Extended Data Fig. 5: EcK2 phosphorylates 20E in blood-fed females and prevents oviposition in virgins.

(a) Expression of EcK2 is high in the lower reproductive tract (LRT) of virgin females and is suppressed after mating. Blood feeding does not affect EcK2 expression at different time points post blood meal (pbm) (0 h: P = 0.024, 3 h: P = 0.031, 24 h: P = 0.029, means ± s.e.m., unpaired t-test, one-sided). (b) 20E22P is detected in the LRT of virgin females after a blood meal (means ± s.e.m.). Quantification was performed using HPLC-MS/MS. Dotted lines connect mean values of the same ecdysteroid at different time points. (c) EcK2 silencing prior to blood feeding significantly increases the ratio of 20E vs 20E22P at 26 pbm in the LRT of virgin females (P = 0.047, means ± s.e.m., unpaired t-test, one-sided). (d) Mating receptivity (assessed by the presence of a mating plug) in blood-fed virgin females is not affected by EcK2 silencing (P = 0.50, Fisher’s exact test, two-sided, data pooled from two replicates). (e) Mating receptivity is not affected in virgins by EcK2 silencing (P = 0.98, Cochran–Mantel–Haenszel test, two-sided). (f) Oviposition is highly induced in virgins when EcK2 is silenced prior to blood feeding (top) (P = 3.30e-06, Cochran–Mantel–Haenszel test, two-sided, data were from four replicates), but not when this kinase is silenced post blood feeding (bottom) (P = 0.94, Cochran–Mantel–Haenszel test, two-sided). (g) EcK2 expression is not suppressed by injection of 20E or 3D20E relative to 10% EtOH-injected controls. Expression values were normalized to the housekeeping gene RpL19 in respective samples before calculating fold changes (means ± s.e.m., unpaired t-tests, two-sided, 20E: P = 0.46, 3D20E: P = 0.85). In all panels: n indicates the number of biologically independent mosquito samples, ns = not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001.Data were from three biological replicates in all panels unless noted otherwise

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