Small brown planthopper infestation enhances it reproduction and insecticide tolerance by manipulating glucose distribution and levels in rice

Reviewer #1 (Public review):

Summary:

The authors investigate how infestation of rice plants by the small brown planthopper (Laodelphax striatellus), an important pest in rice cultivation, alters host plant carbohydrate metabolism and how these changes affect insect physiology and fitness. They show that planthopper infestation leads to a density-dependent increase in glucose levels in rice plants, which the authors suggest results from a redistribution of carbohydrates from roots to shoots. Elevated glucose levels in plants are reflected by increased glucose contents in the insects themselves, an effect that is particularly pronounced in gravid females and associated with enhanced fecundity.

In addition, the authors demonstrate that increased glucose availability enhances tolerance of the small brown planthopper to the neonicotinoid insecticide imidacloprid. These findings suggest that insect-mediated changes in plant carbohydrate allocation may benefit insect fitness in multiple ways, including increased reproductive output and enhanced tolerance to insecticides, both of which are relevant for understanding insect population dynamics in agroecosystems.

Beyond these physiological observations, the authors aim to elucidate the underlying molecular mechanisms. They propose that glucose functions not only as a nutritional resource but also as a signaling molecule. Specifically, they show that increased glucose availability is associated with activation of the Target Of Rapamycin (TOR) pathway, a conserved nutrient-sensing signaling pathway regulating growth and metabolism across eukaryotes. Activation of TOR signaling is linked to increased juvenile hormone levels, which in turn stimulate vitellogenesis and likely contribute to increased fecundity. Furthermore, elevated juvenile hormone levels are associated with increased expression of glutathione S-transferases, suggesting a mechanism contributing to enhanced detoxification capacity. Independent of this pathway, increased glucose availability also leads to higher expression of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione synthesis. Together, these mechanisms provide a non-exclusive explanation for the observed increase in imidacloprid tolerance and form the basis of the authors' proposed mechanistic framework linking glucose availability to reproduction and detoxification.

Strengths:

A major strength of the manuscript is its substantial mechanistic depth and the extensive use of complementary experimental approaches that converge on a coherent mechanistic interpretation. The authors combine plant manipulations, dietary supplementation, injection assays, RNAi-mediated gene silencing, pharmacological inhibition, and rescue experiments to systematically test the role of glucose as a signaling molecule linking plant-derived nutrition to insect reproduction and insecticide tolerance. Results obtained from independent experimental strategies are highly consistent, and the different datasets collectively support the central conclusions of the study.

The role of glucose is supported by multiple lines of evidence demonstrating that increased glucose availability, whether induced by prior planthopper feeding, dietary supplementation, or direct injection, consistently results in elevated glucose levels in insects, increased oviposition, and enhanced expression of vitellogenesis-related genes (LsVg and LsVgR). The specificity of this effect is further strengthened by experiments using alternative carbohydrates that release glucose upon enzymatic cleavage, as well as inhibitor and rescue experiments, supporting the interpretation that glucose acts beyond a purely nutritional role.

The authors further establish a mechanistic link between glucose availability, TOR signaling, juvenile hormone regulation, and vitellogenesis. Activation of TOR signaling by glucose, demonstrated at the level of protein phosphorylation, together with RNAi knockdown and pharmacological inhibition, allows causal placement of TOR upstream of juvenile hormone signaling. Consistent reductions in juvenile hormone titers, vitellogenesis-related gene expression, and oviposition following TOR inhibition, as well as rescue of reproductive output by juvenile hormone analog treatment, provide strong functional support for a glucose-TOR-juvenile hormone axis regulating fecundity. The absence of additive effects following combined knockdown of TOR and juvenile hormone synthesis components further supports the interpretation that these factors act within the same signaling cascade.

Similarly, the authors provide a detailed mechanistic analysis of glucose-mediated effects on imidacloprid tolerance. Functional assays demonstrate that glutathione S-transferases contribute to detoxification in this species and that increased glucose availability enhances GST activity, glutathione synthesis, and overall glutathione levels. Transcriptomic analyses and targeted RNAi experiments further identify specific GSTs contributing to insecticide tolerance and indicate that glucose enhances detoxification through both TOR-dependent and TOR-independent mechanisms. The combined knockdown experiments, which produce additive effects on mortality, provide particularly strong support for the involvement of multiple interacting glucose-dependent pathways.

