The plant metabolome guides fitness-relevant foraging decisions of a specialist herbivore

Abstract

Plants produce complex mixtures of primary and secondary metabolites. Herbivores use these metabolites as behavioral cues to increase their fitness. However, how herbivores combine and integrate different metabolite classes into fitness-relevant foraging decisions in planta is poorly understood. We developed a molecular manipulative approach to modulate the availability of sugars and benzoxazinoid secondary metabolites as foraging cues for a specialist maize herbivore, the western corn rootworm. By disrupting sugar perception in the western corn rootworm and benzoxazinoid production in maize, we show that sugars and benzoxazinoids act as distinct and dynamically combined mediators of short-distance host finding and acceptance. While sugars improve the capacity of rootworm larvae to find a host plant and to distinguish postembryonic from less nutritious embryonic roots, benzoxazinoids are specifically required for the latter. Host acceptance in the form of root damage is increased by benzoxazinoids and sugars in an additive manner. This pattern is driven by increasing damage to postembryonic roots in the presence of benzoxazinoids and sugars. Benzoxazinoid- and sugar-mediated foraging directly improves western corn rootworm growth and survival. Interestingly, western corn rootworm larvae retain a substantial fraction of their capacity to feed and survive on maize plants even when both classes of chemical cues are almost completely absent. This study unravels fine-grained differentiation and combination of primary and secondary metabolites into herbivore foraging and documents how the capacity to compensate for the lack of important chemical cues enables a specialist herbivore to survive within unpredictable metabolic landscapes.

Fig 1. The western corn rootworm prefers to feed on root tissues that are rich in benzoxazinoids and soluble sugars.

(A) Left: Young maize plants produce embryonic primary and seminal roots (orange arrows) and postembryonic crown roots (blue arrows). Right: Western corn rootworm larvae are highly specialized maize root feeders that are very mobile in the second and third instar. A third instar larvae is shown. (B) Average damage score observed on postembryonic and embryonic roots of soil-grown maize plants after 7 days of infestation by western corn rootworm larvae (***p < 0.001, Wilcoxon Signed Rank test, n = 20 plants with 15 larvae each, data from Fig 4D). Dots represent damage scores on individual plants (averaged within root types). For frequency distributions of individual roots, refer to S1 Fig. (C) Preference of western corn rootworm larvae for postembryonic and embryonic roots within the root system (**p < 0.01, ***p < 0.001, FDR-corrected Least Square Mean post hoc tests, n = 16 dishes with 6 larvae each). (D) Preference of western corn rootworm larvae for root pieces of equal size of postembryonic and embryonic roots (***p < 0.001; FDR-corrected Least Square Mean post hoc tests, n = 18 dishes with 5 larvae each). (E) Metabolomics profiles of methanolic extracts of postembryonic and embryonic roots. Orange features are more abundant in embryonic roots, blue features are more abundant in postembryonic roots (min. 2-fold difference, p < 0.05, FDR-corrected Student t tests, n = 9–10). Numbers denote features that were tentatively assigned to structures based on exact mass and fragment information (S1 Table). (F) Relative abundance differences of tentatively identified metabolites between postembryonic and embryonic roots (fold change of peak areas; *p < 0.05, FDR-corrected Student t tests, n = 9–10). Error bars denote SEM. Underlying data can be found in S1 Data. FDR, false discovery rate; SEM, standard errors of means. Picture credits: Ricardo Machado, Lingfei Hu, Cyril Hertz.

Fig 2. A mutation in the Bx1-gene suppresses root-type–specific benzoxazinoid accumulation independently of root sugars.

(A) Concentrations of benzoxazinoids in embryonic and postembryonic roots of WT B73 and bx1 mutant plants (n = 11–18). For an expanded panel of residual benzoxazinoid concentrations in the bx1 mutant, see S2 Fig. (B) Concentrations of glucose, fructose, and sucrose in embryonic and postembryonic roots of WT B73 and bx1 mutant plants (n = 8). Letters indicate significant differences in total amounts between root types and genotypes (p < 0.05, Holm–Sidak post hoc tests). Asterisks indicate significant differences in the concentrations of individual compounds between postembryonic and embryonic roots (p < 0.05, Holm–Sidak post hoc tests). Error bars denote SEM. Underlying data can be found in S1 Data. SEM, standard errors of means; WT, wild-type.

Fig 3. DvvGr43a mediates sugar preference of the western corn rootworm without influencing responsiveness to benzoxazinoids.

