g. minimum temperatures required for ?ight to high temperatures possibly limiting ?ight), thermal tolerance can be a crucial aspect of laboratory-reared moth quality (see discussions in Bloem et al., 2006; Stotter and Terblanche, 2009; Chidawanyika

[()TD$FIG]
1.4 Critical thermal minima (°C)

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A a a

a

1.1

0.8

Table 2 Summarised output of generalized linear models (GLZ) for the effects of low thermal pre-treatments on the survival of Cydia pomonella. First, hardening pretreatment of 0, ?7 and 5 8C for 1 h and plunged immediately thereafter into ?12 8C for 2 h. Second, moths hardened at 5 8C for 1 and 2 h before being plunged into ?9 8C for 2 h. Third, moths hardened at 5 8C for 2 h and given 1 h at 25 8C (gap treatments) before being plunged into ?10 8C for 2 h. Lastly, moths at 25 8C were cooled at the rate of 0.01, 0.1 and 0.5 8C min?1 to 5 8C before being plunged into ?10 8C (cooling rates and plunge treatment combination). Treatment d.f. 1 1 1 1 4 0 1 1 2 0 1 1 1 1 1 0 1 4 14 0.4953 ? 0.4001 1.5964 ? 0.4548 0.6554 ? 0.4001 0.000 ? 0.000 0.9659 ? 0.4202 0.000 ? 0.000 0.4833 ? 0.3722 0.000 ? 0.000 0.7331 ? 0.2953 1.9357 ? 0.4016 Estimate ? S.E. 0.6633 ? 0.1753 28.6110 ? 135530 28.6110 ? 135530 28.6110 ? 135530 Wald x2 14.32 0.00 0.00 0.00 14.32 p 0.0002 0.9998 0.9998 0.9998 0.0063

0.5

Control 1 (25 8C/1 h/?12 8C) 0 8C/1 h/?12 8C ?7/1 h/?12 8C 5 8C/1 h/?12 8C

0.2

0.06

0.12

0.25

Sub-treatment effects Control 2/25 8C/2 h/?9 8C) 5 8C/1 h/?9 8C 5 8C/2 h/?9 8C Sub-treatment effects Control 3 5 8C/2 h/?10 8C

Ramping rate (°C/minute)

Critical thermal maxima (°C)

B
45

6.16 23.23 25.48

0.0131 <0.001 <0.001

a

1.69 1.69 1.53 12.32 2.68

0.1941 0.1941 0.2158 0.0004 0.1014

44

b
43

Sub-treatment effects

b

Control 4 0.01 8C min?1 0.1 8C min?1 Control 5 0.5 8C min?1

5.28 36.70 122.56

0.0215 <0.0001 <0.0001

42

0.06

0.12

0.25

Sub-treatment effects All treatment effects

Ramping rate (°C/minute)
Fig. 4. Effects of cooling and heating rates on (A) critical thermal minima and (B) critical thermal maxima of adult C. pomonella. Data points represent means of n = 20 for each treatment of mixed gender. Error bars represent 95% CL.

and Terblanche, in press) and may also determine survival upon release in the wild. The present study therefore investigated the survival of adult C. pomonella under varying low or high temperatures of different short durations. These results showed that both the magnitude of temperature variation and duration of exposure were important in determining the survival of C. pomonella, as might be expected for insects in general (Chown and Terblanche, 2007). Over a 4-h period, for example, 50% of the population would be killed by temperatures of 42 8C or ?10 8C. By contrast, temperatures which would be lethal for 50% of the population were 45.5 and ?15.0 8C for 1 h. Duration of exposure at sub-lethal or lethal low temperatures is of ecological signi?cance as it determines the limits to activity, severity of tissue injury and possibly the time required for recovery or repair of injury from stress (Chown and Nicholson, 2004; Denlinger and Lee, 2010; Hoffmann, 2010). The absolute temperature tolerance determined in these experiments for adult codling moth raises the issue of their ecological signi?cance. For example, one important question is whether adult C. pomonella are likely to die from thermal stress in their natural or agricultural environments. This can be addressed by combining the microclimate data with the thermal tolerance estimates performed in the laboratory. The short-term ($7 months), high frequency microclimate temperature data we recorded in an apple orchard at Welgevallen farm in Stellenbosch (South Africa) ranged from 4.7 to 42.2 8C and suggests that temperatures potentially causing low temperature mortality never occurred, neither did they even approach lethal levels at any duration (Fig. 7). By contrast, the high temperatures we recorded

