Eur. J. Entomol. 114: 470-480, 2017 | DOI: 10.14411/eje.2017.060

More complex than expected: Cold hardiness and the concentration of cryoprotectants in overwintering larvae of five Erebia butterflies (Lepidoptera: Nymphalidae)

Pavel VRBA1, Oldřich NEDVĚD1,2, Helena ZAHRADNÍČKOVÁ1, Martin KONVIČKA1,2
1 Biology Centre of the Czech Academy of Sciences, Institute of Entomology, Branišovská 31, 370 05 České Budějovice, Czech Republic; e-mails: vrba_pavel@centrum.cz, nedved@prf.jcu.cz, helenaz@entu.cas.cz, konva333@gmail.com
2 Faculty of Sciences, University South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic

Understanding the factors restricting the distribution of some insect species to high altitudes is hindered by poor knowledge of temporal changes in their cold hardiness during overwintering. We studied overwintering larvae of five species of Erebia butterflies (Lepidoptera: Nymphalidae: Satyrinae) differing in altitudinal distribution: lowland E. medusa, submountain E. aethiops, subalpine E. pronoe, alpine E. cassioides, and subnivean E. pluto. We subjected them to three treatments, AutumnWarm (13/8°C), imitating conditions prior to overwintering; AutumnCold (5/0°C), imitating late autumn conditions; and WinterCold (5/0°C), differing from AutumnCold by a shorter photoperiod and longer exposure to zero temperatures. Supercooling points (SCP) did not differ between species in the AutumnWarm treatment, despite large differences in the concentrations of cryoprotectants (CrPC; lowest in E. medusa and E. aethiops). Lowland E. medusa was freeze-tolerant, the subalpine, alpine and subnivean species were freeze-avoidant, whereas submountain E. aethiops displayed a mixed strategy. SCPs diverged in the AutumnCold treatment: it increased in the lowland E. medusa (from -16.5 to -10.8°C) and reached the lowest value in E. cassioides (-21.7°C). In WinterCold, SCP increased in subalpine E. pronoe (from -16.1°C in AutumnWarm and -18.7°C in AutumnCold to -12.6°C). E. medusa decreased and E. aethiops increased their CrPCs between autumn and winter; the highest CrPC was recorded in subnivean E. pluto. CrPC did not correlate with SCP across species and treatments. Cryoprotectant profiles corroborated the difference between lowland and freeze-tolerant E. medusa and the three high altitude freeze-avoidant species, with E. aethiops in an intermediate position. Glycerol was surprisingly rare, trehalose was important in all species, and such rare compounds as monopalmitin and monostearin were abundantly present in E. pronoe, E. cassioides and E. pluto.

Keywords: Lepidoptera, Nymphalidae, Satyrinae, Erebia, larvae, alpine habitats, cold hardiness, cryoprotectant compounds, supercooling point, temperature, overwintering

Received: June 11, 2017; Accepted: October 9, 2017; Published online: November 2, 2017