(A) Phylogenetic relationships between gustatory sugar receptors of different insects and DvvGr43a of the western corn rootworm. The tree is based on protein sequences and drawn to scale, with branch lengths measured in the number of substitutions per site. Asterisks indicate functionally characterized receptors. (B) Protein tertiary structure of DvvGr43a as predicted with the Phyre2 algorithm. (C) DvvGr43a expression in the bodies (thorax and abdomen) or heads of second instar western corn rootworm larvae (*p < 0.05, Student t test n = 10). (D) DvvGr43a expression in western corn rootworm larvae fed with dsRNA targeting GFP (control) or DvvGr43a (Gr43a, ***p < 0.001, Student t test, n = 11). (E) DvvGr2 expression in western corn rootworm larvae fed with dsRNA targeting GFP (control) or DvvGr43a (n = 8). (F) Preference of GFP and DvvGr43a dsRNA fed larvae for buffer or Fe(III)(DIMBOA)₃ on filter discs after 1 hour (***p < 0.001, FDR-corrected Least Square Mean post hoc tests, n = 10 dishes with 6 larvae each). (G–J) Preference of GFP or DvvGr43a dsRNA fed larvae for buffer or glucose, fructose, sucrose, or a mixture of the three on filter discs at different time points (*p < 0.05; **p < 0.01; ***p < 0.001, FDR-corrected Least Square Mean post hoc tests, n = 15 dishes with 6 larvae each). Error bars denote SEM. Underlying data can be found in S1 Data. dsRNA, double-stranded RNA; FDR, false discovery rate; GFP, green fluorescent protein; SEM, standard errors of means.

Fig 4. Benzoxazinoids and sugars play distinct roles in root herbivore foraging in vivo.

(A) Proportion of control (GFP) or DvvGr43a-silenced (Gr43a) larvae found on the roots of WT B73 or bx1 mutant roots 4 hours after their release. Letters indicate statistically significant differences between treatments (Holm–Sidak post hoc tests, p < 0.05, n = 15 dishes with 6 larvae each). (B) Proportion of control (GFP) or DvvGr43a-silenced (Gr43a) larvae found on embryonic (E) and postembryonic (PE) roots within the same experiment. Asterisks indicate significant differences between root types (***p < 0.001, FDR-corrected Least Square Mean post hoc tests, n = 15 dishes with 6 larvae each). (C) Average feeding damage by control (GFP) or DvvGr43a-silenced larvae per root of soil-grown WT B73 or bx1 mutant plants 7 days after infestation. Letters indicate statistically significant differences (p < 0.05, Tukey post hoc tests, n = 20 plants with 15 larvae each). Bubble plots are shown for illustrative purposes. The sizes of the circles are proportional to the relative frequency (% within each treatment) of the different types of observed damage (each column sums up to 100%). For damage scale, refer to Fig 1B. (D) Average feeding damage on embryonic (E) or postembryonic (PE) roots within the same experiment. Asterisks indicate significant differences in root damage between root types (*p < 0.05, ***p < 0.001 Wilcoxon Signed Rank tests, n = 20 plants with 15 larvae each). Bubble plots are shown for illustrative purposes. The sizes of the circles are proportional to the relative frequency (% within each root type) of the different types of observed damage (each column sums up to 100%). Error bars denote SEM. Underlying data can be found in S1 Data. E, embryonic; FDR, false discovery rate; GFP, green fluorescent protein; PE, postembryonic; SEM, standard errors of means; WT, wild-type.

Fig 5. Using benzoxazinoids and sugars as foraging cues improves herbivore growth and survival.

(A) Weight of control (GFP) and DvvGr43a-silenced larvae feeding on WT B73 or bx1 mutant plants for 7 days. Note that in a no-choice situation, neither the bx1 mutation nor DvvGr43a silencing reduce larval performance ([75] and S8 Fig). Letters indicate significant differences (p < 0.05, Holm–Sidak post hoc tests, n = 20 pots with 15 larvae each). (B) Larval mortality within the same experiment. Letters indicate significant differences (p < 0.05, Holm–Sidak post hoc tests, n = 20 pots with 15 larvae each). (C–F) Correlations between cumulative damage per plant and larval performance parameters for postembryonic roots (C, D) and embryonic roots (E, F). Linear regressions are shown for significant correlations (p < 0.05). P values are shown for Spearman Rank Order correlations. Underlying data can be found in S1 Data. dsRNA, double-stranded RNA; GFP, green fluorescent protein; WT, wild-type.