Hemolymph Proteins Involved in Insect Subzero-Temperature Tolerance: Ice Nucleators and Antifreeze Proteins

  • John G. Duman
  • Lei Xu
  • Lisa G. Neven
  • Donald Tursman
  • Ding Wen Wu


Prior to 1976, the majority of published studies dealing with the mechanisms of adaptation to subzero temperatures in cold-tolerant insects were concerned with the roles of low-molecular-weight solutes, mainly polyols and sugars. Since that time numerous examples of the importance of hemolymph proteins in insect cold adaptation have been determined. In this chapter, we discuss two types of hemolymph proteins with functionally opposite effects on the physical state of water at subzero temperatures. These are antifreeze proteins, which inhibit freezing, and ice nucleating proteins, which inhibit supercooling and induce ice formation at subzero temperatures above those at which freezing would normally take place in their absence.


Freezing Point Thermal Hysteresis Short Photoperiod Antifreeze Protein Subzero Temperature 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bakken, H. 1985. Cold hardiness in the alpine beetles Patrobus septentrionis and Calathus melanocephalus. J. Insect Physiol. 31:447–453.CrossRefGoogle Scholar
  2. Bale, J. S., T. N. Hansen, and J. G. Baust. 1989. Nucleators and sites of nucleation in the freeze tolerant larvae of the gallfly Eurosta solidaginis (Fitch). J. Insect Physiol. 35:291–298.CrossRefGoogle Scholar
  3. Baust, J. G. and K. E. Zachariassen. 1983. Seasonably active cell matrix associated ice nucleators in an insect. Cryo-Lett. 5:65–71.Google Scholar
  4. Block, W. and J. G. Duman. 1989. Presence of thermal hysteresis producing antifreeze proteins in the Antarctic mite, Alaskozetes antarcticus. J. Expt. Zool. 250:229–231.CrossRefGoogle Scholar
  5. Bremdal, S. and K. E. Zachariassen. 1988. Thermal hysteresis factors and supercooling of hibernating Rhagium inquisitor beetles. In Endocrinoligical Frontiers in Physiological Insect Ecology, eds.Google Scholar
  6. F. Sehnal, A. Zabza and D. L. Denlinger, pp. 187–191. Wroclaw Tech. Univ. Press, Wroclaw, Poland.Google Scholar
  7. Chino, H. 1985. Lipid transport: Biochemistry of hemolymph lipophorin. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology, Vol. 10, eds. G. A. Kerkut and L. I. Gilbert, pp. 115–135. Pergamon Press, New York.Google Scholar
  8. Davies, P. I., C. L. Hew, and G. L. Fletcher. 1988. Fish antifreeze proteins: Physiology and evolutionary biology. Can. J. Zool. 66:2611–2617.CrossRefGoogle Scholar
  9. DeVries, A. L. 1968. Freezing resistance in some Antarctic fishes. Doctoral dissertation, Stanford University, Stanford, CA.Google Scholar
  10. DeVries, A. L. 1971. Glycoproteins as biological antifreeze agents in Antarctic fishes. Science 172:1152–1155.CrossRefGoogle Scholar
  11. DeVries, A. L., 1983. Antifreeze peptides and glycopeptides in cold-water fishes. Annu. Rev. Physiol. 45:245–260.CrossRefGoogle Scholar
  12. DeVries, A. L. 1984. Role of glycopeptides and peptides in inhibition of crystallization of water in polar fishes. Philos. Trans. R. Soc. Lond. Biol. Sci. 304:575–588.CrossRefGoogle Scholar
  13. DeVries, A. L. 1986. Antifreeze glycopeptides and peptides: interactions with ice and water. Methods Enzymol. 127:293–303.CrossRefGoogle Scholar