Download citation

References

  1. Andreadis S.S., Vryzas Z., Papadopoulou-Mourkidou E. & Savopoulou-Soultani M. 2008: Age-dependent changes in tolerance to cold and accumulation of cryoprotectants in overwintering and non-overwintering larvae of European corn borer Ostrinia nubilalis. - Physiol. Entomol. 33: 365-371. Go to original source...
  2. Atapour M. & Moharramipour S. 2009: Changes of cold hardiness, supercooling capacity, and major cryoprotectants in overwintering larvae of Chilo suppressalis (Lepidoptera: Pyralidae). - Environ. Entomol. 38: 260-265. Go to original source...
  3. Bila K., Sipos J., Kindlmann P. & Kuras T. 2016: Consequences for selected high-elevation butterflies and moths from the spread of Pinus mugo into the alpine zone in the High Sudetes Mountains. - PeerJ 4: e2094, 20 pp. Go to original source...
  4. Brunnhofer V., Nedved O. & Hodkova M. 1991: Methodical improvement for measuring of supercooling point in insects. - Acta Entomol. Bohemoslov. 88: 349-350.
  5. Cha W.H. & Lee D.W. 2016: Identification of rapid cold hardening-related genes in the tobacco budworm, Helicoverpa assulta. - J. Asia-Pac. Entomol. 19: 1061-1066.
  6. Crozier L. 2004: Warmer winters drive butterfly range expansion by increasing survivorship. - Ecology 85: 231-241. Go to original source...
  7. Davies Z.G., Wilson R.J., Coles S. & Thomas C.D. 2006: Changing habitat associations of a thermally constrained species, the silver-spotted skipper butterfly, in response to climate warming. - J. Anim. Ecol. 75: 247-256. Go to original source...
  8. Denlinger D.L. 1991: Relationship between cold hardiness and diapause. In Lee R. E. & Denlinger D.L. (eds): Insects at Low Temperature. Springer US, Boston, MA, pp. 174-198.
  9. Denlinger D.L., Lee R.E., Yocum G.D. & Kukal O. 1992: Role of chilling in the acquisition of cold tolerance and the capacitation to express stress proteins in diapausing pharate larvae of the gypsy moth, Lymantria dispar. - Arch. Insect Biochem. Physiol. 21: 271-280. Go to original source...
  10. Duman J.G. 2001: Antifreeze and ice nucleator proteins in terrestrial arthropods. - Annu. Rev. Physiol. 63: 327-357. Go to original source...
  11. Franco A.M.A., Hill J.K., Kitschke C., Collingham Y.C., Roy D.B., Fox R., Huntley B. & Thomas C.D. 2006: Impacts of climate warming and habitat loss on extinctions at species' low-latitude range boundaries. - Glob. Change Biol. 12: 1545-1553. Go to original source...
  12. Hanson A.A. & Venette R.C. 2013: Thermocouple design for measuring temperatures of small insects. - CryoLetters 34: 261-266.
  13. Horwath K.L. & Duman J.G. 1984: Yearly variations in the overwintering mechanisms of the cold hardy beetle Dendroides canadensis. - Physiol. Zool. 57: 40-45. Go to original source...
  14. Hou M.L., Lin W. & Han Y.Q. 2009: Seasonal changes in supercooling points and glycerol content in overwintering larvae of the Asiatic rice borer from rice and water-oat plants. - Environ. Entomol. 38: 1182-1188. Go to original source...
  15. Kleckova I. & Klecka J. 2016: Facing the heat: Thermoregulation and behaviour of lowland species of a cold-dwelling butterfly genus, Erebia. - PLoS ONE 11: e0150393, 16 pp. Go to original source...
  16. Kleckova I., Konvicka M. & Klecka J. 2014: Thermoregulation and microhabitat use in mountain butterflies of the genus Erebia: Importance of fine-scale habitat heterogeneity. - J. Therm. Biol. 41: 50-58. Go to original source...
  17. Kleckova I., Vrba P. & Konvicka M. 2015: Quantitative evidence for spatial variation in the biennial life cycle of the mountain butterfly Erebia euryale (Lepidoptera: Nymphalidae) in the Czech Republic. - Eur. J. Entomol. 112: 114-119.
  18. Konvicka M., Mihaly C.V., Rakosy L., Benes J. & Schmitt T. 2014: Survival of cold-adapted species in isolated mountains: the population genetics of the Sudeten ringlet, Erebia sudetica sudetica, in the Jesenik Mts., Czech Republic. - J. Insect Conserv. 18: 153-161. Go to original source...
  19. Konvicka M., Benes J., Cizek O., Kuras T. & Kleckova I. 2016: Has the currently warming climate affected populations of the mountain ringlet butterfly, Erebia epiphron (Lepidoptera: Nymphalidae), in low-elevation mountains? - Eur. J. Entomol. 113: 295-301. Go to original source...
  20. Kostal V. & Simek P. 1995: Dynamics of cold hardiness, supercooling and cryoprotectants in diapausing and nondiapausing pupae of the cabbage root fly, Delia radicum (L.). - J. Insect Physiol. 41: 627-634. Go to original source...
  21. Kostal V., Slachta M. & Simek P. 2001: Cryoprotective role of polyols independent of the increase in supercooling capacity in diapausing adults of Pyrrhocotis apterus (Heteroptera: Insecta). - Compar. Biochem. Physiol. (B) 130: 365-374. Go to original source...
