The effects of SBPH infestation on glucose distribution and levels in rice plants

(A, B, C) Glucose levels in aerial tissues of rice plants following SBPH infestation. (A) Rice plants infested with 25 third-instar nymphs/plant for 1, 3, 5 days. (B) Rice plants infested with third-instar nymphs at different density (1–30 insects/plant) for 5 days. (C) Rice plants infested with different developmental stages/sexes (third-instar nymphs, virgin females, gravid females, or males; 25 insects/plant) for 5 days. Uninfested plants served as controls. (D) Glucose levels in root tissues of the rice plants infested with 25 third-instar nymphs/plant for 5 days. (E) Whole-plant glucose levels of the rice plants infested with 25 third-instar nymphs/plant for 5 days. Data are from 4 independent biological replicates and presented as mean ± standard deviation (s.d). (B, C) Significant differences (one-way ANOVA with Tukey’s test; p < 0.05) are indicated by lowercase letters. (A, D, E) Were analyzed by Student’s t-test (*p < 0.05, **p < 0.01, ns = not significant). FW, fresh weight.

Aerial tissues glucose levels induced by SBPH infestation affect the fecundity

(A, B, C) Glucose levels, fecundity, and transcriptional responses of LsVg and LsVgR in SBPH on pre-infested rice plants. Third-instar nymphs were reared on rice plants that had been pre-infested with 25 nymphs for 3 days; control insects were fed on non-infested plants. (A) Glucose levels in nymphs (at 5 days post-infestation) and in females (25 insects/group). (B) Oviposition rate (1♀:1♂ per group). (C) Expression levels of LsVg and LsVgR in females (25 insects/group). (D) Oviposition rate (1♀:1♂ per group) after exogenous glucose injection into nymphs. (E) Expression of LsVg and LsVgR in female that developed from nymphs injected with exogenous glucose (25 insects/group). (F, G) Effects of glucose level on oviposition rate and gene expression in SBPH. Third-instar nymphs were reared on rice plants cultivated in IRRI solution supplemented with 0–2.0% glucose. (F) Oviposition rate (1♀:1♂ per group). (G) LsVg and LsVgR expression in female (25 insects/group). (H) Glucose content in aerial parts of rice plants cultured for 5 days in IRRI solution with 0% (control) or 1.5% glucose (1 plant/group). (I) Glucose levels in nymphs (5 days post-infestation) and adult females reared on 1.5% glucose-supplemented rice; controls fed on plants cultured in standard IRRI solution (25 insects/group). (J) Effect of dietary sugars on SBPH fecundity. Rice roots were irrigated with an IRRI solution containing mannitol, sucrose, or trehalose, each isotonic with 1.5% glucose, and nymphs were reared on these plants. Parallel treatment groups include nymphs from the sucrose– or trehalose-reared groups were injected with the sucrase inhibitor arabinose or the trehalase inhibitor Validamycin A (Val A), respectively, with or without a subsequent glucose injection. After eclosion, adults were paired (1♀:1♂ per group) and continued to be reared on correspondingly treated plants. Fecundity was measured as the total number of eggs laid per female. (K) Glucose levels in nymphs after 5 days of feeding under the treatments described in (J) (25 insects /group). Panel images (A, C, E, G, H, I, K) represent data from four biological replicates, while (B, D, J) and (F) include twelve and thirty replicates, respectively. All data are presented as mean ± s.d. (A-E, H) Were analyzed by Student’s t-test (*p < 0.05, **p < 0.01, ns = not significant). (F, G, I-K) Significant differences (one-way ANOVA with Tukey’s test; p < 0.05) are indicated by lowercase letters. Glu, Glucose.

