Comparative Cold Tolerance Mechanisms in Acorn Pests from Iran and Switzerland: Thermal Hysteresis and Hemolymph Elemental Dynamics

Articles in Press

Document Type : Original Article

Authors
Maryam Atapour 1
Ahmad Bahmani 2
1 Department of Plant Production and Protection, Institute of Agriculture, Iranian Research Organization for Science and Technology (IROST), P. O. Box: 33535111, Tehran, Islamic Republic of Iran.
2 Department of Advanced Materials and New Technologies, Iranian Research Organization for Science and Technology (IROST), P. O. Box: 33535111, Tehran, Islamic Republic of Iran.
Abstract
Oak forests in Iran’s Zagros region are undergoing severe decline due to environmental stress and increasing pest outbreaks. This study examined the overwintering survival mechanisms of two major acorn pests, the beech moth Cydia fagiglandana (Zeller) and an acorn weevil (Curculio sp.), by comparing populations from stressed Zagros forests with those from stable oak forests in Switzerland. We evaluated two key biomarkers of cold tolerance during the coldest months: thermal hysteresis, measured using differential scanning calorimetry (DSC), and hemolymph trace element concentrations (Fe, Cu, Zn, Mg), quantified by ICP-OES. Additionally, EDS–SEM analysis was conducted to assess the spatial distribution of selected internal elements semi-quantitatively. A distinct thermal hysteresis of approximately 4 °C between melting and freezing points was observed exclusively in Zagros Curculio larvae. In most samples, metal ion concentrations increased in January, with Zn showing a nearly fourfold increase in the Zagros acorn weevil (170–585 ppb). Elemental mapping by EDS–SEM further showed higher Cu and Zn contents and a shift from uniform to aggregated elemental distribution in overwintering larvae. This study provides new insights into the overwintering physiology of two major acorn-feeding insects under contrasting climatic conditions. These findings contribute to predicting pest responses to environmental change and support future conservation and management of oak forest regeneration.
Keywords
Acorn pests
Cydia fagiglandana
Curculio sp
DSC
Hemolymph elements
ICP-OES
Overwintering
Thermal hysteresis
Subjects
1.   Arsalani, M., Grießinger, J. and Bräuning, A. 2023. Tree-ring-based seasonal temperature reconstructions and ecological implications of recent warming on oak forest health in the Zagros Mountains, Iran. Int. J. Biometeorol., 67(2), 317–331.https://doi.org/10.1007/s00484-022-02380-5
2.   Beauchemin, D. 2010. Inductively coupled plasma mass spectrometry. Anal. Chem., 82(12), 4786–4810.https://doi.org/10.1021/ac100629x
3.   Block, W. 1990. Cold tolerance of insects and other arthropods. Philos. Trans. R. Soc. Lond. B Biol. Sci., 326(1237), 613–633.https://doi.org/10.1098/rstb.1990.0035
4.   Bowler, K. and Terblanche, J. S. 2008. Insect thermal tolerance and climate change. J. Insect Physiol., 54, 125–136.https://doi.org/10.1016/j.jinsphys.2007.07.009
5.   Butolo, N. P., Azevedo, P., Alencar, L. D., Domingues, C. E. C., Miotelo, L., Malaspina, O. and Nocelli, R. C. F. 2020. A high quality method for hemolymph collection from honeybee larvae. PLoS ONE, 15(6), e0234637.https://doi.org/10.1371/journal.pone.0234637
