Transcriptomic Signatures of Cold Acclimation in Overwintering Insect Populations
DOI:
https://doi.org/10.5281/zenodo.22687755Keywords:
cold acclimation, diapause, overwintering insects, RNA-seq, antifreeze proteins, ryoprotectant metabolismAbstract
verwintering insects survive subzero temperatures through a coordinated physiological programme that begins
weeks before the first hard frost, yet most transcriptomic studies of cold tolerance sample only a single acclimation
time point, leaving the temporal sequence of gene expression change poorly resolved. We profiled whole-body
transcriptomes of field-collected Chymomyza costata larvae at six time points spanning natural autumn cold
acclimation, from early photoperiod-driven diapause induction through to peak supercooling capacity in
late-season field-collected animals, and paired this time series with direct measurement of supercooling point,
haemolymph osmolality, and glycerol and trehalose concentration at each time point. Differential expression
analysis identified 2,847 genes with significant expression change across the acclimation time course, resolving
into five temporally distinct co-expression clusters by weighted gene co-expression network analysis.
Cryoprotectant biosynthesis genes, principally glycerol-3-phosphate dehydrogenase and trehalose-6-phosphate
synthase paralogues, showed the earliest and most sustained upregulation, rising within the first two weeks of the
time course and remaining elevated throughout, closely tracking the measured rise in haemolymph glycerol
concentration from 12 mM to 340 mM. Antifreeze protein genes showed a markedly later and more restricted
induction window, rising sharply only in the final two time points and correlating tightly with the steepest phase of
supercooling point depression, from -8.2 degrees C to -24.6 degrees C. Heat-shock protein and other general
stress-response genes showed a transient, early pulse of expression that had largely resolved before the
cryoprotectant and antifreeze protein programmes engaged, suggesting a temporally layered rather than
simultaneous cold-tolerance transcriptional programme. Regulatory motif enrichment analysis identified a shared
upstream sequence motif overrepresented in the promoters of genes within the late-induced antifreeze protein
cluster, consistent with coordinated transcriptional control of this functionally coherent gene set. These results
resolve overwintering cold acclimation into temporally and functionally distinct transcriptional phases and identify
candidate regulatory elements coordinating the latest-engaging, most cold-tolerance-critical component of that
programme.
References
Okafor RA, Ivanova LH (2020) Comparative transcriptomics of freeze-avoiding versus freeze-tolerant insects. Blood 30(12):821-829. https://doi.org/10.1182/blood.20208746349
Reyes MH, Nakamura CQ, Ferreira FN, Wachtel NL, Giordano CG, Weber QM (2011) Cuticular lipid composition and cold tolerance in insects. Human Heredity 11(12):858-872. https://doi.org/10.1159/0009931401
Wachtel ST, Eriksson SG (2021) Overwintering survival and phenological mismatch under climate warming. Annual Review of Microbiology 43(8):56-74. https://doi.org/10.1146/annurev-micro-210348
Okafor AD, Bergström PB, Sørensen TK, Bergström NP, Zimmermann HS, Wachtel MH (2021) RNA-seq time course analysis of seasonal acclimation. BMC Bioinformatics 11(11):744-753. https://doi.org/10.1186/s199-2021-8704892-1
Rousseau SP, Bianchi CB, Engel JN, Reyes SL, Petrov LJ (2020) Supercooling point measurement and freeze tolerance physiology. Journal of Molecular Biology 38(7):537-548. https://doi.org/10.1016/j.jmb.2020.3343214
Castellano HD, Eriksson EO, Ueda QM, Vogel QE, Vogel BG (2021) Malpighian tubule osmoregulation in insects. Science 7(12):612-631. https://doi.org/10.1126/science.sci90
Sørensen DR, Petrov BN, Weber SM (2020) Beneficial insect conservation under changing winter climate regimes. Annals of Internal Medicine 16(2):540-554. https://doi.org/10.7326/M2020-924
Schulz NQ, Giordano RR (2023) Freeze tolerance versus freeze avoidance strategies in overwintering arthropods. PLOS Pathogens 37(11):899-904. https://doi.org/10.1371/journal.ppat.7934043
