Revealing manduca sextas nicotine metabolism and its ecological consequences using plant mediated RNAi based reverse genetics
Insects have developed a myriad of adaptations to live and evolve. Manduca sexta, a specialist lepidopteran herbivore of Nicotiana attenuata, has developed a remarkable adaptation to tolerate a toxic alkaloid ‘nicotine’ that it ingests from its host plant Nicotiana attenuata. The mechanism of nicotine tolerance in M. sexta larvae is not completely known. In this work I used a reverse genetics based approach to silence M. sexta’s genes involved nicotine tolerance to understand their role and ecological consequences of nicotine adaptation by this insect. Nicotine induced midgut-based M. sexta CYP6B46 was silenced by plant-mediated RNAi. Survivorship of CYP6B46-silenced M. sexta larvae was found to be lowered, similar to that of low-nicotine-foliage feeding larvae in the field (Great Basin desert, Utah). While investigating the reasons for the reduced survivorship, I identified a native wolf-spider that preferred to prey on nicotine-free and CYP6B46-silenced larvae, compared to control larvae. In addition, CYP6B46-silenced larva is impaired in passing nicotine from midgut into hemolymph, thereby reducing the availability of nicotine for emission through spiracles and hence become susceptible to spider attack. Generalist lepidopteran herbivore of N. attenuata, Spodoptera exigua, was found to oxidize nicotine but not M. sexta. Spider predation assay using oxidized forms of nicotine suggests that spider deterrence could be attributed only to nicotine and not to the oxidized forms of nicotine, indicating that modification of nicotine increased the larval vulnerability to spider predation. In addition, there are no physiological advantages of nicotine oxides in terms of rapid excretion and mass gain of M. sexta larvae. I infer that nicotine detoxification in M. sexta by oxidation of nicotine is a physiologically and ecologically expensive strategy. I conclude that ‘use of unmetabolized nicotine against spiders’ is a specialty of M. sexta.