Natural history-driven, plant-mediated RNAi-based study reveals CYP6B46's role in a nicotine-mediated antipredator herbivore defense

Abstract

Manduca sexta (Ms) larvae are known to efficiently excrete ingested nicotine when feeding on their nicotine-producing native hostplant, Nicotiana attenuata. Here we describe how ingested nicotine is co-opted for larval defense by a unique mechanism. Plant-mediated RNAi was used to silence a midgut-expressed, nicotine-induced cytochrome P450 6B46 (CYP6B46) in larvae consuming transgenic N. attenuata plants producing MsCYP6B46 dsRNA. These and transgenic nicotine-deficient plants were planted into native habitats to study the phenotypes of larvae feeding on these plants and the behavior of their predators. The attack-behavior of a native wolf spider (Camptocosa parallela), a major nocturnal predator, provided the key to understanding MsCYP6B46's function: spiders clearly preferred CYP6B46-silenced larvae, just as they had preferred larvae fed nicotine-deficient plants. MsCYP6B46 redirects a small amount (0.65%) of ingested nicotine from the midgut into hemolymph, from which nicotine is exhaled through the spiracles as an antispider signal. CYP6B46-silenced larvae were more susceptible to spider-attack because they exhaled less nicotine because of lower hemolymph nicotine concentrations. CYP6B46-silenced larvae were impaired in distributing ingested nicotine from midgut to hemolymph, but not in the clearing of hemolymph nicotine or in the exhalation of nicotine from hemolymph. MsCYP6B46 could be a component of a previously hypothesized pump that converts nicotine to a short-lived, transportable, metabolite. Other predators, big-eyed bugs, and antlion larvae were insensitive to this defense. Thus, chemical defenses, too toxic to sequester, can be repurposed for defensive functions through respiration as a form of defensive halitosis, and predators can assist the functional elucidation of herbivore genes.

Keywords: Coyote tobacco; Lepidoptera; alkaloid; reverse genetics; tobacco hornworm.

Fig. 1.

Spiders are deterred by nicotine-fed larvae. (A) Nicotiana attenuata growing in the Great Basin Desert, Utah. (B) Nocturnal survival (%) of larvae feeding on nicotine-containing EV and nicotine-deficient (irPMT) plants in the field (n = 50 larvae per line). (C) Spider attacking M. sexta larva. Spider predation (%) in the choice assay (1 h) on second-instar M. sexta larvae feeding on (D) EV (n = 16) and irPMT (n = 16) plants and (E) AD (n = 23) and AD containing 0.1% of nicotine (AD+N) (n = 23). Spider predation (%) in no-choice assays (1 h) with M. sexta larvae feeding on: (F) AD containing 0 (n = 23), 0.03 (n = 20), 0.06 (n = 20), and 0.1% (n = 23) nicotine (AD+N), or (G) water (W) or 1 mM nicotine (N) stem-fed (24 h) EV and irPMT leaves (n = 26 in all of the treatments). Asterisks indicate significant differences (P ≤ 0.05) by Fisher’s exact test on the frequencies as well as percentages. Shading of the bars reflects relative nicotine concentration of the larval diet throughout all figures. Hence, the bar shading provides the important information for the interpretation of transcripts, larval nicotine excretion, and the hemolymph-nicotine data presented in the subsequent figures.

Fig. 2.

Silencing larval CYP6B46 dramatically affects spider predation. (A) CYP6B46 transcript levels (relative to ubiquitin) in midguts of first-instar larvae feeding on WT and irPMT N. attenuata plants (F_1,8 = 9.984, P ≤ 0.05, n = 5). (B) Schematic representation of plant-mediated RNAi: pSOL8 binary vector constructed to express 300-bp dsRNA of MsCYP6B46 in N. attenuata and trophic transfer of CYP6B46 dsRNA from plant to larvae. (C) CYP6B46 transcript levels (relative to ubiquitin) in various tissues (foregut, midgut, hindgut, hemolymph, Malpighian tubules, cuticle with fat body and spiracle) of fourth-instar larvae feeding on EV (E), irCYP (C), and irPMT (P) plants (F_20,84 = 487.2, P ≤ 0.0001, n = 5). (D) Spider predation (%) in no-choice assays on larvae fed EV and irCYP leaves (n= 26). Asterisks and small letters above the bars in A and C indicate significant differences determined by one-way ANOVAs; asterisk in D indicates significant differences (P ≤ 0.05) by Fisher's exact test. See Fig. 1 legend for the codes for the bar-shading.

Fig. 3.

Effect of CYP6B46 silencing on larval nicotine excretion and nicotine flux in larval body. (A) Nicotine excreted (percent of total ingested) by fourth-instar EV- or irCYP-feeding larvae (experimental details in SI Appendix, Fig S4) [(mean ± SE) F_1,12 = 8.77, P ≤ 0.05, n = 8]. (B) Nicotine in hemolymph of fourth-instar EV- or irCYP-feeding larvae (F_1,4 = 106.6, P ≤ 0.01, n = 5). (C) Kinetics of nicotine absorption by (green arrows in larval body and green lines in graph) and discharge from (red arrows and red lines) the hemolymph of control and CYP-silenced fourth-instar larvae (experimental details in SI Appendix, Fig. S5A). (D) Kinetics of nicotine discharge from excised midguts (containing ingested host-plant diet) of control and CYP-silenced fourth instar larvae; nicotine entering the bathing solution (sodium phosphate buffer + 0.3 M sorbitol, pH 7.0) was measured at regular intervals, up to 60 min. (E) Kinetics of nicotine discharge from hemolymph of control and CYP-silenced fourth-instar larvae, after injecting 0.001% nicotine (of FM) into the hemolymph to determine if CYP-silenced larvae discharge nicotine from their hemolymph at rates different from that of controls. Asterisks indicate significant differences determined by one-way ANOVA (P ≤ 0.05). See Fig. 1 legend for the bar-shading codes.

Fig. 4.

CYP6B46 silencing reduces larval nicotine emission and increases spider predation, which can be complemented by volatile nicotine perfuming. (A) Emission of ingested nicotine (Left) [(mean ± SE) F_2,6 = 36.14, P ≤ 0.0005, n = 3] by the fourth-instar larvae feeding on EV and irCYP plants and emission of injected nicotine (Right) [(mean ± SE) n = 3] by the nicotine-free control and CYP-silenced fourth-instar larvae. Nicotine adsorbed on the PDMS tube attached to a spiracle of the fourth-instar control or CYP-silenced larvae (each weighing 7.0 ± 0.25 g): (B) after feeding on nicotine containing leaves for 1 h [(mean ± SE) F_1,9 = 5.82, P ≤ 0.05, n = 5] or (C) after injecting 0.001% nicotine (of FM) [(mean ± SE) n = 5]. (D) M. sexta larva with attached PDMS tubes for the volatile nicotine trapping from the spiracle (Sp) and cuticle (Cu). (E) Spider predation (%) in a no-choice assay on M. sexta larvae fed on EV, irPMT, and irCYP plants with water or nicotine perfuming (n = 15 per treatment); experimental details are given in SI Appendix, Fig. S7B. Asterisks above the bars in A–C indicate significant differences determined by one-way ANOVA; asterisks above the bars in E indicate significant differences (P ≤ 0.05) by Fisher’s exact test. See Fig. 1 legend for the bar-shading codes.