Detoxification of hostplant's chemical defence rather than its anti-predator co-option drives β-glucosidase-mediated lepidopteran counteradaptation

Abstract

The evolutionary plant-herbivore arms race sometimes gives rise to remarkably unique adaptation strategies. Here we report one such strategy in the lepidopteran herbivore Manduca sexta against its hostplant Nicotiana attenuata's major phytotoxins, 17-hydroxygeranyllinalool diterpene glycoside, lyciumoside IV and its malonylated forms. We show that alkalinity of larval regurgitant non-enzymatically demalonylates the malonylated forms to lyciumoside IV. Lyciumoside IV is then detoxified in the midgut by β-glucosidase 1-catalysed deglycosylation, which is unusual, as typically the deglycosylation of glycosylated phytochemicals by insects results in the opposite: toxin activation. Suppression of deglucosylation by silencing larval β-glucosidase 1 by plant-mediated RNAi causes moulting impairments and mortality. In the native habitat of N. attenuata, β-glucosidase 1 silencing also increases larval unpalatability to native predatory spiders, suggesting that the defensive co-option of lyciumoside IV may be ecologically advantageous. We infer that M. sexta detoxifies this allelochemical to avoid its deleterious effects, rather than co-opting it against predators.

Figure 1. N. attenuata's malonylated HGL-DTGs are demalonylated non-enzymatically in M. sexta's alkaline regurgitant.

(a) Structures of core lyciumoside IV (Lyc4) and its singly and dimalonylated forms, nicotianoside I (Nic1) and nicotianoside II (Nic2), respectively. (b) Proportions of Lyc4 and its malonylated forms Nic1 and Nic2 and other HGL-DTGs in uninduced and M. sexta herbivory-induced N. attenuata leaves showing that Lyc4 and its derivatives are the major HGL-DTGs (based on data from Heiling et al.20). (c) Profile of core and malonylated Lyc4 in N. attenuata leaf and in larval regurgitant, midgut content and frass showing that complete demalonylation occurs when the leaf comes into contact with larval regurgitant, at the time of ingestion by larvae (F_3,8=19.81, P≤0.0001; significant differences (threshold: P≤0.05) between means (±s.e.) determined by Games Howell test (Welch's ANOVA); n=3). (d) Analysis to determine whether the demalonylation of Nic1 and Nic2 in larval regurgitant occurs enzymatically or by alkaline hydrolysis in the alkaline pH of the regurgitant; concentration of malonylated Lyc4 (Nic1 and Nic2) in N. attenuata leaf when it was crushed in water, regurgitant, boiled regurgitant, pronase-treated regurgitant, heat-inactivated pronase-treated regurgitant, alkaline buffer, acidic buffer and acidified regurgitant (F_4,20=347.1, P≤0.001; significant differences (threshold: P≤0.05) between means (±s.e.) determined by Fisher's LSD test (one-way ANOVA); n=5). ND, not detected. ANOVA, analysis of variance; LSD, least significant difference.

Figure 2. Lyc4 ingesting M. sexta larvae excrete a novel HGL-DTG.

Ion (m/z 271.24≡HGL) extracted U(H)PLC/ESI-QTOF MS chromatograms showing N. attenuata's major HGL-DTGs in (a) leaf (alkaline extract) and frass of larvae feeding on (b) N. attenuata foliage and (c) artificial diet (AD) containing 3 mM Lyc4 (physiological concentration of Lyc4 in an uninduced leaf). (d) Mass spectrum of the novel compound and prominent losses of functional groups. (e) Structure of the novel compound elucidated by NMR spectroscopy; compound was annotated as 3-O-α-rhamnopyranosyl-(1→4)-β-glucopyranosyl-17-hydroxygeranyllinalool (RGHGL).

Figure 3. Silencing of Lyc4 ingestion-induced, midgut-expressed BG1 impairs larval Lyc4 deglucosylation, moulting and survival.

