The Journal of Experimental Biology 207, 1287-1294 1287 Published by The Company of Biologists 2004 doi:10.1242/jeb.00880 Metabolism of the sub-Antarctic caterpillar Pringleophaga marioni during cooling, freezing and thawing Brent J. Sinclair*, C. Jaco Klok and Steven L. Chown Spatial, Physiological and Conservation Ecology Group, Department of Zoology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa *Author for correspondence (e-mail: [email protected]) Accepted 15 January 2004 Summary Although general models of the processes involved in is hypothesised to be a metabolic manifestation of the insect survival of freezing exist, there have been few latter, possibly a failure of the Na+/K+-ATPase pump. This studies directly investigating physiological processes provides a plausible link between processes at the cellular during cooling, freezing and thawing, without which these level and observations of the action of the CTmin at models remain hypothetical. Here, we use open-flow tissue and whole-organism levels. Caterpillars froze at respirometry to investigate the metabolism of the freeze- –4.6±0.1°C and had detectable metabolism when frozen. tolerant sub-Antarctic caterpillar Pringleophaga marioni Post-thaw, metabolic rates were lower than pre-freezing Viette (Lepidoptera: Tineidae) during cooling, freezing measurements. Post-thaw metabolic rates did not differ and thawing and to compare animals exposed to non- between temperatures that did and did not kill the lethal (–5.8°C) and lethal (–6.0°C, after which caterpillars caterpillars, which suggests that mortality may be a result are moribund for several days, and –18°C, after which of a breakdown in processes at the organismal, rather caterpillars are completely unresponsive) freezing stress. than cellular, level of organisation. We found a large decrease in metabolic rate (that is not associated with freezing) at –0.6±0.1°C and calculated a 3 Q10 of 2.14×10 at this breakpoint. This breakpoint is Key words: freeze tolerance, critical thermal minimum, chill-coma, coincident with the critical thermal minimum (CTmin) and metabolic rate, Pringleophaga marioni. Introduction Although the majority of insects are killed by freezing and a process of osmotic dehydration. Ice forming in the must avoid internal ice formation to survive low temperatures, haemolymph creates concentrated fluid pockets that dehydrate many insect species from a variety of habitats and taxa survive cells and restrict ice to the extracellular spaces (Zachariassen, sub-zero temperatures by withstanding the formation of ice 1985; Sinclair and Wharton, 1997; Ramløv, 2000), although in their bodies and are termed ‘freeze tolerant’ (Lee, intracellular ice formation probably occurs in some species 1991; Ramløv, 2000; Sinclair et al., 2003a). Although the (Salt, 1962; Lee et al., 1993; Wharton and Ferns, 1995; Davis biochemical correlates of this freezing survival are reasonably and Lee, 2001). During thawing, therefore, water must be well known (Lee, 1991; Sømme, 1999; Ramløv, 2000; Sinclair redistributed and ion gradients re-established. In the woodfly et al., 2003b), the actual processes that occur during freezing Xylophaga cincta, re-establishing the ion balance takes and thawing have not been investigated to the same extent (but several hours (Kristiansen and Zachariassen, 2001). see Sinclair and Wharton, 1997; Joanisse and Storey, 1998; Nonetheless, there are currently no explicit data about the Neufeld and Leader, 1998; Kristiansen and Zachariassen, 2001 cause of lower lethal temperatures in freeze-tolerant insects. for studies that do examine the freezing or thawing process Although mechanical stress (for example, ice crystals piercing explicitly). The general model of the response of freeze- cell membranes or lethal recrystallisation of ice) has been tolerant insects to low temperatures is that as the temperature proposed as a cause of mortality in frozen insects (Salt, decreases, the insect will pass through the critical thermal 1961), the relationship between water-to-ice conversion and minimum (CTmin – resulting in chill-coma) and eventually temperature means that the overall proportion of water reach the temperature of crystallisation (Tc – the temperature converted to ice will increase continuously with decreasing at which the body fluids freeze). If the insect continues to temperatures (Zachariassen, 1985), and most hypotheses of cool after freezing, it will eventually reach a lower lethal causes of mortality tend to hinge on an osmotic stress temperature, after which it will not survive when thawed (Lee, threshold being crossed. These hypotheses include the 1991; Ramløv, 2000). inability of individuals to re-establish ion gradients in the Survival of internal ice formation is