Fig. 2

miRNAs involved in the beetle’s cold acclimation. a The procedure of screening for differentially expressed (DE) miRNAs as candidate regulators underlying cold acclimation. b Hierarchical clustering of the expression of the DE miRNAs for AC and control samples. The cluster in red represented the upregulated genes after AC. c The validation of relative expression of upregulated miRNAs after cold acclimation by qPCR (n = 6). miR-31-5p was selected as a candidate miRNA for further functional studies because it exhibits the greatest extent of upregulated expression as well as the highest abundance in larval beetles among the eight upregulated miRNAs. Two-tailed Student’s t test was used to test significant differences, *P < 0.05, **P < 0.01, ***P < 0.001. d Effect of antagomir-control (Anta-control, n = 18) and antagomir-31-5p (Anta-31-5p, n = 18) on supercooling points (SCPs) of larval beetles. Two-tailed Student’s t test was used to test significant differences, ***P < 0.001. e Effect of antagomir-control (Anta-control) and antagomir-31-5p (Anta-31-5p) on mortality of larval beetles (n = 9 for each treatment). Lethal temperature causing 50% mortality (LT₅₀) was determined by probit regression. f Effect of agomir-control (Ago-control, n = 17) and agomir-31-5p (Ago-31-5p, n = 20) on SCPs of larval beetles. Two-tailed Student’s t test was used to test significant differences, *P < 0.05. g Effect of agomir-control (Ago-control) and agomir-31-5p (Ago-31-5p) mortality of larval beetles (n = 10 for each treatment). Lethal temperature causing 50% mortality (LT₅₀) was determined by probit regression. AC, low-temperature acclimation (4 °C); Control (25 °C). The last larval beetles used in miRNA deep sequencing (a, b) were collected at 5 Oct. 2016, qPCR validation (c) and agomir injection (f) at 10 Oct. 2017, antagomir injection (d) at 15 Oct. 2017, and mortality experiments (e, g) at 20–25 Oct. 2019