  14. DeVries, A. L. and Y. Lin. 1977. Structure of a peptide antifreeze and mechanism of adsorption to ice. Biochim. Biophys. Acta, 495:388–392.Google Scholar
  15. DeVries, A. L., J. Vandenheede, and R. E. Feeney. 1971. Primary structure of freezing point depressing proteins. J. Biol. Chem. 246:305–309.Google Scholar
  16. Duman, J. G. 1977a. The role of macromolecular antifreeze in the Darkling beetle Meracantha contracta. J. Comp. Physiol. 115:279–286.Google Scholar
  17. Duman, J. G. 1977b. Variations in macromolecular antifreeze levels in larvae of the Darkling beetle, Meracantha contracta. J. Exp. Zool. 201:85–93.CrossRefGoogle Scholar
  18. Duman, J. G. 1977c. The effects of temperature, photoperiod and relative humidity on antifreeze production in larvae of the Darkling beetle, Meracantha contracta. J. Exp. Zool. 201:333–337.CrossRefGoogle Scholar
  19. Duman, J. G. 1979a. Thermal hystereses factors in overwintering insects. J. Insect Physiol. 25:805–810.CrossRefGoogle Scholar
  20. Duman, J. G. 1979b. Subzero temperature tolerance in spiders: the role of thermal hysteresis factors. J. Comp. Physiol. 131:347–352.Google Scholar
  21. Duman, J. G. 1980. Factors involved in the overwintering survival of the freeze tolerant beetle Dendroides canadensis. J. Comp. Physiol. 136:53–59.Google Scholar
  22. Duman, J. G. 1982. Insect antifreezes and ice nucleating agents. Cryobiol. 19:613–627.CrossRefGoogle Scholar
  23. Duman, J. G. 1984a. Thermal hysteresis antifreeze proteins in the midgut fluid of overwintering larvae of the beetle Dendroides canadensis J. Exp. Zool. 230:355–361.CrossRefGoogle Scholar
  24. Duman, J. G. 1984b. Change in overwintering mechanism in the Cucujid beetle, Cucujus clavipis. J. Insect Physiol. 30:235–239.CrossRefGoogle Scholar
  25. Duman, J. G. and A. L. DeVries. 1972. Freezing behavior of aqueous solutions of glycoproteins from the blood of an Antarctic fish. Cryobiol. 9:469–472.CrossRefGoogle Scholar
  26. Duman, J. G. and A. L. DeVries. 1976. The isolation, characterization and physical properties of protein antifreezes from the winter flounder, Pseudopleuronectes americanus. Comp. Biochem. Physiol. 54B:375–380.CrossRefGoogle Scholar
  27. Duman, J. G. and K. L. Horwath. 1983. The role of hemolymph proteins in the cold tolerance of insects. Annu. Rev. Physiol. 45:261–270.CrossRefGoogle Scholar
  28. Duman, J. G. and J. C. Patterson. 1978. The role of ice nucleators in the frost tolerance of overwintering queens of the bald faced hornet, Vespula maculata. Comp. Biochem. Physiol. 49:69–72.CrossRefGoogle Scholar
  29. Duman, J. G., K. L. Horwath, A. P. Tomchaney, and J. L. Patterson. 1982. Antifreeze agents of terrestrial arthropods. Comp. Biochem. Physiol. A. 45:261–270.Google Scholar
  30. Duman, J. G., J. P. Morris, and F. J. Castellino. 1984. Purification and composition of an ice nucleating protein from queens of the hornet, Vespula maculata. J. Comp. Physiol. B. 154:79–83.CrossRefGoogle Scholar
  31. Duman, J. G., L. G. Neven, J. M. Beal, K. R. Olson, and F. J. Castellino. 1985. Freeze tolerance adaptations, including hemolymph protein and lipoprotein ice nucleators, in the larvae of the cranefly Tipula trivittata. J. Insect Physiol. 31:1–8.CrossRefGoogle Scholar
  32. Farrant, J. 1980. General observations on cell preservation. In Low Temperature Preservation in Biology and Medicine, eds. M. J. Ashwood-Smith and J. Farrant, pp. 1–8. University Park Press, Baltimore.Google Scholar