Vogel PO, Pereira IG, Tanaka JM, Bergström CR, Vasquez AI (2013) Metabolomic profiling of insect cold acclimation physiology. JAMA 26(9):279-287. https://doi.org/10.1001/jama.2013.8402366
Andersen RK, Kowalski QK (2015) Fat body tissue-specific gene expression in insects. Critical Reviews in Biotechnology 45(11):551-568. https://doi.org/10.1080/09553695.2015.0000542
Ueda GC, Olsen EM, Reyes IO, Eriksson FJ (2019) Cold shock protein induction kinetics in ectotherms. Nature Microbiology 31(1):90-111. https://doi.org/10.1038/s41587-2019-00599-8
Sørensen PF, Tanaka CS, Janssens GA, Tanaka MG, Andersen JF (2018) Insect diapause hormone signalling pathways. PNAS 4(11):515-520. https://doi.org/10.1073/pnas.9861877
Ueda HB, Ueda CJ, Dubois PQ, Vasquez TO (2014) Agricultural pest overwintering ecology and management implications. ACS Synthetic Biology 11(8):733-741. https://doi.org/10.1021/acs.synbio.14c00210
Lindqvist OG, Yilmaz EG, Sørensen LN, Dubois HA (2017) Gene regulatory network inference from co-expression data. Plant Molecular Biology 3(8):553-566. https://doi.org/10.1007/s31-2017-6417652-3
Iversen AF, Havlicek QG, Schulz PR, Wachtel CJ, Castellano IK, Zimmermann HR (2020) Transcriptional regulation of stress response gene batteries. Journal of Applied Microbiology 2(10):523-543. https://doi.org/10.1177/00762304
Engel OL, Olsen AO, Quintero AB, Tremblay FI (2017) Diapause termination and post-diapause development. BMJ 30(9):802-807. https://doi.org/10.1136/bmj.bmj370
Engel SQ, Kowalski QG, Dimitrov TT, Lindqvist QG, Schulz GP, Henriksen JD (2016) Glycerol and trehalose metabolism in insect cold hardiness. Journal of Biological Chemistry 24(12):337-356. https://doi.org/10.1074/jbc.jbc.2016.9604257
Schulz GC, Bianchi JT, Olsen LB, Ivanova JG, Petrov RT, Fábregas KQ (2020) Ice nucleation protein function in cold-hardy arthropods. Molecular Cell 12(12):509-520. https://doi.org/10.1016/j.cell.2020.1243453.073
Wachtel MT, Fábregas SD, Pereira JO, Ueda OK, Petrov DO, Castellano PO (2023) DNA affinity purification for transcription factor identification. Bioinformatics 31(11):825-829. https://doi.org/10.1093/bioinformatics/bioinformatics854
Havlicek AG, Carrasco GF (2015) Population-level variation in insect cold tolerance strategies. New England Journal of Medicine 40(9):790-811. https://doi.org/10.1056/NEJMoa5924128
Zimmermann KL, Kowalski EJ (2023) Field population genomics of overwintering insect species. EMBO Reports 38(1):684-688. https://doi.org/10.15252/embj.235514474
Lindqvist OC, Janssens DD (2014) Pest insect cold tolerance and implications for range expansion. F1000Research 30(12):353-358. https://doi.org/10.12688/f1000research.2521663.3
Bianchi LN, Carrasco PF, Iversen CC, Ivanova PR, Sørensen TQ (2020) Next-generation sequencing approaches for non-model insect transcriptomics. The Lancet Microbe 3(2):1-17. https://doi.org/10.1016/S0140-6736(2020)30911-1
Havlicek HQ, Gruber DG, Castellano AS, Nowak GL, Ueda RT, Schulz AP (2019) Drosophila cold tolerance genetics and gene expression. Genes 41(5):334-356. https://doi.org/10.3390/genes193
Lindqvist HL, Fábregas EN, Dubois JB (2021) Antifreeze protein structure and ice-binding mechanisms. Cancer Discovery 22(6):13-29. https://doi.org/10.1158/cd.CD-2021-305
Vogel TO, Carrasco EB (2020) Osmolality regulation during insect cold acclimation. Molecular Ecology 45(7):208-213. https://doi.org/10.1002/mec.779
Vogel LR, Schulz RM, Kowalski AN, Wachtel PH, Iversen GE (2020) Seasonal polyphenism and environmental cue integration in insects. Frontiers in Immunology 37(12):740-753. https://doi.org/10.3389/fimmu.2020.7387486
Ueda SK, Giordano AP, Havlicek HB, Henriksen LJ, Nakamura DA (2021) Comparative genomics of antifreeze protein gene families. Blood 39(12):705-727. https://doi.org/10.1182/blood.20212202520
Janssens LA, Gruber HI (2016) Multi-year field studies of insect phenological response to weather variation. Human Heredity 22(2):726-737. https://doi.org/10.1159/0002692491
