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Freezing survival and cryoprotective dehydration as cold tolerance mechanisms in the Antarctic nematode Panagrolaimus davidi

David A. Wharton^1,*, Gordon Goodall^1,2 and Craig J. Marshall²

¹ Department of Zoology, University of Otago, PO Box 56, Dunedin, New Zealand
² Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand

View larger version (15K): [in a new window]   Fig. 1. The effect of temperature on the freezing (filled circles) and survival (open circles) of P. davidi. Samples were nucleated and held at the test temperature for 30 min. Samples at 0°C were unfrozen controls. Values are means ± S.E.M., and where not visible error bars are contained within the data point; N=4.

View larger version (13K): [in a new window]   Fig. 2. The effect of cooling rate (filled circles, 0.5°C min-1; open circles, 0.2°C min-1; filled squares, 0.1°C min-1) on the freezing of P. davidi during decreasing temperature and after nucleation at -1°C. Values are means ± S.E.M., and where not visible error bars are contained within the data point; N=3.

View larger version (23K): [in a new window]   Fig. 3. The effect of cooling rate (filled bars, 0.5°C min-1; open bars, 0.2°C min-1; hatched bars, 0.1°C min-1) on the freezing of P. davidi after nucleation at -1°C and cooling to -5°C, and on survival. Values are means ± S.E.M., N=3.

View larger version (12K): [in a new window]   Fig. 4. The effect of different media (filled circles, ATW; open circles, dH2O; open squares, 0.1 mol l-1 NaCl dissolved in ATW) on the freezing of P. davidi during cooling from -1°C to -5°C at 0.5°C min-1 after ice nucleation and holding at -1°C for 5 min (A) or 30 min (B). Values are means ± S.E.M., and where not visible error bars are contained within the data point; N=3.

View larger version (25K): [in a new window]   Fig. 5. The effect of different media on the freezing (A) and survival (B) of P. davidi after ice nucleation at -1°C and cooling to -5°C. The samples were held at -1°C for 5 min (open bars) or 30 min (hatched bars) after nucleation. Values are means ± S.E.M., N=3.

View larger version (129K): [in a new window]   Fig. 6. Photomicrographs of P. davidi after cooling to -5°C. Samples were in artificial tapwater (ATW), dH2O or 0.1 mol l-1 NaCl dissolved in ATW. Freezing of the medium was seeded at -1°C and the sample held for 5 min or 30 min before cooling to -5°C at 0.5°C min-1. In ATW and dH2O, nematodes held at -1°C for 5 min tended to freeze upon further cooling (as indicated by darkening and no shrinkage), whilst those held at -1°C for 30 min dehydrated (indicated by them shrinking and remaining clear). In 0.1 mol l-1 NaCl dissolved in ATW, unfrozen nematodes do not show obvious shrinkage. Scale bar, 20 µm.

View larger version (11K): [in a new window]   Fig. 7. The effect of freezing on survival in all samples (nucleation at -1°C to -6°C; A) and on those where nucleation occurred at -1°C (B). The dotted line is the predicted survival if each nematode that had frozen died.