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.