Molecular characteristic of CcBurs-α and CcBurs-β in C. chinensis.

A: Multiple alignments of the amino acid sequences of CcBurs-α with homologs from four other insect species. Black represents 100% identity, red represents 75% identity, and blue represents <75% identity. CcBurs-α (C. chinensis, OR488624), DcBurs-α (Diaphorina citri, XP_008468249.2), ApBurs-α (Acyrthosiphon pisum, XP_001946341.1), MpBurs-α (Myzus persicae, XP_022171710.1), HvBurs-α (Homalodisca vitripennis, XP_046670477.1). The corresponding GenBank accession numbers are as follows. B: Multiple alignments of the amino acid sequences of CcBurs-β with homologs from four other insect species. Black represents 100% identity, red represents 75% identity, and blue represents <75% identity. CcBurs-β (C. chinensis, OR488625), DcBurs-β (D. citri, AWT50591.1), HvBurs-β (H. vitripennis, XP_046671521.1), NvBurs-β (Nezara viridula, AZC86173.1), LsBurs-β (Laodelphax striatellus, AXF48186.1). The corresponding GenBank accession numbers are as follows. C: Predicted protein tertiary structures of CcBurs-α and CcBurs-β. D: Western blot analysis of Bursicon proteins using anti-His-Tag antibody with non-reduced and reduced SDS-PAGE. The left numbers indicate the positions of pre-stained protein markers. Lanes of α, β, and α+β represent separate expression of CcBurs-α, CcBurs-β, or co-expressed of α+β. Monomers were not present under non-reduced conditions. E-F: Relative mRNA expression of CcBurs-α and CcBurs-β after 25 °C or 10 °C treatments at 3, 6, and 10 d (n=3). G-H: Effect of temperature receptor CcTRPM knockdown on the mRNA expression of CcBurs-α and CcBurs-β at 3, 6, and 10 d under 10°C condition (n=3). Data in 1E-1H are shown as the mean ± SE with three independent biological replications, with at least 50 nymphs for each biological replication. Statistically significant differences were determined using pair-wise Student’s t-test in SPSS 26.0 software, and significance levels were denoted by *** (p < 0.001).

Neuropeptide Bursicon was essential for the transition from summer-form to winter-form in C. chinensis.

A-B: RNAi efficiency of CcBurs-α and CcBurs-β after dsRNA treatment at 3, 6, and 10 d by qRT-PCR under 10 °C condition (n=3). C-I: Effect of RNAi-mediated knockdown of CcBurs-α and CcBurs-β on the absorbance of total cuticle pigment, relative cuticle chitin content, cuticle thickness of the thorax, transition percent, and phenotype changes of 1st instar nymphs compared to dsEGFP treatments (n=9). Data in 2A and 2B are shown as the mean ± SE with three independent biological replications, with at least 50 nymphs for each replication. Data in 2C, 2E, and 2G are presented as mean ± SE with three biological replications, with three technical replications for each biological replication. Data in 2H are presented as mean ± SE with nine biological replications. Statistically significant differences were determined using pair-wise Student’s t-test, and significance levels were denoted by *** (p < 0.001). Different letters above the bars indicate statistically significant differences (p < 0.05), as determined by ANOVA followed by a Turkey’s HSD multiple comparison test in SPSS 26.0 software.

CcBurs-R was identified as the Bursicon receptor in C. chinensis.

A: Multiple alignments of the amino acid sequences of CcBurs-R transmembrane domain with homologs from four other insect species. The transmembrane domain from TM1 to TM6 is indicated by blue horizontal lines. CcBurs-R (C. chinensis, OR488626), DcBurs-R (D. citri, KAI5703609.1), MpBurs-R (M. persicae, XP_022172830.1), AgBurs-R (Aphis gossypii, XP_027844917.2), RmBurs-R (Rhopalosiphum maidis, XP_026817427.1). The corresponding GenBank accession number as follows. B: Phylogenetic tree analysis of CcBurs-R with its homologs in six other insect species. BtBurs-R (Bemisia tabaci, XP_018898471.1), NlBurs-R (N. lugens, XP_022198758.2). C: Predicted protein tertiary structure of CcBurs-R and its binding with CcBurs-α and CcBurs-β. D-E: Effect of CcBurs-α and CcBurs-β knockdown on the mRNA expression of CcBurs-R at 3, 6, and 10 d, respectively (n=3). F: CcBurs-α+β heterodimer protein could rescue the CcBurs-R expression after knockdown of CcBurs-α and CcBurs-β together. G: Relative mRNA expression of CcBurs-R after 25°C or 10°C treatments at 3, 6, and 10 d (n=3). H: Effect of temperature receptor CcTRPM knockdown on the mRNA expression of CcBurs-R at 3, 6, and 10 d (n=3). Data in 3D-3H are shown as the mean ± SE with three independent biological replications, with at least 50 nymphs for each replication. Statistically significant differences were determined using pair-wise Student’s t-test in SPSS 26.0 software, and significance levels were denoted by *** (p < 0.001). Different letters above the bars indicated statistically significant differences (p < 0.05), as determined by ANOVA followed by a Turkey’s HSD multiple comparison test in SPSS 26.0 software.