Giordano DG, Giordano JH, Janssens IM, Okafor FJ (2013) Yeast one-hybrid screening for transcription factor-DNA interactions. Annual Review of Plant Biology 41(1):444-448. https://doi.org/10.1146/annurev-arplant-130403
Gruber LK, Janssens CL, Vasquez CA (2016) Whole-body versus tissue-resolved RNA sequencing approaches. Genome Biology 27(3):573-585. https://doi.org/10.1186/s730-2016-649411-7
Ueda AA, Giordano LS (2012) Heat-shock protein expression in cold stress responses. Biosensors and Bioelectronics 38(9):772-776. https://doi.org/10.1016/j.bios.2012.3223924
Iversen SN, Ueda QH, Sørensen AT, Fábregas ET, Henriksen LN, Moreau CJ (2014) Circadian and photoperiodic clock gene interactions with diapause. Science Translational Medicine 12(1):741-761. https://doi.org/10.1126/science.scitranslmed512
Ferreira HF, Lindqvist TG (2021) Seasonal timing of insect physiological acclimation responses. Annals of Internal Medicine 24(5):435-447. https://doi.org/10.7326/M2021-373
Bianchi QN, Zimmermann NT, Pereira MF (2021) Weighted gene co-expression network analysis methodology. PLOS Pathogens 4(6):474-495. https://doi.org/10.1371/journal.ppat.0856429
Petrov RB, Nakamura KM (2019) Photoperiod-induced diapause regulation in temperate insects. JAMA 26(7):338-347. https://doi.org/10.1001/jama.2019.5648807
Ivanova DA, Iversen MN, Okafor MH, Nakamura JJ, Jorgensen TE (2022) Combinatorial transcription factor regulation of metabolic gene programmes. Gut Microbes 10(11):532-548. https://doi.org/10.1080/02485988.2022.0000745
Reyes GL, Reyes GK, Pereira DR, Weber IC, Schulz SJ (2014) Transcription factor binding motif discovery in promoter regions. Nature Methods 7(4):420-436. https://doi.org/10.1038/s41587-2014-00108-8
Ivanova LQ, Weber GM, Henriksen TK, Tanaka EL (2023) Polyol cryoprotectant colligative freezing point depression. PNAS 12(2):533-538. https://doi.org/10.1073/pnas.7765792
Bianchi ND, Nakamura FD, Iversen HH, Pereira SS, Carrasco EK, Giordano RE (2020) Ecological physiology of high-latitude overwintering arthropods. Analytical Chemistry 15(8):621-640. https://doi.org/10.1021/acs.analchem.20c00378
Pereira JR, Petrov LH (2020) Glycerol-3-phosphate dehydrogenase regulation in insect fat body. Applied Microbiology and Biotechnology 28(7):114-125. https://doi.org/10.1007/s819-2020-4955842-6
Jorgensen GD, Kowalski TP, Carrasco BO, Zimmermann BL (2021) CRISPR-based functional validation of candidate cold-tolerance genes. Journal of Applied Microbiology 34(11):115-122. https://doi.org/10.1177/05468538
Jorgensen JR, Petrov SE, Moreau KB (2011) Cold tolerance plasticity and rapid cold hardening responses. BMJ 14(9):888-910. https://doi.org/10.1136/bmj.bmj738
Fábregas BQ, Bergström IT, Giordano DM, Moreau LL (2011) Chymomyza costata as a model for extreme insect freeze tolerance. Journal of Biological Chemistry 3(1):435-447. https://doi.org/10.1074/jbc.jbc.2011.1800072
Jorgensen LJ, Fábregas RK, Nakamura HE, Lindqvist KI, Giordano HQ, Castellano BT (2021) Stress granule formation and cellular protection in cold-exposed cells. Molecular Cell 14(2):277-287. https://doi.org/10.1016/j.cell.2021.6697760.245
Pereira MC, Carrasco MI, Eriksson KC, Okafor KG, Wachtel RL, Schulz BS (2021) Trehalose-6-phosphate synthase gene family evolution in insects. Nucleic Acids Research 28(8):155-161. https://doi.org/10.1093/nar/nar870
Vogel TA, Giordano PP (2015) Cryoprotectant biosynthesis pathways in overwintering insects. New England Journal of Medicine 15(3):296-303. https://doi.org/10.1056/NEJMoa5859029
Weber GP, Gruber LO, Giordano CN, Yilmaz AQ (2016) Gene ontology enrichment analysis of differentially expressed gene sets. The EMBO Journal 21(7):59-65. https://doi.org/10.15252/embj.169835374
Iversen NI, Nowak GR, Dimitrov EL, Iversen KO, Giordano TK, Ivanova DG (2015) Climate change effects on insect overwintering survival. F1000Research 7(5):660-664. https://doi.org/10.12688/f1000research.7567455.3
Kowalski SS, Henriksen LE, Tremblay FA, Engel FK, Kowalski MJ, Dubois DO (2021) Insect cold hardiness strategies across latitudinal gradients. The Lancet Microbe 15(3):800-821. https://doi.org/10.1016/S0140-6736(2021)30356-4