fell within the range of lethal high temperatures and also critical thermal maxima (Fig. 7). This suggests that high temperatures are more likely to cause mortality as compared to low temperatures for the months of October through to early June, particularly for this South African location, and encompasses the period of peak moth activity and abundance (Pringle et al., 2003). Over longer periods ($3 years), absolute minima and maxima recorded in this location at nearby weather stations reached 3.1 and 42.8 8C, further substantiating the view that minimum temperatures are not likely to be lethal, although temperature maxima may well be lethal for large portions of the adult population at certain times of the year (summer). However, other geographic locations, particularly in the Northern Hemisphere, may experience low temperatures that are likely to induce CTmin in C. pomonella adults, especially in autumn (see Bloem et al., 2006), but lethal low temperatures are unlikely given the timing of peaks in adult trap catches. Microclimate temperature recordings (Fig. 7) had an average (?s.d.) heating rate of 0.04 ? 0.01 8C min?1 and average cooling rate of 0.03 ? 0.01 8C min?1 calculated over 12 days. This suggests that the slowest rate of temperature change used to estimate CTLs in the laboratory would probably be best for approximating thermal limits to activity under ?eld conditions. The highest temperature experienced in Stellenbosch over the 7 month recording was 42.2 8C, whereas the CTmax recorded in the laboratory assays was 44.5 ? 0.01 8C at 0.06 8C min?1. The lowest temperature recorded over this period was 4.7 8C which was 4.1 8C higher than the CTmin determined in the laboratory (0.6 ? 0.01 8C at 0.06 8C min?1). This suggests that temperatures eliciting CTmin and CTmax are not frequently encountered in this site over the period recorded and that C. pomonella probably has a greater thermal tolerance and activity range than typically experienced in this habitat.

[()TD$FIG]

114

F. Chidawanyika, J.S. Terblanche / Journal of Insect Physiology 57 (2011) 108–117 Table 4 Summarised output of results of the cross-tolerance experiments in adult Cydia pomonella. Generalized linear models (GLZ) were used to test for the effects of high and low temperature pre-treatments on the survival of C. pomonella at low and high temperatures, respectively. Adult moths were pre-treated at 37 8C for 1 h and given 1 h at 25 8C before exposure to ?9 8C for 2 h. An identical pre-treatment but without the recovery period at 25 8C before plunging into ?9 8C was also undertaken. Similarly, two groups of moths were pre-treated at 5 8C for 2 h and given a gap at 25 8C before being exposed at 45 8C for 2 h. As in the high temperature pretreatments, one group of moths was denied the gap period at intermediate temperatures and instead were plunged directly into 45 8C for 2 h after the 5 8C pretreatment. Treatment Control 1 25 8C/1 h/?9 8C 37 8C 1 h/1 h gap/?9 8C 37 8C 1 h/no gap/?9 8C Treatment effects Control 2 25 8C/2 h/45 8C 5 8C 2 h/1 h gap/45 8C 5 8C 2 h/no gap/45 8C Treatment effects d.f. 1 1 0 2 1 1 0 2 Estimate ? S.E. 0.2412 ? 0.2713 1.0245 ? 0.3005 0.000 ? 0.0000 Wald x2 0.79 11.62 19.12 0.000 ? 0.1996 0.0841 ? 0.2005 0.000 ? 0.000 0.00 0.18 0.23 p 0.3741 0.0007 <0.0001 1.000 0.6750 0.8897

