Despite recent reports that antifreeze glycoproteins (AFGPs) protect mammalian cells during low-temperature preservation, T. Wang, Q. Zhu, X. Yang, J. R. Layne, and A. L. DeVries (Cryobiology 31: 185-192, 1994) reported that AFGPs failed to protect rat hearts during freezing. Rather, the presence of AFGPs exacerbated cardiac damage after freezing. This study examined the effects of freezing (-4 degrees C) in the presence of AFGPs at the cellular level with the use of cryomicroscopy. Large, blunt ice crystals formed in the solutions without AFGPs and excluded most cardiomyocytes from the plane of ice formation. After thawing, cells appeared similar in morphology to unfrozen cells. Ice in 0.5 mg/ml AFGP solution was more dendritic and prismatic than ice formed in the absence of AFGPs. On thawing, many cells exhibited spontaneous contraction, resulting in cell death. Spicular ice formed rapidly in the 10 mg/ml AFGP solution. These needlelike ice crystals appeared to penetrate the cardiomyocytes, resulting in intracellular freezing followed by cell lysis. These AFGP-induced changes in ice crystal structure may account for the injury observed in whole heart and cardiomyocyte experiments.

The wood frog ( Rana sylvatica) is a freeze-tolerant species that encounters subzero temperatures during its winter breeding season, whereas the leopard frog ( R. pipiens) is freeze intolerant and breeds in spring. Osmotic and freezing tolerances of spermatozoa from these species were inferred from spermolysis rate, integrity of the plasma membrane as judged using vital dye assay, and motility rate. Sperm of R. sylvatica became motile in hypotonic media (≤220 mosmol/kg) and tolerated in vitro exposure to osmotic concentrations spanning nearly three orders of magnitude. Relative to sperm from R. sylvatica, which were unaffected by freezing at temperatures of −4°C or greater, R. pipiens sperm were more susceptible to osmotic damage and cryoinjury. These differences likely reflect cellular adaptations to somatic freezing in R. sylvatica. Unprotected sperm from both species were extensively damaged by freezing at −8°C, but the presence of glucose, the cryoprotectant used by R. sylvatica, or the permeant glycerol markedly diminished cryoinjury. These data suggest the feasibility of developing gamete cryopreservation protocols to aid efforts in conserving amphibian populations.

No abstract

The supercooling point (SCP) of an insect model, the lady beetle Hippodamia convergens Guérin‐Menéville (Coleoptera, Coccinellidae) was markedly elevated by treatment with aqueous suspensions of the filamentous, ice nucleation active (INA) fungi Fusarium avenaceum and slightly elevated by Fusarium acuminatum. Addition of the surfactant Tween 80 to the fungal suspensions further reduced the supercooling capacity of adult beetles. When used alone the surfactant Triton X‐100 produced a greater SCP elevation than Tween 20 or Tween 80. The emulsifier gum arabic was ineffective in elevating beetle SCPs when applied alone and when added to INA fungal preparations it decreased their efficacy. Aqueous suspensions of both viable sporulating and viable pleomorphic (a permanent, degenerative, nonsporulating cultural state) forms of both fungal species were more effective in elevating the SCP than killed preparations except for the pleomorphic F. acuminatum suspension in which the killed form was slightly more active. Application of INA fungi applied in combination with surfactants may be useful in the development of methods for the biological control of overwintering freeze‐susceptible insect pests by decreasing their capacity to avoid lethal freezing by supercooling.