Glucose levels induced by SBPH infestation influence fecundity via the JH pathway

(A) Third-instar nymphs were fed for 5 days on rice plants that had been pre-infested with 25 nymphs for 3 days; JH III levels were measured in these nymphs and in the resulting adult females after eclosion. Control insects were fed on non-infested plants (25 insects/group). (B) Expression of JH-related genes (JHAMT, Kr-h1, Met, JHE, JHEH) in nymphs from the treatment described in (A) after 5 days of feeding. (C) Relative mRNA expression of JH pathway genes (JHAMT, Kr-h1, Met, JHE, JHEH) in nymphs after 5-day feeding on rice plants cultured in 1.5% glucose-supplemented IRRI solution. Control plants were grown in standard IRRI solution (25 insects/group). (D) Relative expression of JHAMT, Kr-h1, and Met of the third-instar nymphs were injected with corresponding dsRNAs and reared on rice cultured in standard IRRI solution for 5 days; dsEGFP served as control (25 insects/group). (E) Oviposition rate of adults eclosing from RNAi-treated nymphs in (D) (1♀:1♂ per group). (F) Expression of LsVg and LsVgR in females from RNAi-treated nymphs in (D) (25 insects/group). Panel images (A, B, C, D, F) represent data from four biological replicates, while (E) include thirty replicates. All data are presented as mean ± s.d. (A, B, C, D, F) Were analyzed by Student’s t-test (*p < 0.05, **p < 0.01, ns = not significant). (E) Significant differences (one-way ANOVA with Tukey’s test; p < 0.05) are indicated by lowercase letters.

Glucose levels induced by SBPH infestation regulate fecundity through the TOR–JH-Vg pathway

(A, B, C, D) Relative mRNA expression, protein abundance, and phosphorylation levels (Ser2448) of TOR in nymphs and females. (A, B) Third-instar nymphs were maintained for 5 days or (C, D) until adulthood on rice plants grown in 1.5% glucose-supplemented IRRI solution. Control groups were reared on plants in standard IRRI solution (25 insects/group). (E) Relative TOR expression at 5 days post-injection in third-instar nymphs injected with dsTOR and reared on rice in standard IRRI solution; dsEGFP served as control (25 insects/group). (F) Expression of LsVg and LsVgR in females after injection of third-instar nymphs with dsTOR and rearing on rice in standard IRRI solution; dsEGFP was used as control (25 insects/group). (G, H) JH III titer and relative expression of JH-related genes (JHAMT, Kr-h1, Met, JHE, JHEH) in nymphs at 5 days post-injection with dsTOR and reared on rice cultured in IRRI solution supplemented with 1.5% or 0% glucose; dsEGFP-injected nymphs served as control (25 insects/group). (I) A factorial design was employed to assess fecundity. Third-instar nymphs were first injected with dsTOR and, after 24 hours, subjected to one of three treatments: JHA application, acetone (vehicle control), or no further treatment. These groups were then reared on rice plants supplemented with either 0% or 1.5% glucose. The control cohort received dsEGFP injections followed by the same JHA, acetone, or no-treatment regimens, and was reared exclusively on 0% glucose-supplemented rice. After eclosion, adults were paired (1♀:1♂ per group) and continued to be reared on correspondingly treated plants. Fecundity was measured as the total number of eggs laid per female. (J–M) Gene expression, JH III titer, and oviposition rate following rapamycin treatment. Third-instar nymphs were injected with rapamycin or ethanol (vehicle control) and reared on rice in standard IRRI solution. (J) mRNA levels of JHAMT, Kr-h1, Met, JHE, and JHEH in nymphs at 5 days post-injection (25 insects/group). (K) JH III titer in nymphs at 5 days post-injection (25 insects/group). (L) Oviposition rate (1♀:1♂ per group). (M) Expression of LsVg and LsVgR in females (25 insects/group). (N) Expression of LsVg and LsVgR in females after injection at the third-instar stage with dsEGFP, dsJHAMT, or a mixture of dsTOR and dsJHAMT, and reared on rice plants grown in 1.5% glucose-supplemented IRRI solution (25 nymphs/group; n = 4). Control nymphs were injected with dsEGFP and reared on plants in standard IRRI solution. (O) Oviposition rate of the treated insects in (N) (1♀:1♂ per group). Panel images (A, C, E, F, G, H, J, K, M, N) represent data from four biological replicates, while (B, D), (I), and (L, O) include three, twelve, and thirty replicates, respectively. Data are presented as mean ± s.d. (A, C, E, F, H, J-M) Were analyzed by Student’s t-test (*p < 0.05, **p < 0.01, ns = not significant). (G, I, N, O) Significant differences (one-way ANOVA with Tukey’s test; p < 0.05) are indicated by lowercase letters. JHA, juvenile hormone analog; Glu, Glucose.

