Figure 1.
Nicotine oxidation does not benefit M. sexta larvae.
Larval (a) mass [(mean± SE) F4, 172 = 45.2, P≤0.05, n = 36, 34, 37, 33 and 37 for water, nicotine, NNO, cotinine and CNO, respectively] and (b) mortality (%) after 10 d of feeding on artificial diet containing water (control) or 0.1% (fresh mass) nicotine, NNO, cotinine or CNO (n = 30). (c) Waldbauer assay-based quantification of excreted (%) metabolites by fourth-instar larvae fed artificial diet containing 0.1% (fresh mass) metabolite [(mean± SE) F3, 25 = 4.3, P≤0.05, n = 6 for nicotine, NNO and cotinine and 8 for CNO]; (n. a.≡ not applicable). (d) Nicotine, NNO, cotinine or CNO are not degraded in frass over the 24 h period of the Waldbauer assays. Fresh frass was spiked with each metabolite to attain the final concentration of 0.5%; the spiked frass was extracted and analyzed after zero and 24 h of incubation to quantify the recovered metabolite. Every bar represents data from 3 replicates (n = 3). (e) Melanization of cotinine-fed (right) larva. (f) Discharge kinetics of hemolymph-injected (70±2.5 µg≡ 0.001% of larval fresh mass) nicotine, NNO, cotinine or CNO (n = 5). Lower-case letters and asterisks in (a), (c) and (e) indicate significant differences (P≤0.05) by one-way ANOVA; in (b), asterisk indicates significant differences (P≤0.05) in frequencies (and displayed percentages) by Fisher's exact test.
Figure 2.
Transcript regulation of M. sexta CYPs in response to ingestion of nicotine and nicotine oxides.
(a) CYP6B46 [(mean± SE) F4, 19 = 94.5, P≤0.05, n = 5] (b) CYP4M1 [(mean± SE) F4, 20 = 9.5, P≤0.05, n = 5] and (c) CYP4M3 [(mean± SE) F4, 20 = 11.1, P≤0.05, n = 5] transcript levels (relative to ubiquitin) in midguts of 48 h old first-instar M. sexta larvae fed artificial diet containing water (control) or 0.1% (fresh mass) nicotine, NNO, cotinine or CNO. Asterisks indicate significant differences (P≤0.05) by one-way ANOVA.
Figure 3.
S. exigua oxidizes nicotine, but M. sexta does not.
U(H)PLC/ESI-QTOF-MS-based quantitative analysis of nicotine, NNO, cotinine and CNO in (a) frass (b) hemolymph and (c) headspace of third-instar M. sexta (n = 5) and S. exigua (n = 5) larvae fed artificial diet containing 0.1% (fresh mass) nicotine. Lower-case letters above the S. exigua bars indicate significant differences (P≤0.05) among them, by one-way ANOVA. Asterisks above the M. sexta nicotine bars indicate that they differ significantly (P≤0.05) from the S. exigua nicotine values, as determined by one-way ANOVA. Nicotine oxides were not detected in M. sexta. The detection limit of nicotine was 0.25 ng and 0.5 ng for cotinine, CNO and NNO; efficiency of extraction was >90% for all these compounds.
Figure 4.
S. exigua oxidizes nicotine, but M. sexta does not.
U(H)PLC/ESI-QTOF-MS-based quantitative analysis of nicotine, NNO, cotinine and CNO in (a) frass (b) hemolymph and (c) headspace of third-instar M. sexta (n = 5) and S. exigua (n = 5) larvae fed N. attenuata leaves. Lower-case letters above the S. exigua bars indicate significant differences (P≤0.05) among them by one-way ANOVA. Asterisks above the M. sexta nicotine bars indicate that they are significantly different (P≤0.05) from the S. exigua nicotine bars, as determined by one-way ANOVA. Nicotine oxides were not detected in M. sexta. The detection limit of nicotine was 0.25 ng and 0.5 ng for cotinine, CNO and NNO; efficiency of extraction was >90% for all these compounds [1].
Figure 5.
Volatility analysis of nicotine and nicotine oxides.
(a) Schematic of setup used for the collection of evaporated nicotine, NNO, cotinine and CNO placed in the collection vial (1 µg/5 µL methanol). (b) Percentage of evaporated and residual nicotine, NNO, cotinine and CNO (n = 4); one µg of nicotine, NNO, cotinine or CNO (in 5 µL methanol) was incubated for 1 h.
Figure 6.
Nicotine is more toxic than the nicotine oxides to S. exigua larvae.
(a) Larval mass of S. exigua [(mean± SE) F4, 154 = 6.03, P≤0.05, n = 32, 27, 33, 30 and 33 for water, nicotine, NNO, cotinine and CNO, respectively] and (b) mortality (%) of S. exigua larvae during 10days of feeding artificial diet containing water (control) or 0.1% (fresh mass) nicotine, NNO, cotinine or CNO; each bar represents data from 30 larvae. In (a), lower-case letters above the bars indicate significant differences (P≤0.05) by one-way ANOVA; in (b) asterisk indicates significant differences (P≤0.05) in frequencies (and displayed percentages) by Fisher's exact test.
Figure 7.
Nicotine deters C. parallela but nicotine oxides do not.
C. parallela's predation (%) (in 1 h no-choice assay) on second-instar (a) M. sexta and (b) S. exigua larvae fed artificial diet containing water (control) or 0.1% (fresh mass) nicotine, NNO, cotinine or CNO. C. parallela predation (%) (in 1 h no-choice assay) on second-instar artificial diet fed (c) M. sexta and (d) S. exigua larvae coated with water (control) or 0.2% aqueous nicotine, NNO, cotinine or CNO. Each bar represents data from 30 larvae. Asterisks indicate significant differences (P≤0.05) in frequencies (and displayed percentages) by Fisher's exact test.
Figure 8.
C. parallela's preference of S. exigua larvae over M. sexta larvae is diminished by topically coating larvae or supplementing their headspace with nicotine.
C. parallela's predation (%) (in 1 h choice assay) on second-instar M. sexta and S. exigua larvae fed (a) artificial diet or artificial diet containing 0.1% (fresh mass) nicotine (b) artificial diet and coated with water (control) or nicotine, and (c) artificial diet containing water (control) or 0.1% (fresh mass) nicotine and having the assay environment nicotine-perfumed using 500 µL of 1 mM nicotine on a cotton swab. Schematics in right panels of (a), (b) and (c) show the effects of various modes of nicotine supplementation to M. sexta and S. exigua larvae on the spider predation behavior. Each bar represents data from 30 larvae. Asterisks indicate significant differences (P≤0.05) in frequencies (and displayed percentages) by Fisher's exact test.