  33. Feeney, R. E. and T. S. Burcham. 1986. Antifreeze glycoproteins from polar fish blood. Annu. Rev. Biophys. Biophys. Chem. 15:59–78.CrossRefGoogle Scholar
  34. Fields, P. G. and J. N. McNeil. 1986. Possible dual cold-hardiness strategies in Cisseps fulvicollis (Lepidoptera: Arctiidae). Can. Entomol. 118:1309–1311.CrossRefGoogle Scholar
  35. Gehrken, U. 1984. Winter survival of an adult bark beetle Ips acuminatus. J. Insect Physiol. 30:421–429.CrossRefGoogle Scholar
  36. Gehrken, U. and L. Sømme. 1987. Increased cold hardiness in eggs of Arcynopteryx compacta (Plecoptera) by dehydration. J. Insect Physiol. 33:987–991.CrossRefGoogle Scholar
  37. Gordon, M. S., B. H. Amdur, and P. F. Scholander. 1962. Freezing resistance in some northern fishes. Biol. Bull. 122:52–62.CrossRefGoogle Scholar
  38. Green, R. L. and G. Warren. 1985. Physical and functional repetition in a bacterial ice nucleation gene. Nature 317:645–648.CrossRefGoogle Scholar
  39. Grimstone, A. V., A. M. Mullinger, and J. A. Ramsay. 1968. Further studies on the rectal complex of the mealworm, Tenebrio molitor (Coleoptera, Tenebrionidae). Philos. Trans. R. Soc. Biol.Sci 253:343–382.CrossRefGoogle Scholar
  40. Hansen, T. N. and J. G. Baust. 1988. Differential scanning calorimetric analysis of antifreeze protein activity in the common mealworm, Tenebrio molitor. Biochim. Biophys. Acta 957:217–221.CrossRefGoogle Scholar
  41. Hew, C. L., M. H. Kao, and Y. P. So. 1983. Presence of cystine-containing antifreeze proteins in the spruce budworm, Choristoneura fumiferana. Can. J. Zool. 61:2324–2328.CrossRefGoogle Scholar
  42. Hirsh, A. G., R. J. Williams, and G. T. Meryman. 1985. A novel method of natural protection: Intracellular glass formation in deeply frozen Populus. Plant Physiol. 79:41–56.CrossRefGoogle Scholar
  43. Horwath, K. L. and J. G. Duman. 1982. Involvement of the circadian system in photoperiodic regulation of insect antifreeze proteins. J. Exp. Zool. 219:267–270.CrossRefGoogle Scholar
  44. Horwath, K. L. and J. G. Duman. 1983a. Photoperiodic and thermal regulation of antifreeze protein levels in the beetle Dendroides canadensis. J. Insect Physiol. 29:907–917.CrossRefGoogle Scholar
  45. Horwath, K. L. and J. G. Duman. 1983b. Induction of antifreeze protein production by juvenile hormone in larvae of the beetle Dendroides canadensis. J. Comp. Physiol. 151:233–240.Google Scholar
  46. Horwath, K. L. and J. G. Duman. 1984a. Further studies on the involvement of the circadian system in photoperiodic control of antifreeze protein production in the beetle Dendroides canadensis. J. Insect Physiol. 30:947–955.CrossRefGoogle Scholar
  47. Horwath, K. L. and J. G. Duman. 1984b. Yearly variations in the overwintering mechanism of the cold hardy beetle Dendroides canadensis. Physiol. Zool. 57:40–45.Google Scholar
  48. Horwath, K. L. and J. G. Duman. 1986. Thermoperiodic involvement in antifreeze protein production in the cold hardy beetle Dendroides canadensis. Implications for photoperiodic time measurement. J. Insect Physiol. 32:799–806.CrossRefGoogle Scholar
  49. Husby, J. A. and K. E. Zachariassen. 1980. Antifreeze agents in the body fluid of winter active insects and spiders. Experientia 36:963–964.CrossRefGoogle Scholar
