Diurnal rhythmicity of aphid feeding behaviors.

a, b The number of honeydew droplets produced by aphids was measured at the end of each 12h day and night cycle for 48h. Average number (±SE) of honeydew droplets produced per aphid on wheat plants (a, n=10) and artificial diet (b, n=10). Significant differences between day and night were tested by the two-tailed Mann-Whitney test (wheat: ***p=0.0006 and diet: *p=0.0411). c, d Aphid feeding activity was monitored every 4h over a 24h period, 0zt (n=25), 4zt (n=21), 8zt (n=26), 12zt (n=23), 16zt (n=29), and 20zt (n=16). The total time (±SE) spent by aphids in phloem salivation (E1) and phloem ingestion (E2) are shown in (c) and (d), respectively. Solid lines, boundaries, and whiskers indicate the median, 25th, and 75th percentiles and 1.5 interquartile ranges, respectively. Points indicate outliers. Different letters above the boxes denote significant differences (Kruskal-Wallis test followed by Dunn’s multiple comparisons adjusted by Bonferroni for p-values, adj. p<0.05). Diurnal rhythmicity of E1 (c) and E2 (d) waveforms over 24h was assessed with Cosinor model. The curve line and shaded area represent the fitted periodic sinusoidal curve and 95% confidence band, respectively. Significance is only established for a diurnal rhythm if the Cosinor p-value is <0.05 (E1: p=0.0119 and E2: p=0.7313). Yellow and black bars at the x-axis mirror the day and night. ZT = Zeitgeber time. Source data and full statistical summary are provided in the Source Data file.

Diurnal rhythmicity of gene expression in R. padi and their roles in aphid feeding.

a Acrophase frequency of diurnally rhythmic transcripts in R. padi. Each bar represents the number of rhythmic sequences with acrophase values in each 24h period. Numbers around the outer sphere indicate zeitgeber time (ZT). b Soft-clustering of diurnally rhythmic transcripts. High, medium, and low membership scores are color-coded with red, yellow, and blue lines. c Top-10 enriched KEGG pathways for diurnally rhythmic transcripts in each cluster. Circle size and color represent enriched gene number in a pathway and Benjamini-Hochberg adjusted p-value, respectively. Gene ratio indicates the percentage of diurnally rhythmic transcripts of a cluster. d, e Expression profile of two diurnally rhythmic osmoregulatory genes, aquaporin 1 (AQP1, d) and gut sucrase 1 (SUC1, e). Solid lines, boundaries, and whiskers of each box indicate median, 25th, and 75th percentiles and 1.5 interquartile ranges. Diurnal rhythmicity of AQP1 (p=0.031) and SUC1 (p=0.013) was determined with Cosinor model. The curve line and shaded area represent the fitted periodic sinusoidal curve and 95% confidence band, respectively. f Kaplan-Meier survival curves and g fecundities of aphids after treatment with dsRNA AQP1 (dsAQP1, n=3), dsRNA SUC1 (dsSUC1, n=3), and dsRNA GFP (dsGFP, n=3, negative control). Significant differences in survivorship and fecundity were tested using a log-rank test with Benjamini-Hochberg adjustment (dsAQP1 vs dsGFP: *p=0.0272 and dsSUC1 vs dsGFP: ***p=0.0003) and a repeated measures ANOVA with Dunn’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), respectively. h Average number (±SE) of honeydew droplets produced per aphid after treatment with dsAQP1 (n=5), dsSUC1 (n=3), and dsGFP (n=5) on wheat. Yellow and black bars represent the average number of droplets produced at the end of each 12:12h day and night for 4 days (Mann-Whitney test, **p<0.01, ****p<0.0001, NS=no significant difference). Source data and full statistical summary are provided in the Source Data file.

Diurnally rhythmic putative salivary effectors in R. padi.

a Heatmap showing temporal gene expression patterns of 42 diurnally rhythmic putative salivary effectors. Each row and column represents the salivary effectors and zeitgeber time (ZT). The hierarchical clustering of gene expression data was generated using one minus Spearman correlation metric based on the average linkage method. Color scale represents the standardized expression values (Z-score) across samples. b Enriched gene ontology (GO) terms associated with diurnally rhythmic putative salivary effectors. Node color corresponds to the clade number in the heatmap (a). Source data and full statistical summary are provided in the Source Data file.

Aphid performance on wheat and artificial diet after silencing diurnally rhythmic salivary effectors.

a Key salivary effector identification for functional analysis. Yellow and black dots represent diurnally rhythmic effectors with acrophases during the day and night. b Expression levels of C002 and E8696 in R. padi heads (n=3) and bodies (n=3, without heads and nymphs) are compared using a two-tailed Welch’s t-test (C002: t=6.532, df=3.218, *p=0.0059 and E8696: t=8.528, df=2.072, **p<0.0121). c, d Expression profiles of C002 (c) and E8696 (d) over a 24h period (n=3 per time point). Solid lines, boundaries, and whiskers of each box indicate the median, 25th, and 75th percentiles, and 1.5 interquartile ranges, respectively. Diurnal rhythmicity of C002 (p=0.008) and E8696 (p=0.001) was determined by Cosinor model. The curve line and shaded area represent the fitted periodic sinusoidal curve and 95% confidence band, respectively. e Kaplan-Meier survival curves and f fecundities of aphids on wheat plants after treatment with dsRNA C002 (dsC002, n=9), dsRNA E8696 (dsE8696, n=9), and dsRNA GFP (dsGFP, n=12, negative control). g Kaplan-Meier survival curves and h fecundities of aphids on diets after treatment with dsC002 (n=9), dsE8696 (n=11), and dsGFP (n=12). Significant differences in survivorship and fecundity were tested using a log-rank test with Benjamini-Hochberg adjustment and a repeated measures ANOVA with Dunn’s multiple comparisons test, respectively (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). i, j The number of honeydew droplets produced per aphid after treatment with dsC002, dsE8696, and dsGFP on wheat plant (i, n=6 per treatment group) and diet (j, dsC002: n=6, dsE8696: n=8, and dsGFP: n=6). Yellow and black bars represent the mean (±SE) number of honeydew droplets produced at the end of each 12h day and night cycle for 4 days (Mann-Whitney test, *p<0.05, **p<0.01, ****p<0.0001). k Aphid feeding activity after treatment with dsE8696 (n=13) and dsGFP (n=15). Solid lines, boundaries, and whiskers indicate the median, 25th, and 75th percentiles and 1.5 interquartile ranges, respectively. Points indicate outliers. Asterisk(s) denote significant differences (Mann-Whitney test, E1: **p=0.0071 and E2: *p=0.0195). Source data and full statistical summary are provided in the Source Data file.

a. Diurnal behavioral activities of R. padi on wheat plant and artificial diet.

Five age-synchronized adult females were placed on a wheat leaf or diet under 12h:12h, light:dark cycles. The number of honeydew drops and nymphs produced by aphids was recorded at the end of each night and day for two days. Each experiment on wheat or diet contains a total of 10 biological replicates. b. Diurnal time-course experiment. Aphid samples were collected every four hours over a 24h day-night cycle (n = 3 per timepoint).