Abstract
The springtail, Megaphorura arctica, is freeze-avoiding and survives sub-zero temperatures by cryoprotective dehydration. At the onset of dehydration there is some supercooling of body fluids, and the danger of inoculative freezing, which would be lethal. To see if the springtails are protected by antifreeze proteins in this pre-equilibrium phase, we examined extracts from cold-acclimated M. arctica and recorded over 3 °C of freezing point depression. Proteins responsible for this antifreeze activity were isolated by ice affinity. They comprise isoforms ranging from 6.5 to 16.9 kDa, with an amino acid composition dominated by glycine (>35 mol%). Tryptic peptide sequences were used to identify the mRNA sequence coding for the smallest isoform. This antifreeze protein sequence has high similarity to one characterized in Hypogastrura harveyi, from a different springtail order. If these two antifreeze proteins are true homologs, we suggest their origin dates back to the Permian glaciations some 300 million years ago.
Highlights
The springtail, Megaphorura arctica Tullberg 1876, is widely distributed in the northern parts of the Palaearctic region where it is common and abundant along sea shores[1]
An initial extraction of M. arctica (2.3 mg) with buffer (18.4 μL) categorically showed the presence of an antifreeze proteins (AFPs) in the crude homogenate based on ice crystal shaping and high thermal hysteresis activity (Fig. 1)
AFP-mediated freezing point depression in the spruce budworm (Choristoneura fumiferana) of 6 °C is an important component in overwintering of first-instar larvae remaining unfrozen and viable in the boreal forest at temperatures of −20 to −30 °C28
Summary
The springtail, Megaphorura arctica Tullberg 1876, is widely distributed in the northern parts of the Palaearctic region where it is common and abundant along sea shores[1]. Other springtails inhabiting deeper layers of the soil (hemi- and eu-edaphic species), have little cuticular resistance to desiccation, and base their freeze-avoiding capacity on an alternative strategy termed cryoprotective dehydration In this strategy, the difference in water vapor pressure between ice in the soil and the supercooled hemolymph drives a net outflux of water vapor[8,9]. Even though supercooling is limited to a few °C, and of short duration, physical contact between springtails and ice crystals in the habitat potentially may result in inoculative spread of external ice to hemolymph through hydrophilic surfaces or openings of the animal such as mouth or ventral tube These considerations prompted us to look for antifreeze proteins (AFPs) which are well-known for their ability to limit ice growth in freeze-avoiding fish and insects[12,13,14]. Preliminary characterization of Gomphiocephalus hodgsoni from Antarctica suggests it contains an AFP with a distinct amino acid composition that is rich is Cys and His[22]
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