  50. Katagiri, C. 1985. Structure of lipophorin in insect blood: location of phospholipid. Biochim. Biophys. Acta 834:139–143.Google Scholar
  51. Knight, C. A. 1967. The Freezing of Supercooled Liquids. Van Nostrand, New York.Google Scholar
  52. Knight, C. A. 1979. Ice nucleation in the atmosphere. Adv. Coll. Int. Sci. 10:369–395.CrossRefGoogle Scholar
  53. Knight, C. A. and J. G. Duman. 1986. Inhibition of recrystallization of ice by insect thermal hysteresis proteins: a possible cryoprotective role. Cryobiol. 23:256–262.CrossRefGoogle Scholar
  54. Knight, C. A., A. L. DeVries, and L. D. Oolman. 1984. Fish antifreeze protein and the freezing and recrystallization of ice. Nature 308:295–296.CrossRefGoogle Scholar
  55. Knight, C. A., J. Hallett, and A. L. DeVries. 1988. Solute effects on ice recrystallization: an assessment technique. Cryobiol. 25:55–60.CrossRefGoogle Scholar
  56. Kozloff, L. M., M. Lute, and D. Westaway. 1984. Phosphatidylinositol as a component of the ice nucleating site of Pseudomonas syringae and Erwinia herbicola. Science 226:845–846.Google Scholar
  57. Kozloff, L. M., M. Lute, and F. Arellano. 1987. Role of phosphatidylinositol in ice nucleation. Paper delivered at the Third International Conference on Biological Ice Nucleation, October 1987, Newport, Oregon.Google Scholar
  58. Kukal, O., A. S. Serianni, and J. G. Duman. 1988. Glycerol production in a freeze tolerant arctic insect, Gynaephora groenlandica: an in vivo 13C NMR study. J. Comp. Physiol. 158:175–183.Google Scholar
  59. Levenbook, L. 1985. Insect storage proteins. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 10, eds. G. A. Kerkut and L. I. Gilbert, pp. 307–346. Pergamon Press, New York.Google Scholar
  60. Lin, Y., J. G. Duman, and A. L. DeVries. 1972. Studies on the structure and activity of low molecular weight glycoproteins from an Antarctic fish. Biochem. Biophys. Res. Comm. 46:87–92.CrossRefGoogle Scholar
  61. Lindow, S. E. 1983. The role of bacterial ice nucleation in frost injury to plants. Annu. Rev. Phytopath. 21:363–384.CrossRefGoogle Scholar
  62. Loomis, S. H. 1987. Freezing in intertidal invertebrates: An update. Cryo-Lett. 8:186–195.Google Scholar
  63. Manavalan, P. and P. K. Ponnuswamy. 1978. Hydrophobic character of amino acid residues in globular proteins. Nature 275:673–674.CrossRefGoogle Scholar
  64. Mazur, P. 1984. Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247:C125–C142.Google Scholar
  65. Miller, L. K. 1982. Cold hardiness strategies of some adult and immature insects overwintering in interior Alaska. Comp. Biochem. Physiol. 73A:595–604.CrossRefGoogle Scholar
  66. Neven, L. G., J. G. Duman, J. M. Beals, and F. J. Castellino. 1986. Overwintering adaptations of the stag beetle, Ceruchus piceus: removal of ice nucleators in the winter to promote supercooling. J. Comp. Physiol. 156:707–716.Google Scholar
  67. Neven, L. G., J. G. Duman, M. G. Low, L. C. Sehl, and F. J. Castellino. 1989. Purification and characterization of an insect hemolymph lipoprotein ice nucleator: evidence for the importance of phosphatidylinositol and apolipoprotein in the ice nucleator activity. J. Comp. Physiol. 159:71–82.Google Scholar
  68. Parody-Morreale, A., G. Bishop. R. Fall, and S. J. Gill. 1986. A differential scanning calorimeter for ice nucleation distribution studies—application to bacterial nucleators. Anal. Biochem. 154:682–690.CrossRefGoogle Scholar