Glucose levels induced by SBPH infestation influence imidacloprid tolerance in SBPH by regulating GST activity

(A) GST activity in nymphs treated with the LC₅₀ dose of imidacloprid (2.26 mg/L) as third-instars and detected 5 days later. Control nymphs were treated with an imidacloprid-free solution under identical conditions (25 nymphs/group). (B) Nymph mortality treated with a combination of imidacloprid and the GST inhibitor DEM as third-instars and recorded at 5 days post-treatment (50 nymphs/group). (C) GST activity in nymphs reared on rice plants supplemented with 0–2.0% glucose as third-instars and assayed 5 days later (25 nymphs/group). (D) GCL expression and (E)GSH content in nymphs reared on rice supplemented with 0–2.0% glucose as third-instars and assayed 5 days later (25 insects/group). (F) GSH content in nymphs injected with dsGCL as third-instars and assayed 5 days later (25 insects/group). (G) GST activity of nymphs injected with dsGCL as third-instars, then reared on rice seedlings cultured in 0% or 1.5% glucose IRRI solution and assayed 5 days later; dsEGFP-injected nymphs served as controls (25 nymphs/group). (H) Mortality rate of insects treated like (G) exposure with LC50 imidacloprid for 5 days (50 nymphs/group). (I) Relative expression of 9 GST genes in nymphs treated with the LC₅₀ dose of imidacloprid as third-instars and detected 5 days later (25 nymphs/group). (J) Relative expression of 9 GST genes in nymphs reared on rice plants supplemented with 0% or 1.5% glucose as third-instars and assayed 5 days later (25 nymphs/group). (K) Mortality of nymphs injected with dsRNA targeting GSTe1, GSTo1, or both as third-instars, then exposed to the LC₅₀ dose of imidacloprid and recorded 5 days later (50 nymphs/group). (L) Mortality of nymphs with co-knockdown of GSTe1 and GSTo1 treated with the LC₅₀ dose of imidacloprid as third-instars, then reared on rice irrigated with 0% or 1.5% glucose solution and recorded 5 days later (50 nymphs/group). (M) Mortality of nymphs subjected to RNAi-mediated TOR knockdown as third-instars, then exposed to the LC₅₀ dose of imidacloprid and reared for 5 days on rice cultured in IRRI solution supplemented with 0% or 1.5% glucose; dsEGFP-injected nymphs served as the control (50 nymphs/group). (N) Mortality of nymphs at 5 days post-treatment with the LC₅₀ dose of imidacloprid, following RNAi-mediated knockdown of TOR, JHAMT, Kr-h1, Met, combined TOR and JHAMT, initiated at the third-instar stage (50 nymphs/group). (O) Mortality of nymphs, treated with the LC₅₀ of imidacloprid as third-instars and assayed 5 days later, following RNAi-mediated TOR knockdown; TOR knockdown with JHA rescue; or combined TOR and GCL knockdown; compared to the dsEGFP-injected control (50 nymphs/group). (P) GST activity in nymphs 5 days after undergoing RNAi-mediated TOR knockdown; TOR knockdown with JHA rescue; or combined TOR and GCL knockdown, compared to the dsEGFP-injected control (25 nymphs/group). (Q) Relative expression of LsGSTe1 and LsGSTo1 in nymphs 5 days after RNAi-mediated TOR knockdown or TOR knockdown with JHA rescue, compared to the dsEGFP-injected control (25 nymphs/group). Panel images (A, C) represent data from six biological replicates, while (B, D, E, F, G, H, I, J, K, L, M, N, O, P, Q) include four replicates. Data are presented as mean ± s.d. (A, B, F, I, J, L) Were analyzed by Student’s t-test (*p < 0.05, **p < 0.01, ns = not significant). (C, D, E, G, H, K, M-Q) Significant differences (one-way ANOVA with Tukey’s test; p < 0.05) are indicated by lowercase letters. IM, imidacloprid; DEM, diethyl maleate; JHA, juvenile hormone analog; Glu, Glucose.