[()TD$FIG]
100

A

***
b

80

Survival (%)

60

a

a

40

Fig. 5. Mean survival of Cydia pomonella at ?9, ?10 and ?12 8C for 2 h (A) and at 43 and 45 8C for 2 h (B) after receiving a range of pre-treatments. Detailed statistical results are given in Table 2. (***p < 0.001) (**p < 0.005) (NS: non-signi?cant). Data points for the ?gures represent means of n = 50 per treatment. Error bars represent 95% CL.

20

0

Control 1

37°C/ 1hr/ no gap 37°C/ 1hr/ 1 hr gap

Treatment
100

B

80

Survival (%)

Table 3 Summarised output of generalized linear models (GLZ) for the effects of high temperature pre-treatments on the survival of C. pomonella at 43 and 45 8C for 2 h. Pre-treatments were done at 35 8C for 1 or 2 h and 37 8C for 1 h before being exposed to 43 8C test temperature. Similarly, moths which were exposed at 45 8C test temperature were pre-treated at 32 8C for 1 or 2 h. Treatment Control 1 25 8C/1 h/43 8C 35 8C/1 h/43 8C 37 8C/1 h/43 8C 35 8C/2 h/43 8C Sub-treatment effects Control 1 25 8C/2 h/45 8C 32 8C/1 h/45 8C 32 8C/2 h/45 8C Sub-treatment effects All treatment effects d.f. 1 1 1 1 3 1 1 1 2 6 Estimate ? S.E. 0.0000 ? 0.0000 0.5225 ? 0.3493 3.8602 ? 0.5044 0.4734 ? 0.3515 Wald x2 10.88 58.56 1.81 59.30 p 0.1347 <0.0001 0.1781 <0.0001

60

NS c c c

40

20

0

control 2

5 °C 2hrs/1hr gap

5 °C 2hrs/no gap

0.0000 ? 0.0000 0.1301 ? 0.2831 0.0614 ? 0.2749

Treatment
0.21 0.05 0.48 85.29 0.6460 0.8233 0.7869 <0.0001 Fig. 6. Mean survival of Cydia pomonella at ?9 8C for 2 h (A) and at 45 8C for 2 h (B) after receiving a range of pre-treatments. Detailed statistical results are given in Table 4. (***p < 0.001) (NS: non-signi?cant). Data points for the ?gures represent means of n = 50 per treatment. Error bars represent 95% CL.

[()TD$FIG]

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115

Fig. 7. (A) Microclimate temperature recordings by Thermocron iButtons (Model DS 1920; Dallas Semiconductors, Dallas, TX) (0.5 8C precision; 1 h sampling frequency) located at ground level, mid and canopy level of apple tree in an orchard at Welgevallen farm, Stellenbosch, South Africa (338560 88400 S, 188520 37300 E) which hosts Cydia pomonella. (Sampling was done from November 2009–June 2010). Heating and cooling rates were calculated using Expedata software, version 1.1.25 (Sable Systems, Las Vegas, NV). (B) Frequency distribution of the recorded temperatures over the same period. Arrows indicate critical temperatures for rapid cold-hardening (RCH), rapid heat-hardening (RHH), lower critical thermal limits for activity (CTmin), and upper critical thermal limits for activity (CTmax).