6.   Chapman, R. F. 2013. The Insects: Structure and Function. 5th Ed., Cambridge University Press, Cambridge.
7.   Denlinger, D. L. 2002. Regulation of diapause. Annu. Rev. Entomol., 47, 93–122. https://doi.org/10.1146/annurev.ento.47.091201.145137
8.   Dow, J. A. T. 2017. The multifunctional Drosophila melanogaster Malpighian tubule. J. Insect Physiol., 106, 44–54.https://doi.org/10.1016/j.jinsphys.2017.05.003
9.   Duman, J. G. 1979. Thermal hysteresis factors in overwintering insects. J. Insect Physiol., 25(10), 805–810.https://doi.org/10.1016/0022-1910(79)90083-0
10.  Duman, J. G. 2001. Antifreeze and ice nucleator proteins in terrestrial arthropods. Annu. Rev. Physiol., 63, 327–357.https://doi.org/10.1146/annurev.physiol.63.1.327
11.  Duman, J. G. 2015. Animal ice-binding (antifreeze) proteins and glycolipids: An overview with emphasis on physiological function. J. Exp. Biol., 218(12), 1846–1855. https://doi.org/10.1242/jeb.118503
12.  Duman, J. G. and Newton, S. S. 2020. Antifreeze and ice-recrystallization-inhibition proteins in insects. Curr. Opin. Insect Sci., 41, 1–8.https://doi.org/10.1016/j.cois.2020.04.003
13.  Felton, G. W. and Summers, C. B. 1995. Antioxidant systems in insects. Arch. Insect Biochem. Physiol., 29, 187–197.https://doi.org/10.1002/arch.940290207
14.  Fialkowski, W., et al. 2013. Elemental microanalysis of insect cuticle using SEM-EDS. Micron, 45, 186–192.https://doi.org/10.1016/j.micron.2013.01.001
15.  Gabbott, P. 2008. Principles and Applications of Thermal Analysis. Royal Society of Chemistry, Cambridge.
16.  Gao, Y., Li, S., Zhang, Y. and Wang, X. 2016. Determination of trace metals in biological tissues using ICP-OES following nitric acid digestion. Biol. Trace Elem. Res., 172(2), 380–388.https://doi.org/10.1007/s12011-015-0579-3
17.  Grewal, P. S. 2012. Magnesium-dependent metabolic regulation in insects. Insect Biochem. Mol. Biol., 42, 1–10.https://doi.org/10.1016/j.ibmb.2011.10.002
18.  Hahn, D. A. and Denlinger, D. L. 2011. Energetics of insect diapause. Annu. Rev. Entomol., 56, 103–121.https://doi.org/10.1146/annurev-ento-112408-085436
19.  Hansen, M., Hyldig, G. and Larsen, E. H. 2013. Quantification of trace metals in biological matrices using ICP-OES. Food Chem., 141(3), 1976–1981. https://doi.org/10.1016/j.foodchem.2013.05.050
20.  Hou, X. and Jones, B. T. 2000. Inductively coupled plasma optical emission spectrometry. In: Meyers, R. A. (Ed.), Encyclopedia of Analytical Chemistry, pp. 9468–9485. Wiley, Chichester.
https://doi.org/10.1002/9780470027318.a0918
21.  IPCC. 2023. Climate Change 2023: Synthesis Report. Intergovernmental Panel on Climate Change, Geneva, Switzerland.
22.  Kokot, S. and Matysiak, J. 2008. Determination of trace elements in biological samples by ICP-OES after acid digestion. J. Anal. At. Spectrom., 23(8), 1101–1106.
https://doi.org/10.1039/b803501a
23.  Košťál, V. 2006. Eco-physiological phases of insect diapause. J. Insect Physiol., 52(2), 113–127.https://doi.org/10.1016/j.jinsphys.2005.09.008
24.  Košťál, V., Vambera, J. and Bastl, J. 2004. On the nature of pre-freeze mortality in insects: Water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus. J. Exp. Biol., 207(9), 1509–1521.https://doi.org/10.1242/jeb.00923
25.  Košťál, V., Zahradníčková, H. and Šimek, P. 2011. Hyperactive antifreeze proteins of a freeze-avoiding insect enhance supercooling capacity and prevent ice recrystallization. Proc. Natl. Acad. Sci. U.S.A., 108(10), 4042–4047.https://doi.org/10.1073/pnas.1017305108
26.  Lee, R. E. Jr. 2010. A primer on insect cold-tolerance. In: Denlinger, D. L. and Lee, R. E. (Eds.), Low Temperature Biology of Insects, pp. 3–34. Cambridge University Press, Cambridge.https://doi.org/10.1017/CBO9780511675997.003
27.  Li, J. M., Su, Y. L., Gao, X. L., He, J., Liu, S. S. and Wang, X. W. 2011. Molecular characterization and oxidative stress response of an intracellular Cu/Zn superoxide dismutase (CuZnSOD) of the whitefly, Bemisia tabaci. Arch. Insect Biochem. Physiol., 77(3), 118–133.
https://doi.org/10.1002/arch.20428
28.  MacMillan, H. A. and Sinclair, B. J. 2011. Mechanisms underlying insect low-temperature tolerance. J. Insect Physiol., 57 (1), 12-20. https://doi.org/10.1016/j.jinsphys.2010.10.004.
