These authors contributed equally to this work. SUMMARYInduced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for 15 N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/ oxygenase (RuBisCO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites, irLOX3 showed a similar, albeit attenuated, pattern, whereas irMYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBisCO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, 15 N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBisCO turnover.

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

The continuous and indiscriminate use of insecticides has been responsible for the emergence of insecticide resistant vector insect populations, especially in Aedes aegypti. Thus, it is urgent to find natural insecticide compounds with novel mode of action for vector control. The goal of this study was to investigate the larvicidal activity of essential oils (EOs) from Piper species against A. aegypti characterized as resistant and susceptible strains to pyrethroids. The EOs from leaves of 10 Piper species were submitted to the evaluation of larvicidal activity in populations of A. aegypti in agreement with the (World Health Organization, 2005) guidelines. The resistance of the strains characterized by determining the lethal concentrations (LCs) with the insecticide deltamethrin (positive control). The major compounds of the EOs from Piper species was identified by GC-MS. The EOs from Piper aduncum, P. marginatum, P. gaudichaudianum, P. crassinervium, and P. arboreum showed activity of up to 90% lethality at 100 ppm (concentration for screening). The activities of the EOs from these 6 species showed similar LCs in both susceptible strain (Rockefeller) and resistant strains (Pampulha and Venda Nova) to pyrethroids. The major compounds identified in the most active EO were available commercially and included β-Asarone, (E)-Anethole, (E)-β-Caryophyllene, γ-Terpinene, p-Cymene, Limonene, α-Pinene, and β-Pinene. Dillapiole was purified by from EO of P. aduncum. The phenylpropanoids [Dillapiole, (E)-Anethole and β-Asarone] and monoterpenes (γ-Terpinene, p-Cymene, Limonene, α-Pinene, and β-Pinene) showed larvicidal activity with mortality between 90 and 100% and could account for the toxicity of these EOs, but the sesquiterpene (E)-β-Caryophyllene, an abundant component in the EOs of P. hemmendorffii and P. crassinervium, did not show activity on the three populations of A. aegypti larvae at a concentration of 100 ppm. These results indicate that Piper's EOs should be further evaluated as a potential larvicide, against strains resistant to currently used pesticides, and the identification of phenylpropanoids and monoterpenes as the active compounds open the possibility to study their mechanism of action.

SummaryThe defensive chemistry and persistence of plant tissues determine their suitability and apparency -the likelihood of being discovered -to insect herbivores. As consumers of plant tissues with transient apparency, florivores and seed-feeders must frequently migrate between host plants to synchronize colonization with plant phenology. Aggregation pheromones could provide information-based solutions to finding ephemeral hosts, but little is known about plant-influenced variation in this form of chemical communication.Combining analytical chemistry, de novo synthesis and field ecology, we investigated the change in colonization of two sympatric host plants, Nicotiana attenuata and Nicotiana obtusifolia, which differ in apparency-related life history traits, by a heteropteran seed-feeder, Corimelaena extensa.We identified a novel pheromone released by C. extensa males -(5Z,8Z)-tetradeca-5,8-dienal -and performed field assays with the synthetic pheromone, showing that it stimulates the formation of feeding aggregations on the post-fire annual N. attenuata. Corimelaena extensa pheromone emission was 40-fold higher when feeding on N. attenuata compared with the perennial N. obtusifolia, as were adult fecundity and seed capsule content of the putative biosynthetic precursor, linoleic acid.Higher pheromone emission increases the apparency and colonization of the ephemeral, high-quality host N. attenuata. This plant-specific variation in insect signaling could facilitate host-finding by seed-feeders migrating between plant patches.

Aristolochic acids (AAs) are thought to be responsible for the chemical protection of the aposematic larvae Battus polydamas (L.) (Papilionidae: Troidini) against predators. These compounds are sequestered by larvae from their Aristolochia (Aristolochiaceae) host plants. Studying the role of the chemical protection of the second and fifth instars of B. polydamas against potential predators, we found that the consumption of larvae by the carpenter ant Camponotus crassus Mayr and young chicks Gallus gallus domesticus was dependent on larval developmental stage. Second instars were more preyed upon than fifth instars; however, the assassin bug Montina confusa Stål was not deterred by chemical defences of the fifth instar B. polydamas. Laboratory bioassays with carpenter ants and young chicks using palatable baits topically treated with a pure commercial mixture of AAs I and AAs II in concentrations up to 100 times those previously found in B. polydamas larvae showed no activity. Similar results were found in field bioassays, where palatable baits treated as above were exposed to the guild of predators that attack B. polydamas larvae and were also consumed irrespective of the commercial AA concentration used. These results suggest that the mixture of AAs I and AAs II have no defensive role against predators, at least against those investigated in the present work. Other compounds present in Aristolochia host plants such as O-glycosylated AAs; benzylisoquinoline alkaloids; and mono-, sesqui-, di-, and triterpenes, which can be sequestered by Troidini, could act as deterrents against predators.