4.2. Rapid thermal responses The present work found evidence for both rapid heat-hardening and rapid cold-hardening responses in adult C. pomonella. The maximum survival improvement that could be induced at low temperatures was following the slow cooling (0.01 8C/min) protocol, and in that case improved survival from $35% to 80% (Fig. 5a). Using direct plunge protocols, the most survival improved was from 55% to 88% after 2 h at 5 8C, with many treatments not improving survival. This suggests restricted responses to low temperature, and when survival responses could be induced, they were of a low magnitude by comparison with other species showing rapid cold-hardening (e.g. fruit ?ies, Nyamukondiwa et al., 2010; Lee and Denlinger, 2010). Nevertheless, RCH responses in C. pomonella were similar to responses documented for some other Lepidoptera species to date (e.g. Larsen and Lee, 1994; Kim and Kim, 1997; but see also Sinclair and Chown, 2003; Stotter and Terblanche, 2009). At high temperatures, a marked RHH response was detected following 37 8C for 1 h, improving survival from 20% to $90%

(Fig. 5b). However, several treatments which were explored failed to elicit any RHH response. This is typical of RHH responses of insects more generally. For example, in D. melanogaster and some other insects, RHH is limited to only a small range of pre-treatment conditions (discussed in Denlinger et al., 1991; and see e.g. Chen et al., 1991; Overgaard and S?renson, 2008; Nyamukondiwa et al., 2010). To our knowledge, no studies have explicitly examined the biochemical responses underlying changes in heat tolerance of our study organism. However, it is highly likely that C. pomonella employs a heat shock protein responses as is the case in several other insect species (MacMillan et al., 2009; Zi-wen et al., 2009; Kalosaka et al., 2009). Moreover, the results of the cross tolerance experiments further support this possibility since the high temperature pre-treatment improved survival at ?9 8C (and see Chen et al., 1991). The brief gap at 25 8C after the pre-treatment seemed to be important, possibly allowing full expression of Hsps. The results of the cross-tolerance experiments for C. pomonella are similar to responses documented for Sarcophaga crassipalpis ?esh ?ies which improved low temperature survival in response to high temperature pre-treatment but not the opposite way around (Chen et al., 1991; but see also Sinclair and Chown, 2003 for a similar Lepidopteran example). In the context of ?eld Ta in South Africa, the ecological signi?cance of RCH is questionable given the microclimate recordings. Temperatures eliciting RCH virtually never occurred, although slow cooling conditions may occur more frequently (see discussion of CTLs). By contrast, 1 h at 37 8C was recorded relatively frequently (Fig. 7b) and thus, RHH responses may play an important role in increasing survival and perhaps also enhancing ?ight and other behaviours (e.g. mating and feeding) at high temperatures. Investigation of CTLs and the effects of varying rates of temperature change on CTLs also yielded novel insights into adult codling moth thermal biology. We found that slower rates of cooling did not signi?cantly reduce the CTmin of C. pomonella, as might be expected for a species with a pronounced rapid coldhardening response (e.g. Chown et al., 2009; Nyamukondiwa and Terblanche, 2010). However, similar CTmin recorded across different cooling rates might also be viewed as a form of plasticity since duration of exposure varied among these treatments (see discussions in Terblanche et al., 2007; Chown et al., 2009). Although a trend seems evident in the results perhaps indicating low statistical power (Fig. 4a), this result is likely also a consequence of a limited RCH response or perhaps the somewhat limited range of rates we employed. Indeed, the lack of a rate effect on CTmin contrasts with the survival assay results (cf. Fig. 4a with Fig. 5a). For CTmax the rate effect was more marked, with slowheated moths having signi?cantly higher tolerance (by about 1.5 8C) and also reinforces the notion that RHH is probably relatively more important under natural diel ?uctuations compared to the RCH response. Indeed, a longer duration during temperature increase allowed moths to better withstand high temperatures, but not particularly well at low temperatures. Moreover, the limited low temperature plasticity of adults con?rms the notion that temperate insects generally show more plasticity than their tropical counterparts since tropical environments are less variable than temperate environments (e.g. Hazell et al., 2010b; reviewed in Chown and Terblanche, 2007). This pattern seems to hold true in comparison between C. pomonella and false codling moth, Thaumatotibia leucotreta rapid coldhardening responses. False codling moth is native to tropical regions (Stofberg, 1954; Catling and Aschenborn, 1974) while C. pomonella is native to temperate regions (Wearing et al., 2001). Indeed, C. pomonella has a more pronounced RCH response, albeit rather limited, than T. leucotreta which shows virtually no responses to a range of low or high pre-treatments (Stotter and

