Cold-hardening during long-term acclimation in a freeze-tolerant woolly bear caterpillar, Pyrrharctia isabella

Abstract

The banded woolly bear caterpillar, Pyrrharctia isabella (Lepidoptera: Erebidae), overwinters in leaf litter and survives freezing under natural conditions. Following 18 weeks of cold acclimation at 5°C, all caterpillars could survive 1 week of continuous freezing at −20°C or seven cycles of freezing-thawing at −20°C, but none survived freezing at −80°C. Field-collected caterpillars had a temperature of crystallization of −7.7±0.5°C that decreased significantly to −9.5±0.6°C after 12 weeks of acclimation at 5°C. Hemolymph levels of free proline, total amino acids and proteins reached a peak during the first 4 weeks of acclimation; concomitantly, hemolymph osmolality increased markedly during this interval (from 364 to 1282 mosmol kg −1). In contrast, hemolymph pH decreased during the first 4 weeks of acclimation before this trend reversed and pH values gradually returned to initial values. However, pH reached its peak value following 1 week at −20°C, but decreased after longer periods of freezing. During cold acclimation, cholesterol levels decreased in the hemolymph and the membrane fraction of fat body but not in other tissues. Lethal freezing at −80°C reduced cell survival in foregut tissue and caused leakage of free proline, total amino acids and proteins from tissues into the hemolymph. The addition of glycerol to the bathing medium reduced freezing injury in fat body cells, as evidenced by reduced leakage of amino acids and proteins.

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

“…Similarly, high exogenous glycerol concentrations increase freezing survival in less than 10% of (normally chill-susceptible) D. melanogaster larvae [9]. Glycerol also protected freezeintolerant G. veletis fat body cells frozen ex vivo, similar to ex vivo experiments with C. suppressalis fat body cells frozen in 250-750 mM glycerol [19] and P. isabella foregut cells frozen with 1 M glycerol [20]. Our combination of in vivo and ex vivo functional assays suggest that, in insects that accumulate it, high concentrations of glycerol may contribute to whole animal freeze tolerance by improving cell survival.…”

“…In vitro, trehalose and proline reduce membrane disruption during freezing [15]; and sugars [16], amino acids [17] and glycerol [18] can help maintain protein structure by stabilizing their hydration shell. Glycerol also improves survival of Chilo suppressalis fat body [19] and P. isabella foregut cells [20] frozen ex vivo. However, there is currently limited support for either hypothesis in vivo.…”

Lipid Metabolism in Response to Cold

“…However, in the gall fly E. solidaginis even if cholesterol levels increase in the hemolymph during winter acclimatization, the proportion of cholesterol embedded in membranes from Malpighian tubules does not increase (Yi and Lee, 2005). Similarly, in the woolly bear caterpillar Pyrrhactia isabella cholesterol levels remain stable during cold acclimation in tissues such as Malphighian tubules and cells from the midgut, but decrease both in the hemolymph, and in fat body cell membranes (Yi and Lee, 2016). As insect sterols originated from their diet, other sterols such as phytosterols can replace the role of cholesterol in insect membranes, as in D.…”

Effects of rapid cold-hardening and cold acclimation on egg survival and cryoprotectant contents in Ceracris kiangsu (Orthoptera: Arcypteridae)

“…However, Mohammadzadeh and Izadi (2018) found that the myo-inositol content was elevated during RCH and ACC in Trogoderma granarium , whereas trehalose was only elevated during ACC. In addition, low temperature acclimation increased the protein levels in the hemolymph of overwintering Pyrrharctia isabella larvae (Yi and Lee 2016). These findings suggest that the biochemical mechanisms associated with RCH and ACC induced cold tolerance may vary among species.…”