generally attributed to nervous system and transgression of a minimum cell volume 1288 B. J. Sinclair, C. J. Klok and S. L. Chown resulting in lethal Ca2+ concentrations (Meryman, 1971; not present metabolic data from during the freezing, thawing Kristiansen and Zachariassen, 2001). or post-thawing processes. Here, we use open-flow Although the hypothesised causes of lower lethal respirometry to explore the metabolic changes that occur temperature act at the sub-organismal level, it is reasonable during cooling, freezing and thawing, using the sub-Antarctic to assume that the results are manifest at higher levels of caterpillar Pringleophaga marioni as a model freeze-tolerant organisation. However, aside from the progression of ice organism, and compare metabolic patterns in individuals formation (see Ramløv, 2000 for a summary), there is little exposed to lethal and non-lethal freezing stress. information about processes at the organismal level during freezing and thawing of insects. Kozhantsikov (1938) suggested that cold-hardy insects are those that are able to Materials and methods maintain a detectable level of metabolism below 0°. This idea Study animals was challenged on methodological grounds by Scholander et Pringleophaga marioni Viette (Lepidoptera: Tineidae) is a al. (1953), who pointed out that Kozhantsikov heated his key decomposer on Marion Island (46°54′ S 37°45′ E; Crafford animals to above 0°C to perform measurements. Scholander et et al., 1986). The long-lived (several years) larvae occur in al. (1953) and Salt (1958) both showed that the rates of change most of the habitats on the island, from sea level to ~1000·m, of metabolic rates with temperature (i.e. Q10) in frozen and where they are likely to experience multiple freeze–thaw supercooled insects were extremely high at sub-zero events during their lifetime (Crafford, 1990; Crafford and temperatures and that the rate of change in metabolic rate Chown, 1992). Klok and Chown (1997) studied the increases logarithmically as the 0° threshold is crossed. In later environmental physiology of P. marioni larvae and determined work, Zachariassen et al. (1979) directly addressed the that caterpillars have a critical thermal minimum (CTmin) of manifestation of freezing injuries by investigating the post- –0.6±0.2°C and that they are freeze tolerant, with a mean thaw metabolism of freeze-tolerant Eleodes blanchardi beetles temperature of crystallisation (Tc) of –5.0±0.2°C and mortality exposed to lethal and sub-lethal temperatures. They concluded at temperatures below approximately –6°C to –10°C. Recently, that metabolic rate was elevated after freezing. Moreover, Sinclair and Chown (2003) have shown that P. marioni larvae whole-organism metabolic rate did not differ between those do not exhibit a rapid cold-hardening response but that cold beetles that survived and those that were killed by freezing, hardiness may be enhanced by prior desiccation or heat stress, suggesting that (at least initially) mortality does not occur suggesting that heat shock proteins may play some role in the at the cellular level. Most recently, Block et al. (1998) freezing survival of this species. investigated the metabolic rates of freeze-tolerant larvae and Pringleophaga marioni larvae were collected from adults of the perimylopid beetles Hydromedion sparsutum and abandoned albatross nests in the vicinity of the Research Perimylops antarcticus on sub-Antarctic South Georgia. They Station on Marion Island during the April 2002 relief voyage. found that, while metabolic rates of both species did not differ Caterpillars were placed in groups of five in 250·ml plastic jars pre- and post-chilling (exposure to non-freezing sub-zero filled with nest material and were returned under refrigeration temperatures), metabolic rates of both larvae and adults of P. to the laboratory in Stellenbosch within 10·days, where they antarcticus were significantly higher after a brief freezing were housed in the dark in a refrigerator (6.0±0.4°C) until they event but did not change after freezing in H. sparsutum. They were used for experiments. Animals housed in this manner fed concluded that the elevated metabolic rate of the former and moulted normally, and several individuals pupated. species is indicative of a rapid repair of slight freeze-induced injuries that explains its greater cold tolerance but did not Freezing and respirometry experiments investigate the effects of freezing injury or mortality within the Caterpillars (0.202±0.016·g, mean mass ± S.E.M., N=29) frozen beetles. were frozen individually in a 3·cm3 plastic respirometry All five of the preceding studies were conducted using cuvette. A 44-SWG type-T thermocouple was affixed to the closed-system respirometry, which integrates