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Terblanche, 2009). However, such a comparison is limited by a lack of information on ?eld Tb for both these nocturnal species, and due consideration of the peak activity times relative to Ta commonly encountered. 5. Conclusions This study reports adult C. pomonella thermal tolerance and the plasticity thereof. Thermal ?uctuations experienced over diurnal scales likely play a signi?cant role in the survival of adult C. pomonella, especially at high temperatures but probably to a much lesser extent at low temperatures, at least for the geographic region investigated here. At longer timescales, within-generation changes in thermal tolerance have also been demonstrated (Chidawanyika and Terblanche, in press), suggesting an important role for thermal history in modifying future tolerance of adults over moderate (sub-lethal) and extreme thermal conditions. Knowledge of the temperatures (and time–temperature combinations) which can elicit RCH or RHH is also important for postharvest protocols for fruit and disinfestations of harvest bins as some protocols may induce hardening giving C. pomonella the capacity to resist potentially lethal thermal treatments, although this would likely be more critical for larvae and pupae in these cases. Nevertheless, apart from a handful of studies (e.g. Wang et al., 2002, 2004) such rapid responses have not been well examined for developing C. pomonella. This is particularly important for quarantine treatments of fruits where thermal treatments might impact on fruit quality (Hallman, 2000). However, non-lethal temperature treatments may indeed improve survival of adult C. pomonella at lethal temperatures. Hence, postharvest treatment protocols will need to be mindful of the capacity of C. pomonella adults to rapidly cold- and heat-harden as this may in?uence the ef?cacy, or time required to achieve ef?cacy, of a post-harvest treatment. The present results may also be of importance to C. pomonella SIT programmes because it is clear that short-term ?uctuations (diel thermal history), rate of temperature change, magnitude and duration of temperature exposure affects survival of the adult moths. The implications of these effects for ?tness and performance, and the impact of the observed rapid thermal responses on behaviour, are however not clear and further investigation would be valuable. It would also be useful to know if these results for the laboratory-reared moths are comparable to wild-caught moth’s thermal tolerance in the context of laboratory adaptation (see discussions in Stotter and Terblanche, 2009). Although low temperatures seem unlikely to be a direct cause of mortality in this species, low Ta may still contribute to reducing population abundance due to impacts on activity, growth rates, reproduction and fecundity (Bloem et al., 2006; Chidawanyika and Terblanche, in press). High temperatures appeared much more likely to in?uence survival in two main ways. First, high temperatures in some months were similar to those that could induce rapid heathardening making it more likely that moth’s could tolerate subsequent lethal high temperatures. Second, the high temperatures recorded (Fig. 7) were probably high enough to cause direct mortality. Therefore, high temperatures potentially experienced during SIT releases undertaken in mid-summer, are likely to be negatively affected and may limit the wild C. pomonella population, although such a speculation requires further information regarding use of microclimates and behavioural thermoregulation. One positive implication of the close relationship between temperature maxima and lethal limits of adult moths is that increased temperatures predicted under climate change scenarios may aid in controlling C. pomonella populations (and see Bale and Hayward, 2010). Nevertheless, future work could incorporate the thermal responses reported here into predictive models of C. pomonella

population dynamics, phenology, and potential agricultural impacts. Acknowledgements We thank Stellenbosch DFPT Sterile Insect Rearing Facility for providing C. pomonella. Financial support for this research was provided by DFPT and a National Research Foundation THRIP award to Pia Addison. Water baths and iButtons were purchased with funding support from Stellenbosch University‘s Sub-Committee B to J.S.T. We are grateful for the comments provided by M. Addison and two anonymous referees on this work. References
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