Intracellular freezing and survival in the freeze tolerant alpine cockroach Celatoblatta quinquemaculata

Abstract

The alpine cockroach Celatoblatta quinquemaculata is common at altitudes of around 1500 m on the Rock and Pillar range of Central Otago, New Zealand where it experiences freezing conditions in the winter. The cockroach is freeze tolerant, but only to c. −9 °C. The cause of death at temperatures below this is unknown but likely to be due to osmotic damage to cells (shrinkage). This study compared the effect of different ice nucleation temperatures (−2 and −4 °C) on the viability of three types of cockroach tissue (midgut, Malpighian tubules and fat body cells) and cooling to three different temperatures (−5, −8, −12 °C). Two types of observations were made (i) cryomicroscope observations of ice formation and cell shrinkage (ii) cell integrity (viability) using vital stains.

Cell viability decreased with lower treatment temperatures but ice nucleation temperature had no significant effect. Cryomicroscope observations showed that ice spread through tissue faster at −4 than −2 °C and that intracellular freezing only occurred when nucleated at −4 °C. From temperature records during cooling, it was observed that when freezing occurred, latent heat immediately increased the insect’s body temperature close to its melting point (c. −0.3 °C). This “rebound” temperature was independent of nucleation temperature.

Some tissues were more vulnerable to damage than others. As the gut is thought to be the site of freezing, it is significant that this tissue was the most robust. The ecological importance of the effect of nucleation temperature on survival of whole animals under field conditions is discussed.

Introduction

Cockroaches are commonly thought of as being temperate or tropical insects. However, Celatoblatta quinquemaculata (Dictyoptera: Blattoidea) is one of the exceptions. This particular species is common in the subalpine and alpine regions of New Zealand at altitudes of up to 1500m on the Rock and Pillar Range of Central Otago (Johns, 1966) where winter temperatures are unpredictable and regularly fall below freezing.

Insects which have adapted to survive subzero temperatures are normally classified as being either freeze tolerant or freeze avoiding (Cannon and Block, 1988). The cold tolerance strategy of C. quinquemaculata has been the subject of several studies (Block et al., 1998, Sinclair, 1997, Sinclair, 1999), which have shown that its extracellular body water freezes at a relatively high temperature due to the presence of ice nucleators in the gut and haemolymph (Worland et al., 1997). The whole body freezing point, commonly termed the supercooling point, of adult C. quinquemaculata varies with season between −4.2 °C in the autumn to −3.4 °C in the winter (Sinclair, 1997). However, it has been shown to survive “frozen” at −5 °C for 4 days under laboratory conditions and to have a lower lethal temperature in winter of about −9 °C (Sinclair, 1997). This level of freeze tolerance, whereby animals survive freezing of some body fluids at a relatively high temperature (often −2 to −5 °C) but die when their body temperature is further reduced (usually to between −8 and −15 °C) has been termed “moderately freeze tolerant” (Sinclair, 1999). In comparison, “strongly freeze tolerant” insects such as the larval stage of the fly Heliomyza borealis survive frozen to −60 °C for long periods (Worland et al., 2000). The survival strategy evolved by C. quinquemaculata is adequate to allow the majority of overwintering animals to survive normal winter conditions in it’s cold, wet, alpine habitat under slabs of schist where it will experience many freeze-thaw cycles (Sinclair, 1999). Several other insects have been classified as moderately freeze tolerant including the subantarctic beetle, Hydromedion sparsutum, from South Georgia which freeze at c. −2.5 °C and survives frozen to c. −8 °C (Worland and Block, 2003). However, the cause of mortality at this particular temperature has not been established.

When extracellular water freezes, water is drawn from cells due to the osmotic gradient, which is established across the membrane. This will eventually result in dehydration and shrinkage of cells which may cause permanent physical damage and death (Meryman, 1971). At the same time, cryoprotective compounds will diffuse into cells until a concentration equilibrium is established. Cells of freeze tolerant organisms may have adapted to be tolerant to such effects by being able to survive a high degree of cell shrinkage and/or by the production of low molecular weight cryoprotectants such as glycerol, which reduce the amount of ice formed at subfreezing temperatures (Zachariassen, 1991). Both freeze avoiding and freeze tolerant insects produce a range of cryoprotective compounds which can be any osmotically active substance which is nontoxic in high concentrations.

Survival of freezing is affected by the rate at which the animal is cooled (Baust, 1980) perhaps because polyols such as glycerol become more viscous at low temperatures and take time to enter cells. The temperature at which freezing first occurs (the nucleation temperature) affects the rate at which ice forms which in turn will affect the likelihood of intracellular freezing and consequently cell survival.

Although intracellular freezing in animals is said to be lethal, this hypothesis is based mainly on studies of mammalian cells (see, Lee et al., 1993). Salt, 1959, Salt, 1962 observed intracellular freeze tolerance in fat body cells from larvae of the Goldenrod gall fly. This has been confirmed in a more recent study by Lee et al. (1993) who found that while 60% of fat body cells survived freezing to −80 °C, no larvae survived and concluded that other types of cells must be more susceptible to freezing and responsible for the death of the larvae. The only whole organism shown to be able to survive intracellular freezing is the Antarctic nematode, Panagrolaimus davidi (Wharton and Ferns, 1995).

Many laboratory determinations of both whole body freezing point (SCP) and lower lethal temperature have been conducted with cooling rates of 1 °C min⁻¹ which is a convenient rate, easily achieved by most types of laboratory cooling systems and allows some comparison of results. However, cooling rates within a hibernaculum in the field are likely to be much slower and, as ice nucleation is a stochastic event, ice formation may be initiated at a relatively high temperature during a period of slowly reducing, or constant subzero temperature, particularly if ice nucleators are present. Under these conditions, freezing will occur rapidly at first but due to the production of latent heat of freezing, the body temperature will increase towards the melting point slowing the formation of ice (Zachariassen, 1991).

The aim of this study is to compare the freeze tolerance of whole cockroaches with excised tissue frozen under different conditions. As it has been predicted (Lee et al., 1993) that different types of tissue may survive better than others, the study examines three different types of cells (fat body, gut and Malpighian tubules). To investigate the effect of freezing temperature on cell survival, freezing was initiated at two different temperatures (−2 and −4 °C) by inoculating the sample with an ice crystal. The effect on survival was tested by further cooling the cells to three different minimum temperatures (−5, −8 and −12 °C). The study uses two different techniques to examine the effects of these treatments i) vital stains to count the proportion of living and dead cells ii) a cryomicroscope stage to observe the spread of ice through the tissue after inoculation and during the subsequent cooling phase.