BG1 transcripts (relative to ubiquitin) in midguts of fourth-instar larvae feeding on (a) Lyc4-containing EV and Lyc4-depleted irGGPPS plants (F_1,10=833.5, P≤0.0001; significant difference (P≤0.05) between means (±s.e.) determined by Fisher's LSD test (one-way ANOVA); n=6) and (b) AD containing water (control), 6 mM Lyc4 or 6 mM RGHGL (F_2,15=5812.13, P≤0.0001; significant differences (P≤0.05) between means (±s.e.) determined by Games Howell test (Welch's ANOVA); n=6). (c) Plant-mediated RNAi: pSOL8 binary vector constructed to express 301 bp MsBG1 dsRNA in N. attenuata and the trophic transfer of dsRNA from plant to larvae. (d) BG1 transcripts (relative to ubiquitin) in various tissues of fourth-instar larvae feeding on EV, irBG1 and irGGPPS plants (midgut: F_2,15=9.53 P≤0.002; hindgut: F_2,15=248, P≤0.0001; significant differences (P≤0.05) between means (±s.e.) determined by Games Howell test (Welch's ANOVA, separately conducted for each tissue); n=6 in all bars). (e) RGHGL formed in vitro enzyme assays containing midgut extracts of fourth-instar EV- and irBG1-feeding larvae and either (1 mM) Lyc4 (F_3,8=104.9, P≤0.0001; significant differences (P≤0.05) between means (±s.e.) determined by Fisher's LSD test (one-way ANOVA); n=3) or 1 mM Lyc4+1 mM BG inhibitor, δ-gluconolactone (n=3). Excretion (% of total Lyc4 ingested) of (f) Lyc4 (F_{1, 25}=13.95, P≤0.001; significant difference (P≤0.05) between means (±s.e.) determined by Fisher's LSD test (one-way ANOVA); n=13 (EV) and 14 (irBG1)) and (g) RGHGL (F_{1, 25}=62.65, P≤0.0001; significant difference (P≤0.05) between means (±s.e.) determined by Fisher's LSD test (one-way ANOVA); n=13 (EV) and 14 (irBG1)) by fourth-instar EV- or irBG1-feeding larvae. (h) Phenotype (in irBG1-feeding larva) and (i) percentage of moulting impairment (significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=30) and (j) mortality (%) in larvae feeding (for 12 d) on EV or irBG1 plants (significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=30). ANOVA, analysis of variance; d, days; LSD, least significant difference.

Figure 4. Moulting impairment and mortality in BG1-silenced larvae is associated with suppressed Lyc4 metabolism.

(a) Schematic showing the motivation for the hypothesis that suppressed Lyc4 metabolism in irBG1-feeding larvae causes moulting impairment and mortality. (b) Generation of MsBG1-containing and Lyc4-depleted transgenic N. attenuata plants (B × G) by crossing irBG1 with irGGPPS plants. (c) Moulting impairment (%) (significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=30) and (d) mortality (%) (significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=30) in larvae after 12 d feeding on water coated EV, irBG1, irGGPPS and B × G leaves, Lyc4 coated (final concentration 6 mM) irGGPPS and B × G leaves and RGHGL-coated (final concentration 6 mM) irGGPPS and B × G leaves. d, days.

Figure 5. In the native habitat, spiders capture and kill but do not ingest the BG1-silenced M. sexta larvae.

To test (a) whether Lyc4 or RGHGL is defensively co-opted by larvae against the natural enemies in the native habitat, (b) EV, irBG1 and irGGPPS plants were grown in the Great Basin Desert, Utah. (c) Diurnal and nocturnal survival (%) of larvae feeding on these plants (n=32 larvae per line). (d) Native C. parallela spider attacking M. sexta larva. (e) Three stages of spider's predation behaviour. Spider's (f) prey capture and killing (%; n=28) and (g) prey ingestion (% of total killed; significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=28) in a no-choice assay (1 h) on second-instar M. sexta larvae feeding on EV and irBG1 and irGGPPS plants.

Figure 6. High larval body Lyc4 concentration hinders spiders' prey ingestion and topically coated Lyc4 deters spiders.

Spider's (a) prey capture and killing (%; n=25) and (b) prey ingestion (% of total killed; significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=25) in a no-choice assay (1 h) using second-instar M. sexta larvae feeding on EV and irBG1 leaves coated with water (control) and irGGPPS and B × G leaves coated with water (control), Lyc4 (final concentration 6 mM) and RGHGL (final concentration 6 mM). (c) Schematic of a no-choice assay. (d) Spider's prey capture and killing (%; significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=30) and (e) prey ingestion (% of total killed; significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=30) in no-choice assays (1 h) using second-instar water-, Lyc4- or RGHGL-coated (final concentration 6 mM for both Lyc4 and RGHGL) M. sexta larvae feeding on irGGPPS plants and (f) schematic of a choice assay. Spider's prey capture and killing (%) in choice assays (1 h) using irGGPPS-feeding second-instar M. sexta larvae coated with (g) water or Lyc4 (final concentration 6 mM; significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=21), (h) water or RGHGL (final concentration 6 mM; n=21) and (i) Lyc4 (final concentration 6 mM) or RGHGL (final concentration 6 mM; significant difference (P≤0.05) determined by Fisher's exact test of frequencies; n=21).