Sawfly Sex Pheromones: Analysis of Their Impact on Pine Odor Attractive to Egg Parasitoids.

Pinus sylvestris trees are known to efficiently defend themselves against eggs of the herbivorous sawfly Diprion pini. Their direct defense against eggs is primable by prior exposure to the sex pheromones of this species and their indirect defense involves attraction of egg parasitoids by egg-induced pine needle odor. But it is unknown whether exposure of pine to D. pini sex pheromones also affects pine indirect defense against sawfly eggs. In this study, we investigated the influence of exposure of P. sylvestris trees to the sex pheromones of D. pini on indirect defense mediated by egg parasitoids. Behavioral assays with Closterocerus ruforum, a key parasitoid of sawfly eggs, revealed no significant attraction to odor from egg-free pines pre-exposed to pheromones. Chemical analyses of odor from egg-free pines showed no pheromone-induced change in the emission rates of the known key terpenoids promoting parasitoid attraction. Further comparative analyses of odor from egg-laden pines pre-exposed to the sex pheromones and of odor from egg-laden pines unexposed to pheromones neither revealed significant differences in the emission rates of terpenoids relevant for parasitoid attraction. The results suggest that a pheromone-induced or pheromone-primed, egg-induced pine indirect defense seems to be redundant in addition to the known pheromone-primable pine direct defense against the eggs and the known egg-inducible indirect defense.

Plumbagin, a Potent Naphthoquinone from Nepenthes Plants with Growth Inhibiting and Larvicidal Activities.

Some plant species are less susceptible to herbivore infestation than others. The reason for this is often unknown in detail but is very likely due to an efficient composition of secondary plant metabolites. Strikingly, carnivorous plants of the genus Nepenthes show extremely less herbivory both in the field and in green house. In order to identify the basis for the efficient defense against herbivorous insects in Nepenthes, we performed bioassays using larvae of the generalist lepidopteran herbivore, Spodoptera littoralis. Larvae fed with different tissues from Nepenthes x ventrata grew significantly less when feeding on a diet containing leaf tissue compared with pitcher-trap tissue. As dominating metabolite in Nepenthes tissues, we identified a naphthoquinone, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone). When plumbagin was added at different concentrations to the diet of S. littoralis larvae, an EC50 value for larval growth inhibition was determined with 226.5 µg g-1 diet. To further determine the concentration causing higher larval mortality, sweet potato leaf discs were covered with increasing plumbagin concentrations in no-choice-assays; a higher mortality of the larvae was found beyond 60 µg plumbagin per leaf, corresponding to 750 µg g-1. Plant-derived insecticides have long been proposed as alternatives for pest management; plumbagin and derivatives might be such promising environmentally friendly candidates.

Pine Response to Sawfly Pheromones: Effects on Sawfly's Oviposition and Larval Growth.

Insect pheromones have been intensively studied with respect to their role in insect communication. However, scarce knowledge is available on the impact of pheromones on plant responses, and how these in turn affect herbivorous insects. A previous study showed that exposure of pine (Pinus sylvestris) to the sex pheromones of the pine sawfly Diprion pini results in enhanced defenses against the eggs of this sawfly; the egg survival rate on pheromone-exposed pine needles was lower than that on unexposed pine. The long-lasting common evolutionary history of D. pini and P. sylvestris suggests that D. pini has developed counter-adaptations to these pine responses. Here, we investigated by behavioral assays how D. pini copes with the defenses of pheromone-exposed pine. The sawfly females did not discriminate between the odor of pheromone-exposed and unexposed pine. However, when they had the chance to contact the trees, more unexposed than pheromone-exposed trees received eggs. The exposure of pine to the pheromones did not affect the performance of larvae and their pupation success. Our findings indicate that the effects that responses of pine to D. pini sex pheromones exert on the sawfly eggs and sawfly oviposition behavior do not extend to effects on the larvae.

Carnivorous Nepenthes x ventrata plants use a naphthoquinone as phytoanticipin against herbivory.

Carnivorous plants feed on animal prey, mainly insects, to get additional nutrients. This carnivorous syndrome is widely investigated and reported. In contrast, reports on herbivores feeding on carnivorous plants and related defenses of the plants under attack are rare. Here, we studied the interaction of a pitcher plant, Nepenthes x ventrata, with a generalist lepidopteran herbivore, Spodoptera littoralis, using a combination of LC/MS-based chemical analytics, choice and feeding assays. Chemical defenses in N. x ventrata leaves were analyzed upon S. littoralis feeding. A naphthoquinone, plumbagin, was identified in Nepenthes defense against herbivores and as the compound mainly responsible for the finding that S. littoralis larvae gained almost no weight when feeding on Nepenthes leaves. Plumbagin is constitutively present but further 3-fold increased upon long-term (> 1 day) feeding. Moreover, in parallel de novo induced trypsin protease inhibitor (TI) activity was identified. In contrast to TI activity, enhanced plumbagin levels were not phytohormone inducible, not even by defense-related jasmonates although upon herbivory their level increased more than 50-fold in the case of the bioactive jasmonic acid-isoleucine. We conclude that Nepenthes is efficiently protected against insect herbivores by naphthoquinones acting as phytoanticipins, which is supported by additional inducible defenses. The regulation of these defenses remains to be investigated.