  22. Kostal V., Tamura M., Tollarova M. & Zahradnickova H. 2004: Enzymatic capacity for accumulation of polyol cryoprotectants changes during diapause development in the adult red firebug, Pyrrhocoris apterus. - Physiol. Entomol. 29: 344-355. Go to original source...
  23. Kostal V., Zahradnickova H., Simek P. & Zeleny J. 2007: Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus. - J. Insect Physiol. 53: 580-586. Go to original source...
  24. Kostal V., Miklas B., Dolezal P., Rozsypal J. & Zahradnickova H. 2014: Physiology of cold tolerance in the bark beetle, Pityogenes chalcographus and its overwintering in spruce stands. - J. Insect Physiol. 63: 62-70. Go to original source...
  25. Kukal O. & Duman J.G. 1989: Switch in the overwintering strategy of 2 insect species and latitudinal differences in cold hardiness. - Can. J. Zool. 67: 825-827. Go to original source...
  26. Kukal O., Ayres M.P. & Scriber J.M. 1991: Cold tolerance of the pupae in relation to the distribution of swallowtail butterflies. - Can. J. Zool. 69: 3028-3037. Go to original source...
  27. Kuras T., Benes J. & Konvicka M. 2000: Differing habitat affinities of four Erebia species (Lepidoptera : Nymphalidae, Satyrinae) in the Hruby Jesenik Mts, Czech Republic. - Biologia 55: 169-175.
  28. Kuras T., Benes J., Fric Z. & Konvicka M. 2003: Dispersal patterns of endemic alpine butterflies with contrasting population structures: Erebia epiphron and E. sudetica. - Popul. Ecol. 45: 115-123. Go to original source...
  29. Lee R.E. 1991: Principles of insect low temperature tolerance. In Lee R.E. & Denlinger D.L. (eds): Insects at Low Temperature. Springer US, Boston, MA, pp. 17-46. Go to original source...
  30. MacLean H.J., Higgins J.K., Buckley L.B. & Kingsolver J.G. 2016: Morphological and physiological determinants of local adaptation to climate in Rocky Mountain butterflies. - Conserv. Physiol. 4(1): cow035, 10 pp. Go to original source...
  31. Marshall K.E. & Sinclair B.J. 2011: The sub-lethal effects of repeated freezing in the woolly bear caterpillar Pyrrharctia isabella. - J. Exp. Biol. 214: 1205-1212. Go to original source...
  32. Nedved O., Hodkova M., Brunnhofer V. & Hodek I. 1995: Simultaneous measurement of low temperature survival and supercooling in a sample of insects. - CryoLetters 16: 108-113.
  33. Pena C., Witthauer H., Kleckova I., Fric Z. & Wahlberg N. 2015: Adaptive radiations in butterflies: evolutionary history of the genus Erebia (Nymphalidae: Satyrinae). - Biol. J. Linn. Soc. 116: 449-467. Go to original source...
  34. Pere C., Jactel H. & Kenis M. 2013: Response of insect parasitism to elevation depends on host and parasitoid life-history strategies. - Biol. Lett. 9: 20130028, 4 pp. Go to original source...
  35. Polic D., Fiedler K., Nell C. & Grill A. 2014: Mobility of ringlet butterflies in high-elevation alpine grassland: effects of habitat barriers, resources and age. - J. Insect Conserv. 18: 1153-1161. Go to original source...
  36. Pullin A.S. & Bale J.S. 1989a: Effects of low temperature on diapausing Aglais urticae and Inachis io (Lepidoptera, Nymphalidae) - cold hardiness and overwintering survival. - J. Insect Physiol. 35: 277-281. Go to original source...
  37. Pullin A.S. & Bale J.S. 1989b: Effects of low temperature on diapausing Aglais urticae and Inachis io (Lepidoptera, Nymphalidae) - overwintering physiology. - J. Insect Physiol. 35: 283-290. Go to original source...
  38. Pullin A.S. & Bale J.S. 1989c: Influence of diapause and temperature on cryoprotectant synthesis and cold hardiness in pupae of Pieris brassicae. - Compar. Biochem. Physiol. (A) 94: 499-503. Go to original source...
  39. Ramlov H. 2000: Aspects of natural cold tolerance in ectothermic animals. - Human Reprod. 15: 26-46. Go to original source...
  40. Roland J. & Matter S.F. 2016: Pivotal effect of early-winter temperatures and snowfall on population growth of alpine Parnassius smintheus butterflies. - Ecol. Monogr. 86: 412-428. Go to original source...
  41. Scalercio S., Bonacci T., Mazzei A., Pizzolotto R. & Brandmayr P. 2014: Better up, worse down: bidirectional consequences of three decades of climate change on a relict population of Erebia cassioides. - J. Insect Conserv. 18: 643-650. Go to original source...
  42. Schmitt T. 2002: The biology of Erebia medusa ([Denis & Schiffermüller],1775) in Central Europe (Lepidoptera). - Acta Biol. Debr. 24: 113-129.
  43. Schmitt T., Hewitt G.M. & Muller P. 2006: Disjunct distributions during glacial and interglacial periods in mountain butterflies: Erebia epiphron as an example. - J. Evol. Biol. 19: 108-113. Go to original source...