  69. Parody-Morreale, A., K. P. Murphy, E. Di Cera, R. Fall, A. L. DeVries, and S. J. Gill. 1988. Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins. Nature 333:782–783.CrossRefGoogle Scholar
  70. Patterson, J. L. and J. G. Duman. 1978. The role of thermal hysteresis producing proteins in the low temperature tolerance and water balance of larvae of the mealworm, Tenebrio molitor. J. Exp. Biol. 74:37–45.Google Scholar
  71. Patterson, J. L. and J. G. Duman. 1979. Composition of a protein antifreeze from larvae of the beetle Tenebrio molitor. J. Exp. Zool. 210:361–367.CrossRefGoogle Scholar
  72. Patterson, J. C. and J. G. Duman. 1982. Purification and composition of protein antifreezes with high cysteine contents from larvae of the beetle Tenebrio molitor. J. Exp. Zool. 219:381–384.CrossRefGoogle Scholar
  73. Patterson, J. L., T. J. Kelly, and J. G. Duman. 1981. Purification and composition of a thermal hysteresis producing protein from the milkweed bug, Oncopeltus fasciatus. J. Comp. Physiol. 142:539–542.Google Scholar
  74. Ramsay, J. A. 1964. The rectal complex of the mealworm, Tenebrio molitor L. (Coleoptera, Tenebrionidae). Philos. Trans. R. Soc. Biol. Sci. 248:279–314.CrossRefGoogle Scholar
  75. Ramsay, J. A. and R. A. Brown. 1955. Simplified apparatus and procedure for freezing point determinations upon small volumes of fluid. J. Scient. Instrum. 32:372–375.CrossRefGoogle Scholar
  76. Rancourt, D. E., V. K. Walker, and P. L. Davies. 1987. Flounder antifreeze protein synthesis under heat shock control in transgenic Drosophila melanogaster. Mol. Cell Biol. 7:2188–2195.Google Scholar
  77. Raymond, J. A. and A. L. DeVries. 1977. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc. Natl. Acad. Sci. USA 74:2589–2593.CrossRefGoogle Scholar
  78. Raymond, J. A., P. Wilson, and A. L. DeVries. 1989. Inhibition of growth of nonbasal planes in ice by fish antifreezes. Proc. Natl. Acad. Sci. USA 86:881–885.CrossRefGoogle Scholar
  79. Ring, R. A. 1982. Freezing tolerant insects with low supercooling points. Comp. Biochem. Physiol. 73A:605–612.CrossRefGoogle Scholar
  80. Schneppenheim, R. and H. Theede. 1980. Isolation and characterization of freezing point depressing peptides from larvae of Tenebrio molitor. Comp. Biochem. Physiol. 67:561–568.Google Scholar
  81. Scholander, P. F., L. van Dam, J. W. Kanwisher, H. T. Hammel, and M. S. Gordon. 1957. Supercooling and osmoregulation in Arctic fish. J. Cellular Comp. Physiol. 49:5–24.CrossRefGoogle Scholar
  82. Shapiro, J. P., P. S. Keim, and J. H. Law. 1984. Structural studies on lipophorin, an insect lipoprotein. J. Biol. Chem. 259:3680–3685.Google Scholar
  83. Shapiro, J. P., M. A. Wells, and J. H. Law. 1988. Lipid transport in insects. Annu. Rev. Entomol. 33:297–318.CrossRefGoogle Scholar
  84. Shier, W. T., Y. Lin, and A. L. DeVries. 1975. Structure of the carbohydrate of antifreeze glycoproteins from an Antarctic fish. FEBS Lett. 54:135–138.CrossRefGoogle Scholar
  85. Slaughter, D., G. L. Fletcher, V. S. Ananthanarayanan, and C. L. Hew. 1981. Antifreeze proteins from the sea raven, Hemitripterus americanus. J. Biol. Chem. 256:2022–2026.Google Scholar
