Metabolic cost of freeze-thaw and source of CO2 production in the freeze-tolerant cricket Gryllus veletis

Freeze-tolerant insects can survive the conversion of a substantial portion of their body water to ice. While the process of freezing induces active responses from some organisms, these responses appear absent from freeze-tolerant insects. Recovery from freezing likely requires energy expenditure to repair tissues and re-establish homeostasis, which should be evident as elevations in metabolic rate after thaw. We measured carbon dioxide (CO2) production in the spring field cricket (Gryllus veletis) as a proxy for metabolic rate during cooling, freezing and thawing and compared the metabolic costs associated with recovery from freezing and chilling. We hypothesized that freezing does not induce active responses, but that recovery from freeze-thaw is metabolically costly. We observed a burst of CO2 release at the onset of freezing in all crickets that froze, including those killed by either cyanide or an insecticide (thiacloprid), implying that the source of this CO2 was neither aerobic metabolism or a coordinated nervous system response. These results suggest that freezing does not induce active responses from G. veletis, but may liberate buffered CO2 from hemolymph. There was a transient ‘overshoot’ in CO2 release during the first hour of recovery, and elevated metabolic rates at 24, 48 and 72 hours, in crickets that had been frozen compared to crickets that had been chilled (but not frozen). Thus, recovery from freeze-thaw and the repair of freeze-induced damage appears metabolically costly in G. veletis, and this cost persists for several days after thawing.

Evidence for non-colligative function of small cryoprotectants in a freeze-tolerant insect

Freeze tolerance, the ability to survive internal ice formation, facilitates survival of some insects in cold habitats. Low-molecular-weight cryoprotectants such as sugars, polyols and amino acids are hypothesized to facilitate freeze tolerance, but their in vivo function is poorly understood. Here, we use a combination of metabolomics and manipulative experiments in vivo and ex vivo to examine the function of multiple cryoprotectants in the spring field cricket Gryllus veletis . Cold-acclimated G. veletis are freeze-tolerant and accumulate myo -inositol, proline and trehalose in their haemolymph and fat body. Injecting freeze-tolerant crickets with proline and trehalose increases survival of freezing to lower temperatures or for longer times. Similarly, exogenous myo -inositol and trehalose increase ex vivo freezing survival of fat body cells from freeze-tolerant crickets. No cryoprotectant (alone or in combination) is sufficient to confer freeze tolerance on non-acclimated, freeze-intolerant G. veletis . Given that each cryoprotectant differentially impacts survival in the frozen state, we conclude that small cryoprotectants are not interchangeable and likely function non-colligatively in insect freeze tolerance. Our study is the first to experimentally demonstrate the importance of non-colligative cryoprotectant function for insect freeze tolerance both in vivo and ex vivo , with implications for choosing new molecules for cryopreservation.

Playing to an audience: the social environment influences aggression and victory displays

Animal behaviour studies have begun to incorporate the influence of the social environment, providing new opportunities for studying signal strategies and evolution. We examined how the presence and sex of an audience influenced aggression and victory display behaviour in field-captured and laboratory-reared field crickets ( Gryllus veletis ). Audience type, rearing environment and their interaction were important predictors in all model sets. Thus, audience type may impose different costs and benefits for competing males depending on whether they are socially experienced or not. Our results suggest that field-captured winners, in particular, dynamically adjust their contest behaviour to potentially gain a reproductive benefit via female eavesdropping and may deter future aggression from rivals by advertising their aggressiveness and victories.

The calling songs of male spring field crickets (Gryllus veletis) change as males age

Sexual traits are typically thought to convey information about a male's quality or condition. Female preference for older males has been documented in many taxa, but the evidence that males signal their age is inconclusive. We investigated lifetime patterns of acoustic mate attraction signalling in a longitudinal study of the spring field cricket, Gryllus veletis. We recorded males continuously throughout their lives, such that every pulse of sound produced by every male was analyzed. Our study answers two main questions: (1) Do calls change as males age? Our results reveal that the calls of male spring field crickets change with age; the calls of older males were quieter, with more silent periods within and between chirps, and produced less often than those of younger males. As males aged most of the changes in call structure reflect decreased calling effort. (2) What is the relationship between calling effort and longevity? Lifetime calling effort was positively related to longevity, such that males that called the most over their life also lived longer than males that called less. Together, our findings provide the most thorough exploration of lifetime signalling patterns in crickets to date.