CcBurs-R directly mediated the transition from summer-form to winter-form in C. chinensis.

A: RNAi efficiency of CcBurs-R after dsRNA treatment at 3, 6, and 10 d by qRT-PCR under 10 °C condition (n=3). B-H: Effect of RNAi-mediated knockdown of CcBurs-R on the absorbance of total cuticle pigment, relative cuticle chitin content, cuticle thickness of the thorax, transition percent, and phenotype changes of 1st instar nymphs compared to dsEGFP treatments under 10 °C condition (n=9). I-J: Relative mRNA expression of CcTre1 and CcCHS1 after knockdown of CcBurs-α, CcBurs-β, and CcBurs-R at 10 d, separately (n=3). Data in 4A, 4I, and 4J are shown as the mean ± SE with three independent biological replications, with at least 50 nymphs for each replication. Data in 4B, 4C, and 4E are presented as mean ± SE with three biological replications, with three technical replications for each biological replication. Data in 4G are presented as mean ± SE with nine biological replications. Statistically significant differences were determined using pair-wise Student’s t-test, and significance levels were denoted by ** (p < 0.01) and *** (p < 0.001). Different letters above the bars indicate statistically significant differences (p < 0.05), as determined by ANOVA followed by a Turkey’s HSD multiple comparison test in SPSS 26.0 software.

miR-6012 directly targeted CcBurs-R to inhibit its expression.

A: Predicted binding sites of four miRNAs in the 3’UTR of CcBurs-R. B: In vitro confirmation of the target relationship between miR-6012 and CcBurs-R using dual luciferase reporter assays. C: In vivo validation of miR-6012 directly targeting CcBurs-R using RNA-binding protein immunoprecipitation (RIP) assay. D: Co-localization of miR-6012 and CcBurs-R in different development stages of C. chinensis using FISH. E: Effect of different temperature treatments on the expression of miR-6012 by qRT-PCR. F: Effect of miR-6012 agomir and antagomir treatments on the mRNA level of CcBurs-R at 6 d under 10 °C conditions. Data in 5B and 5F are presented as the mean ± SE with nine biological replicates. Results of 5C and 5E are indicated as the mean ± SE with six or three biological replicates. Statistically significant differences were determined using pair-wise Student’s t-test, and significance levels were denoted by *** (p < 0.001). Different letters above the bars represent statistically significant differences (p < 0.05), as determined by ANOVA followed by a Turkey’s HSD multiple comparison test in SPSS 26.0 software.

miR-6012 targeted CcBurs-R to mediate the seasonal polyphenism in C. chinensis.

A: Expression of miR-6012 after agomir-6012 treatment at 3, 6, and 10 d by qRT-PCR under 10 °C condition (n=3). B-H: Effect of agomir-6012 treatment on the absorbance of total cuticle pigment, relative cuticle chitin content, cuticle thickness of the thorax, transition percent, and phenotype changes of 1st instar nymphs compared to agomir-NC treatments under 10 °C condition (n=9). I: Relative mRNA expression of CcTre1 and CcCHS1 after agomir-6012 treatment at 6 d, separately (n=3). Data in 6A and 6I are shown as the mean ± SE with three independent biological replications, with at least 50 nymphs for each replication. Data in 6C and 6E are presented as mean ± SE with three biological replications of three technical replications for each biological replication. Data in 6B and 6G are presented as mean ± SE with nine biological replications. Statistically significant differences were determined using pair-wise Student’s t-test, and significance levels were denoted by ** (p < 0.01) and *** (p < 0.001).

Schematic model of the novel functions of Bursicon signaling in the seasonal polyphenism of C. chinensis in response to low temperature.

Under 10°C condition, low temperature significantly upregulated the expression of Bursicon signaling pathway. CcBurs-α and CcBurs-β then formed a heterodimeric neuropeptide to activate their receptor CcBurs-R, which mediated the transition from summer-form to winter-form in C. chinensis by acting on the chitin biosynthesis pathway. Furthermore, low temperature inhibited the expression of miR-6012, relieving its inhibitory effects on CcBurs-R. miR-6012 directly targeted CcBurs-R, contributing to the novel function of Bursicon signaling in seasonal polyphenism. Finally, the 1st instar nymphs of summer-form developed into 3rd instar nymphs of winter-form in C. chinensis.