  86. Sømme, L. 1978. Nucleating agents in the haemolymph of the third instar larvae of Eurosta solidaginis (Fitch) (Diptera: Tephritidae). Norw. J. Entomol. 25:187–188.Google Scholar
  87. Sømme, L. 1982. Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. 73:519–543.CrossRefGoogle Scholar
  88. Storey, K. B. and J. M. Storey. 1988. Freeze tolerance in animals. Physiol. Rev. 68:27–84.Google Scholar
  89. Theede, H., R. Schneppenheim, and L. Bevess. 1976. Frostschutz—Glycoproteine bei Mytilus edulis? Mar. Biol. 36:183–189.CrossRefGoogle Scholar
  90. Tomchaney, A. P., J. P. Morris, S. H. Kang, and J. G. Duman. 1982. Purification, composition and physical properties of a thermal hysteresis antifreeze protein from larvae of the beetle, Tenebrio molitor. Biochem. 21:716–721.Google Scholar
  91. Vali, G. 1971. Quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids. J. Atmos. Sci. 28:402–409.CrossRefGoogle Scholar
  92. Wasylyk, J. M., A. R. Tice, and J. G. Baust. 1988. Partial glass formation: a novel method of insect cryoprotection: Cryobiol. 25:451–458.CrossRefGoogle Scholar
  93. Wolber, P. and G. Warren. 1989. Bacterial ice-nucleation proteins. TIBS 14:179–182.Google Scholar
  94. Wolber, P. K., C. A. Derninger, M. W. Southworth, J. Vandekerchove, M. van Montagu, and G. Warren. 1986. Identification and purification of a bacterial ice nucleation protein. Proc. Natl. Acad. Sci. USA 83:7256–7260.CrossRefGoogle Scholar
  95. Wu, D. W., J. G. Duman and L. Xu. 1991. Enhancement of insect antifreeze protein activity by antibodies. Biochem. Biophys. Acta (in press).Google Scholar
  96. Xu, L., L. G. Neven, and J. G. Duman. 1990. Hormonal control of hemplymph lipoprotein ice nucleators in overwintering freeze susceptible larvae of the stag beetle Ceruchus piceus: Adipokinetic hormone and juvenile hormone. J. Comp. Physiol. 160: 51–59.Google Scholar
  97. Xu, L., and J. G. Duman. 1991. Involvement of juvenile hormone in the induction of antifreeze protein production by the fat body of larvae of the beetle Dendroides canadensis. J. Exp. Zool. (in press).Google Scholar
  98. Yang, D. S. C., M. Sax, A. Chakrabarthy, and C. L. Hew. 1988. Crystal structure of an antifreeze polypeptide and its mechanistic implications. Nature 333:232–237.CrossRefGoogle Scholar
  99. Zachariassen, K. E. 1982. Nucleating agents in cold hardy insects. Comp. Biochem. Physiol. 73:557–562.CrossRefGoogle Scholar
  100. Zachariassen, K. E. 1985. Physiology of cold tolerance in insects. Physiol. Rev. 65:799–832.Google Scholar
  101. Zachariassen, K. E. and H. T. Hammel. 1976. Nucleating agents in the haemolymph of insects tolerant to freezing. Nature 262:285–287.CrossRefGoogle Scholar
  102. Zachariassen, K. E. and J. A. Husby. 1982. Antifreeze effect of thermal hysteresis agents protects highly supercooled insects. Nature 298:865–867.CrossRefGoogle Scholar
  103. Zachariassen, K. E., J. G. Baust, and R. E. Lee. 1982. A method for the quantitative determination of ice nucleating agents in insect hemolymph. Cryobiol. 19:180–184.CrossRefGoogle Scholar
  104. Zettel, J. 1984. Cold hardiness strategies and thermal hysteresis in Collembola. Rev. Ecol. Biol. Sol. 21:189–203.Google Scholar

Copyright information

© Chapman and Hall 1991

Authors and Affiliations

  • John G. Duman
  • Lei Xu
  • Lisa G. Neven
  • Donald Tursman
  • Ding Wen Wu

There are no affiliations available

Personalised recommendations