Weaknesses:

While I am impressed by the mechanistic depth of the study and the clarity with which the authors dissect the underlying physiological pathways, I am less convinced by the current conceptual framing of the phenomenon as a sophisticated adaptive strategy "co-opted" by the small brown planthopper. The data convincingly demonstrate that glucose availability activates conserved nutrient-sensing and endocrine pathways, including TOR signaling and juvenile hormone regulation, which in turn affect reproduction and detoxification capacity. However, these pathways are deeply conserved and likely operate in many insects in response to nutritional status. As such, the results may reflect a general physiological response to elevated carbohydrate availability rather than a species-specific, evolved strategy. Relatedly, herbivory-induced changes in plant carbohydrate allocation appear to be relatively common across plant-insect systems, and it would be helpful to discuss how specific (or general) the observed phenomenon is likely to be.

In particular, I encourage the authors to more clearly distinguish between (i) a conserved nutrient-responsive signaling cascade and (ii) an adaptive mechanism that evolved specifically under selection imposed by insecticide exposure. The presented data strongly support the former interpretation, whereas evidence for the latter is less clear. The increased tolerance to imidacloprid appears to arise as a consequence of enhanced metabolic and detoxification capacity under elevated glucose conditions, rather than as a trait shaped directly by insecticide-driven selection. Framing this phenomenon as an adaptation to insecticide stress may therefore overextend the conclusions that can be drawn from the data. A more cautious discussion acknowledging that glucose-mediated activation of conserved metabolic and endocrine pathways may incidentally enhance insecticide tolerance, without necessarily having evolved under insecticide selection, would strengthen the conceptual clarity of the manuscript.

Reviewer #2 (Public review):

Summary:

Zhang and colleagues investigate the molecular mechanisms by which the small brown planthopper (SBPH, Laodelphax striatellus) manipulates host rice carbohydrate metabolism to enhance its own fitness. Using a combination of molecular, pharmacological, and biochemical approaches, they demonstrate that SBPH infestation induces systemic glucose reallocation in rice, as evidenced by the upregulation of glucose levels in aerial tissues and a simultaneous reduction in root glucose levels. Notably, host-derived glucose acts as a central signaling molecule, driving two key adaptive traits: enhanced fecundity via the glucose-TOR-JH-Vg signaling cascade, and increased imidacloprid tolerance through synergistic metabolic (GCL-GSH) and regulatory (TOR-JH-GST) pathways targeting GST activity. These findings uncover a sophisticated resource-manipulation strategy in SBPH and identify nutrient-sensing and detoxification pathways as potential targets for pest control.

Strengths:

(1) The study addresses a gap in plant-insect coevolution research by identifying glucose as a dual-function signaling molecule that coordinates SBPH reproduction and insecticide tolerance, providing valuable insights into how herbivores exploit host nutritional signals.

(2) The experimental design is well structured and multifaceted, integrating RNAi, RT-qPCR, Western blotting, pharmacological inhibition, and biochemical assays. The use of appropriate controls (e.g., osmotic controls with mannitol and hydrolase-inhibitor rescue experiments) strengthens the causal interpretation of the results.

(3) The mechanistic framework is clear and well-supported. The authors delineate two interconnected molecular cascades (glucose-TOR-JH-Vg for fecundity and GCL-GSH/TOR-JH-GST for tolerance) with hierarchical validation (e.g., rescue experiments with JHA), ensuring the reliability of conclusions.

Weaknesses:

(1) The study focuses exclusively on SBPH without validating whether the observed phenomena and mechanisms are conserved in closely related planthopper species (e.g., brown planthopper Nilaparvata lugens). This limitation restricts the generalizability of the findings to other economically important rice pests.

(2) The specific upstream signals that trigger glucose reallocation in rice (e.g., SBPH salivary effectors or oviposition-associated factors) are not identified. Although this represents a complex and independent research direction, the absence of such information limits the depth and completeness of the mechanistic framework and leaves open questions regarding the initiation of host metabolic manipulation.

(3) Insecticide tolerance assays are limited to imidacloprid. Extending these analyses to one or two additional commonly used insecticides (e.g., thiamethoxam) would help determine whether the glucose-mediated detoxification pathway is specific to imidacloprid or reflects a broader resistance mechanism, thereby strengthening conclusions regarding the generality of the GST activation cascade.

(4) Given the study's potential implications for pest management, the manuscript would benefit from a brief discussion of possible practical applications, such as manipulating rice glucose metabolism through breeding strategies or developing small-molecule inhibitors targeting the TOR-JH axis. Including such perspectives would enhance the translational relevance of the work by linking mechanistic insights to real-world pest control strategies.