Abstract
The exposure to subzero temperatures, and the associated risk of freezing, is a major challenge faced by insects in alpine areas. Those insects that do not move to warmer areas to avoid cold temperatures have evolved a range of physiological adaptations, traditionally grouped in two main strategies, to cope with the risk of freezing: the avoidance of ice in their bodies or the ability to survive such ice forming. The cold tolerance strategy of the New Zealand alpine wētā, Hemideina maori, falls into the second category. It is the world’s largest known freezing-tolerant insect, making the details of how it survives freezing an interesting question.
Freezing-tolerant insects cope with the cold through a variety of adaptations, including the accumulation of low molecular weight cryoprotectants and the production of ice-active proteins. These proteins include varieties that initiate the formation of ice at higher temperatures than would otherwise occur, inhibit the formation of ice (and, according to recent studies, other crystals) and inhibit the growth of already formed crystals. While good theories exist, exactly how these adaptations allow the survival of ice formation is not yet known at all levels, with many potential applications waiting to be unlocked through more thorough understanding.
The Rock and Pillar Range of southern New Zealand is one of the natural habitats of H. maori. Temperatures can drop to freezing levels here even in the summer, which is likely why H. maori is freezing tolerant throughout the year. While the wētā has been investigated in previous studies, this thesis was an attempt to investigate more factors involved in the freeze tolerance over a wider range of tissues throughout the year, to get as complete a picture as possible of its cold tolerance strategy.
Various activities and substances associated with cold tolerance were investigated on a tissue-specific and seasonal basis using a variety of methods including gas chromatography, high performance liquid chromatography, vapour pressure osmometry, protein assays, ice nucleation spectrometry, optical recrystallometry, nanolitre osmometry, and the splat freezing assay. Various patterns of change were observed on a seasonal and tissue basis, ranging from no change at all to many-fold changes in activities and the accumulation of substances. An unexpected pattern of the change in trehalose concentration led to the suggestion of a different role for this substance, desiccation survival, in H. maori than what was previously thought. The major amino acid accumulated by the wētā was proline, although its role in cold tolerance remains unclear.
No thermal hysteresis activity was found in any tissues at any time of year, apart from after extensive concentration through dehydration of the tissue. Strong recrystallisation inhibition activity was found throughout the year in the wētā’s gut, but its activity was absent in the haemolymph in autumn. This discovery led to the suggestion of a novel function in H. maori, possibly related to the control of crystallisation of trehalose. Ice nucleation activity was also found throughout the year, with the hindgut the likely site of ice formation in the wētā’s body.
Ice-active agents produced by H. maori were not successfully purified, but indications that the factors involved either do not bind to ice or depend on an activating compound remain intriguing. An investigation into the location of ice within the wētā’s body revealed interesting patterns that hint at the possible ability to survive intracellular freezing. A new imaging technique was introduced to insect cryobiology, with potentially useful applications.
Some questions surrounding the details of the cold tolerance strategy of H. maori, and the underlying mechanisms for insects in general, remain unanswered, but promise rewarding directions for future research.