Through the winter months, decreasing temperature induces a range of direct and indirect effects on insect physiology. During the occurrence of low temperature episodes, there is the potential for freezing to occur, which imposes additional stressors. Winter is also a time of low nutrient availability, so insects frequently fuel overwintering metabolism on the energetic resources that they gathered through the growing season. Since metabolic rate is directly dependent on body temperature, increased temperatures can increase resource use. Both low and high temperatures during overwintering can therefore be stressors for insects.
1.3.1 Cold-induced damage
Since insect body temperature lowers linearly with decreasing environmental temperature, rates of chemical reactions within their bodies decline with decreasing environmental temperature following Arrhenius kinetics (Hochachka and Somero, 2002). This temperature dependence has important consequences for insects at low
temperatures: the biological activities of important macromolecules such as phospholipids and enzymes slow in cold conditions. At low enough temperatures, this can disrupt the function of these macromolecules, and lead to chill coma and chilling injury (Chen and Walker, 1994; Lee, 2010; MacMillan and Sinclair, 2011a). Chill coma is the reversible cessation of movement in an insect that occurs when temperatures drop (MacMillan and Sinclair, 2011a), and the temperature this occurs at (the CTmin) is plastic
on both evolutionary (Huey, 2010) and individual (Ransberry et al., 2011) time-scales. Chilling injury is the damage, unrelated to freezing of water, induced by low temperature exposure, and may be divided into two categories based on the speed of damage accumulation (Lee, 2010). Direct chilling injury occurs on the scale of minutes, while indirect chilling injury may take days or weeks to accumulate. Damage caused by either type of chilling injury usually accumulates exponentially over time, with increasing intensity, or with the interaction between the two (Nedvĕd et al., 1998).
The mechanisms of both direct and indirect chilling injury remain under active investigation (Koštál and Tollarová-Borovanská, 2009; Lee, 2010; MacMillan and Sinclair, 2011b; Powell and Bale, 2004). Proteins can be denatured following low temperature exposure, such as in Antarctic notothenoid fish (Todgham et al., 2007). Similarly, membrane fluidity decreases at lower temperatures, which can result in disrupted cell homeostasis (Chown and Terblanche, 2007; Hochachka and Somero, 2002; Koštál et al., 2007a). Together, cold inactivation or denaturation of membrane proteins and decreased membrane fluidity can severely disrupt ion homeostasis (Koštál et al., 2004; Koštál et al., 2007a; MacMillan and Sinclair, 2011a). This disruption of ion homeostasis may cause neuromuscular damage or loss of function. For example, cold shock damages behaviour (and likely nervous system tissue) in the flesh fly Sarcophaga crassipalpis (Diptera: Sarcophagidae), (Yocum et al., 1994). At low temperatures, antioxidant enzymes can be less effective and allow damage from reactive oxygen species to accumulate, causing oxidative damage (Lalouette et al., 2011; Rojas and Leopold, 1996). Cold-induced oxidative damage is problematic for many biologically important macromolecules. For example, in the intertidal mussels Mytilus galloprovincialis and M. californianus (Mytiloidea: Mytilidae), low temperature exposure causes both single and double-stranded breaks in DNA (Yao and Somero,
2012). Similarly, low temperature exposure can oxidize the double bonds within unsaturated lipids within living insects (Lalouette et al., 2011). There is much less known about the mechanisms of indirect chilling injury. This type of chilling injury takes much longer to accrue, and is often associated with mortality that occurs much later in development (Rojas and Leopold, 1996). While many of the mechanisms appear to be similar between direct and indirect chilling injury, selection for survival of direct chilling injury in Drosophila melanogaster (Drosophila: Drosophilidae) does not confer resistance to indirect chilling injury (Chen and Walker, 1994). Despite the ongoing research in this area, the majority of studies have only focused on the physiological effects of a single exposure to low temperature (Chown and Sinclair, 2010).
1.3.2 Freezing-induced damage
Pure water is capable of supercooling (cooling below its melting point without the nucleation of ice) to -40 ºC (Zachariassen and Kristiansen, 2000; Zachariassen et al., 2004). Due to their small size and high concentration of solutes, even very cold- intolerant insects do not risk freezing until well below 0 ºC, despite having relatively high melting points (Lee, 2010; Lee and Costanzo, 1998; Renault et al., 2002; Strachan et al., 2011; Zachariassen, 1985). Ice forms in insects in one of two ways: either by heterogeneous or homogeneous nucleation (Zachariassen, 1985; Zachariassen et al., 2004). Homogeneous nucleation occurs when, at low enough temperatures, water molecules aggregate spontaneously into a lattice structure. By contrast, heterogeneous nucleation occurs when water molecules aggregate around another substance (Zachariassen et al., 2004). Regardless of the method of nucleation, ice formation in insects can cause lethal damage to biological tissues.
Formation of ice crystals can induce mechanical damage to membranes and proteins (Storey and Storey, 1988). In addition, ice is often nucleated extracellularly (Zachariassen and Kristiansen, 2000), and as liquid water joins the growing ice lattice, both intra and extracellular solutes concentrate (Lee, 2010). This concentration increase can also denature proteins which disrupts metabolism. Reduction of cell volume in response to efflux of cellular water content can also induce additional damage to cytoskeleton and membranes (Storey and Storey, 1988). These effects can significantly
disrupt ion homeostasis (Boardman et al., 2011), and damage nervous tissue (Collins et al., 1997). In addition, animals are hypoxic while frozen (Joanisse and Storey, 1996). During thawing, reperfusion of oxygen can generate reactive oxygen species, and therefore insects and may face oxidative damage during reperfusion during thawing (Storey and Storey, 2010). Despite study of these effects, in insects that can survive freezing it remains unclear which process results in the most damage: the freezing event itself, the physiological effects of remaining frozen, or the events during thawing.
1.3.3 Overwintering energetics
Another important stress during overwintering is energetic drain. Nutrient resources are usually unavailable through the winter months due to the senescence of plant tissues in temperate environments as well as the dormancy of prey species. As a result, insects must overwinter on the lipid and carbohydrate resources they have stored during the growing season (Hahn and Denlinger, 2011). Since body temperature is directly linked to environmental temperature, and metabolic rate is directly related to body temperature, temperature regimes can therefore greatly alter the nutrient usage of overwintering insects. For example, increased overwintering temperature raises the metabolic rate of
Eurosta solidaginis (Diptera: Tephritidae), which results in fewer eggs per individual in the spring (Irwin and Lee, 2003). Many species deeply depress their metabolic rates during diapause and thereby reduce energetic demand (Hahn and Denlinger, 2011). In addition to the effects of increased mean temperature, increased variation in temperature can also increase metabolic rate due to the nonlinear relationship between metabolic rate and temperature (“Jensen’s Inequality”; Ruel and Ayres, 1999). As a result, some species have adapted by reducing the sensitivity of their metabolic rate to temperature (Williams
et al., 2012). While the predominant overwintering energy source for insects is triacylglycerols (Hahn and Denlinger, 2011), some species such as C. fumiferana fuel overwintering metabolism with glycogen reserves (Han and Bauce, 1998). Since carbohydrate cryoprotectants such as glycerol and sorbitol are synthesized from glycogen (Storey and Storey, 1983), this could lead to a conflict between the need to conserve glycogen and the need to synthesize cryoprotectants in these species (Sinclair et al., 2013).