  44. Schmitt T., Louy D., Zimmermann E. & Habel J.C. 2016: Species radiation in the Alps: multiple range shifts caused diversification in Ringlet butterflies in the European high mountains. - Org. Divers. Evol. 16: 791-808. Go to original source...
  45. Sformo T., Walters K., Jeannet K., Wowk B., Fahy G.M., Barnes B.M. & Duman J.G. 2010: Deep supercooling, vitrification and limited survival to-100 degrees C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae. - J. Exp. Biol. 213: 502-509. Go to original source...
  46. Shimada K. 1988: Seasonal changes of supercooling points, haemolymph carbohydrate contents and freezing tolerance in overwintering pupae of two common swallowtails Papilio machaon and P. xuthus. - Kontyu 56: 678-686.
  47. Sinclair B.J., Addo-Bediako A. & Chown S.L. 2003: Climatic variability and the evolution of insect freeze tolerance. - Biol. Rev. 78: 181-195. Go to original source...
  48. Sinclair B.J., Alvarado L.E.C. & Ferguson L.V. 2015: An invitation to measure insect cold tolerance: Methods, approaches, and workflow. - J. Therm. Biol. 53: 180-197. Go to original source...
  49. Slamova I., Klecka J. & Konvicka M. 2011: Diurnal behavior and habitat preferences of Erebia aethiops, an aberrant lowland species of a mountain butterfly clade. - J. Insect Behav. 24: 230-246. Go to original source...
  50. Slamova I., Klecka J. & Konvicka M. 2013: Woodland and grassland mosaic from a butterfly perspective: habitat use by Erebia aethiops (Lepidoptera: Satyridae). - Insect Conserv. Divers. 6: 243-254. Go to original source...
  51. Somme L. 1982: Supercooling and winter survival in terrestrial arthropods. - Compar. Biochem. Physiol. (A) 73: 519-543. Go to original source...
  52. Sonderegger P. 2005: Die Erebien der Schweiz (Lepidoptera: Satyrinae, Genus Erebia). Peter Sonderegger, Biel / Bienne, 712 pp.
  53. Storey K.B. & Storey J.M. 1991: Biochemistry of cryoprotectants. In Lee R.E. & Denlinger D.L. (eds): Insects at Low Temperature. Chapman & Hall, New York and London, pp. 64-93.
  54. Stuhldreher G. & Fartmann T. 2015: Oviposition-site preferences of a declining butterfly Erebia medusa (Lepidoptera: Satyrinae) in nutrient-poor grasslands. - Eur. J. Entomol. 112: 493-499. Go to original source...
  55. Ter Braak C.J.F. & Smilauer P. 2013: Canoco 5, Windows Release (5.00). URL: http://www.canoco5.com
  56. Trudeau M., Mauffette Y., Rochefort S., Han E. & Bauce E. 2010: Impact of host tree on forest tent caterpillar performance and offspring overwintering mortality. - Environ. Entomol. 39: 498-504. Go to original source...
  57. Turnock W.J. & Fields P.G. 2005: Winter climates and coldhardiness in terrestrial insects. - Eur. J. Entomol. 102: 561-576. Go to original source...
  58. Vernon P. & Vannier G. 2002: Evolution of freezing susceptibility and freezing tolerance in terrestrial arthropods. - C. R. Biol. 325: 1185-1190. Go to original source...
  59. Vesala L., Salminen T.S., Kostal V., Zahradnickova H. & Hoikkala A. 2012: Myo-inositol as a main metabolite in overwintering flies: seasonal metabolomic profiles and cold stress tolerance in a northern drosophilid fly. - J. Exp. Biol. 215: 2891-2897. Go to original source...
  60. Voituron Y., Mouquet N., de Mazancourt C. & Clobert J. 2002: To freeze or not to freeze? An evolutionary perspective on the cold-hardiness strategies of overwintering ectotherms. - Am. Nat. 160: 255-270.
  61. Vrba P., Konvicka M. & Nedved O. 2012: Reverse altitudinal cline in cold hardiness among Erebia butterflies. - CryoLetters 33: 251-258.
  62. Vrba P., Dolek M., Nedved O., Zahradnickova H., Cerrato C. & Konvicka M. 2014a: Overwintering of the boreal butterfly Colias palaeno in central Europe. - CryoLetters 35: 247-254.
  63. Vrba P., Nedved O. & Konvicka M. 2014b: Contrasting supercooling ability in lowland and mountain European Colias butterflies. - J. Entomol. Sci. 49: 63-69. Go to original source...
  64. Vrba P., Nedved O., Zahradnickova H. & Konvicka M. 2017: Temporal plasticity in cold hardiness and cryoprotectant contents in northern versus temperate Colias butterflies (Lepidoptera: Pieridae). - CryoLetters 38: 330-338.
  65. Williams C.M., Nicolai A., Ferguson L.V., Bernards M.A., Hellmann J.J. & Sinclair B.J. 2014: Cold hardiness and deacclimation of overwintering Papilio zelicaon pupae. - Compar. Biochem. Physiol. (A) 178: 51-58. Go to original source...
  66. Zakharova E.Y. & Tatarinov A.G. 2016: Chrono-geographical approach to analysis of variability of bicyclic Erebia ligea (L.) (Lepidoptera: Satyridae) species in the Urals. - Contemp. Probl. Ecol. 9: 272-281. Go to original source...