Overwintering insects are categorized either as freeze tolerant or freeze avoiding (supercooling) based on their ability or inability, respectively, to tolerate the formation of ice in their body. The freeze tolerant insects set their supercooling point (SCP) higher for winter to stimulate freezing at higher temperatures, while freeze avoiding insects survive winter in a supercooled state by depressing their SCP. Some supercooling insects, however, were found to survive in frozen state when freezing occurred through inoculation by external ice at mild subzero temperatures. Here, we assessed the potential relevance of inoculative freezing and freeze tolerance strategy in an insect that was so far considered as a classical example of a 'supercooler', the linden bug (Pyrrhocoris apterus). Microclimatic conditions of the overwintering microhabitat of P. apterus (leaf litter layer with buffered temperature fluctuations, mild sub-zero extremes, high humidity, and presence of ice) present a potentially high risk of inoculative freezing. We found that P. apterus is highly susceptible to inoculation by external ice. The temperature at which inoculative freezing occurred (above -3°C) was much higher compared to SCP (-16 °C to -20 °C in winter). The insects were inoculated through body openings and across cuticle and were able to survive after freezing. There was, however, a distinct critical ice fraction, corresponding to 38.7-42.8% of total body water, beyond which survival rapidly decreased to zero. We found that P. apterus adaptively reduces the actual ice fraction below critical ice fraction for winter season. Since many insect species overwinter in habitats similar to that of P. apterus, the ability to tolerate freezing after inoculation by external ice crystals could be much more common among 'supercooling' insects than it is currently appreciated.

Biology Centre CAS Institute of Entomology České Budějovice Czech Republic

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