29.  MacMillan, H. A., Baatrup, E. and Overgaard, J. 2015. Concurrent effects of cold and hyperkalemia cause insect chilling injury. Proc. R. Soc. B, 282(1817), 20151483.
https://doi.org/10.1098/rspb.2015.1483
30.  Mao, Y., Liu, X., Wang, Y. and Chen, L. 2011. Measurement of thermal hysteresis activity of antifreeze proteins using differential scanning calorimetry. Cryobiology, 63(3), 236–242.
https://doi.org/10.1016/j.cryobiol.2011.06.005
31.  Missirlis, F. and Oliveira, P. L. 2022. Metal metabolism and oxidative stress in insects. Curr. Opin. Insect Sci., 49, 1–7.https://doi.org/10.1016/j.cois.2021.10.003
32.  Montaser, A. (Ed.). 1998. Inductively Coupled Plasma Mass Spectrometry. Wiley-VCH, New York.
33.  Nation, J. L. 2002. Insect Physiology and Biochemistry. CRC Press, Boca Raton.
34.  Overgaard, J. and MacMillan, H. A. 2017. The integrative physiology of insect chill tolerance. Annu. Rev. Physiol., 79, 187–208.https://doi.org/10.1146/annurev-physiol-022516-034142
35.  Polilov, A. A., Makarova, A. A., Pang, S., Xu, C. S. and Hess, H. 2021. Protocol for preparation of heterogeneous biological samples for 3D electron microscopy. Sci. Rep., 11, 2993.
https://doi.org/10.1038/s41598-021-83936-0
36.  Pröfrock, D. and Prange, A. 2012. ICP-MS for quantitative analysis in environmental and life sciences. Appl. Spectrosc., 66(8), 843–868.https://doi.org/10.1366/12-06681
37.  Rozsypal, J. and Košťál, V. 2018. Supercooling and freezing as alternative strategies. J. Insect Physiol., 111, 53–62.https://doi.org/10.1016/j.jinsphys.2018.10.006
38.  Rozsypal, J., Košťál, V., Zahradníčková, H. and Šimek, P. 2013. Overwintering strategy and cold tolerance in Cydia pomonella. PLoS ONE, 8(4), e61745.
https://doi.org/10.1371/journal.pone.0061745
39.  Sabo, R., Staroň, M., Sabová, L., Jančo, I., Tomka, M. and Árvay, J. 2024. Toxic and essential elements in honeybee venom from Slovakia: Potential health risk to humans. Heliyon, 10(20), e39282.https://doi.org/10.1016/j.heliyon.2024.e39282
40.  Sadeghian, B., Dordaei, A. A. and Nikdel, M. 2007. Damage assessment of acorn pests in Arasbaran forests. Iran. J. For. Range Prot. Res., 4(2), 113–118.
41.  Steinbrecht, R. A. 1993. Freeze-substitution for insect studies. Microsc. Res. Tech., 24(6), 488–504.https://doi.org/10.1002/jemt.1070240605
42.  Storey, K. B. and Storey, J. M. 2012. Insect cold hardiness. Can. J. Zool., 90(4), 456–475.
https://doi.org/10.1139/z2012-011
43.  Thomas, R. 2013. Practical Guide to ICP-MS: A Tutorial for Beginners. 3rd Ed., CRC Press, Boca Raton.
44.  Vukašinović, E. L., Popović, Ž. D., Ninkov, J., Čelić, T. V., Uzelac, I., Kojić, D. and Purać, J. 2024. Management of inorganic elements by overwintering physiology of cold hardy larvae of European corn borer (Ostrinia nubilalis, Hbn.). J. Comp. Physiol. B, 194(2), 145–154.
https://doi.org/10.1007/s00360-024-01537-5
45.  Xikeranmu, Z., Abdunasir, M., Ma, J., Tusong, K. and Liu, X. 2019. Characterization of two copper/zinc superoxide dismutases (Cu/Zn-SODs) from the desert beetle Microdera punctipennis and their activities in protecting Escherichia coli cells against cold. Cryobiology, 87, 15–27.https://doi.org/10.1016/j.cryobiol.2019.03.006
46.  Zachariassen, K. E., Kristiansen, E. and Pedersen, S. A. 2004. Inorganic ion homeostasis in cold-acclimated insects. J. Exp. Biol., 207(23), 4215–4223.https://doi.org/10.1242/jeb.01289
47.  Zargaran, M. Z., Najafi, M. R., Mirab-Balou, M. and Tavakoli, M. 2018. Identification of oak seed insects in Ilam forests. Iran. J. For., 10(1), 55–65.
Journal of Agricultural Science and Technology

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Available Online from 18 August 2026