Fig. 5

miR-31-5p-acox1 promotes beetle’s asc-C9 accumulation and cold acclimation. a ACOX1 is the rate-limiting enzyme in peroxisomal β-oxidation cycles which function in ascaroside side chain shortening in C. elegans. Modification from Zhang et al. [32]. b The amount of asc-C9 after RNAi by injecting dsAcox1 and dsGFP (Control) in larval beetles (n = 8). ***, P < 0.001; Student’s t test. c Comparison of the concentration of asc-C9 secreted by the wild type (WT, N2 strain), acox1 loss of function mutant (MT, VC1785 strain), and rescue type (RT, acox1 (ok2257):: Cel-p:: Mal-acox1, line 1 and line 2) of C. elegans using HPLC-MS/MS. The rescue types were constructed from the acox1 mutant C. elegans with heterologous expression of Mal-acox1 through transforming the pPD95_77 plasmid cloned with the promoter of Cel-acox1 (Cel-p) and CDS of Mal-acox1 into C. elegans mutant. The bars are means ± s.e.m. and differ significantly from each other with different letters (n = 8, one-way ANOVA with Tukey’s HSD multiple comparison, P < 0.05). d, e The amount of asc-C9 after injecting the agomir-31-5p (Ago-31-5p) (d) and antagomir-31-5p (Anta-31-5p) (e) in larval beetles (n = 8). **, P < 0.01; Student’s t test. f The supercooling points (SCPs) of larval beetles after RNAi by injecting dsAcox1 (n = 26) and dsGFP (Control) (n = 20). **, P < 0.01; Student’s t test. g Probit regression between mortality rate and temperature after injecting dsGFP and dsAcox1 (n = 10 for each treatment). h The supercooling points (SCPs) of wild type larval beetles (25 °C) (n = 17), antagomir-31-5p injected beetles (n = 11), and the rescue beetles by injecting dsAcox1 (n = 14) or low-temperature acclimation (n = 14). The bars with different letters differ significantly from each other (one-way ANOVA with Tukey’s HSD multiple comparison, P < 0.05). i Probit regression between mortality rate and temperature for antagomir-control-injected beetles, antagomir-31-5p (Anta-31-5p)-injected beetles, and the rescue beetles by injecting dsAcox1 (Anta-31-dsacox1) or low-temperature acclimation (Anta-31-AC) (n = 12 for each treatment). Control (25 °C); AC, low-temperature acclimation (4 °C). The last larval beetles used in b were collected at 15 Oct. 2017, d, f at 5 Oct. 2017, e, h at 6 Nov. 2017, and g, i at 20–25 Oct. 2019