Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)

Abstract

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 significantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between −20 °C to −5 °C and 32 °C to 47 °C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were −12 °C for 2 h and 44 °C 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 °C improved survival at −9 °C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 °C for 1 h markedly improved survival at 43 °C 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 °C for 1 h) significantly improved low temperature survival (−9 °C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not significantly affected by cooling at rates of 0.06, 0.12 and 0.25 °C min⁻¹ (CTmin range: 0.3–1.3 °C). By contrast, critical thermal maxima (CTmax) were significantly affected by heating at these rates and ranged from 42.5 to 44.9 °C. 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 significant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects.

Introduction

Temperature plays a key role in the life of insects. Over longer periods, temperature influences seasonality and evolutionary responses of insects (Bale, 2002, Lee and Denlinger, 2010, Chown and Nicholson, 2004). However, the ability of diel temperature fluctuations 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 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 influenced 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 (T_a) (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 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 benefit 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 (T_b), or the extremes they can withstand, using either physiological or behavioural mechanisms or some combination of both. For example, an insect experiencing adverse high T_a can lower its T_b by avoidance of sunny hot spots and vice versa (e.g. Kührt et al., 2006, Huey and Pascual, 2009). However, behavioural adjustments only act as the first line of defence against sub-optimal T_a and depend to a large degree on microsite opportunities in their habitat (Kührt et al., 2006). If unfavourable T_a 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 Sørenson, 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 Sørenson, 2005). Here, we use ‘hardening’ to refer to the physiological responses which are of primary interest. Rapid cold-hardening (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 fitness (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 significant 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, Lee and Denlinger, 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 sugars and polyols (e.g. Koštál 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 Rehfield-Ray, 2006, Neven and Hansen, 2010) or overwintering physiology (Khani et al., 2007, Khani and Moharramipour, 2010). When other life-stages 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 specifically 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 fluctuations more generally, may influence 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 influence performance and survival as this could help improve quarantine or post-harvest control procedures, or alternatively, may be used to enhance SIT efficacy (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 fitness 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.