(+)-Catechin, epicatechin and epigallocatechin gallate are important inducible defensive compounds against Ectropis grisescens in tea plants.

The tea plant, Camellia sinensis (L.) O. Kuntze, is an economically important, perennial woody plant rich in catechins. Although catechins have been reported to play an important role in plant defences against microbes, their roles in the defence of tea plants against herbivores remain unknown. In this study, we allowed the larvae of Ectropis grisescens, a leaf-feeding pest, to feed on the plants, and alternatively, we wounded the plants and then treated them with E. grisescens oral secretions (WOS). Both approaches triggered jasmonic acid-, ethylene- and auxin-mediated signalling pathways; as a result, plants accumulated three catechin compounds: (+)-catechin, epicatechin and epigallocatechin. Not only was the mass of E. grisescens larvae fed on plants previously infested with E. grisescens or treated with WOS significantly lower than that of larvae fed on controls, but also artificial diet supplemented with epicatechin, (+)-catechin or epigallocatechin gallate reduced larval growth rates. In addition, the exogenous application of jasmonic acid, ethylene or auxin induced the biosynthesis of the three catechins, which, in turn, enhanced the resistance of tea plants to E. grisescens, leading to the coordination of the three signalling pathways. Our results suggest that the three catechins play an important role in the defences of tea plants against E. grisescens.

Reciprocal mutations of two multifunctional ß-amyrin synthases from Barbarea vulgaris shift α/ß-amyrin ratios.

In the wild cruciferous wintercress (Barbarea vulgaris), ß-amyrin-derived saponins are involved in resistance against insect herbivores like the major agricultural pest diamondback moth (Plutella xylostella). Enzymes belonging to the 2,3-oxidosqualene cyclase family have been identified and characterized in B. vulgaris G-type and P-type plants that differ in their natural habitat, insect resistance and saponin content. Both G-type and P-type plants possess highly similar 2,3-oxidosqualene cyclase enzymes that mainly produce ß-amyrin (Barbarea vulgaris Lupeol synthase 5 G-Type; BvLUP5-G) or α-amyrin (Barbarea vulgaris Lupeol synthase 5 P-Type; BvLUP5-P), respectively. Despite the difference in product formation, the two BvLUP5 enzymes are 98% identical at the amino acid level. This provides a unique opportunity to investigate determinants of product formation, using the B. vulgaris 2,3-oxidosqualene cyclase enzymes as a model for studying amino acid residues that determine differences in product formation. In this study, we identified two amino acid residues at position 121 and 735 that are responsible for the dominant changes in generated product ratios of ß-amyrin and α-amyrin in both BvLUP5 enzymes. These amino acid residues have not previously been highlighted as directly involved in 2,3-oxidosqualene cyclase product specificity. Our results highlight the functional diversity and promiscuity of 2,3-oxidosqualene cyclase enzymes. These enzymes serve as important mediators of metabolic plasticity throughout plant evolution.

The Jasmonic Acid Pathway Positively Regulates the Polyphenol Oxidase-Based Defense against Tea Geometrid Caterpillars in the Tea Plant (Camellia sinensis).

Polyphenol oxidases (PPOs) as inducible defense proteins, contribute to tea (Camellia sinensis) resistance against tea geometrid larvae (Ectropis grisescens), and this resistance has been associated with the jasmonic acid (JA) signaling by testing geometrid performance in our previous work. However, the regulation of PPO-based defense by JA and other hormone signaling underlying these defense responses is poorly understood. Here, we investigated the role of phytohormones in regulating the PPO response to tea geometrids. We profiled levels of defense hormones, PPO activity and CsPPO genes in leaves infested with tea geometrids. Then, hormone levels were manipulated by exogenous application of methyl jasmonate (MeJA), gibberellin acid (GA3), abscisic acid (ABA), JA biosynthesis inhibitors (sodium diethyldithiocarbamate trihydrate, DIECA and salicylhydroxamic acid, SHAM) and GA inhibitor (uniconazole, UNI). Upon geometrid attack, JA levels significantly increased, whereas GA levels notably decreased and ABA level was slightly decreased. And the PPO activity significantly increased in line with the transcript levels of CsPPO2 and CsPPO4 but not CsPPO1. There were an obvious antagonistic cross-talk between JA and GA signals and an association among JA signals, PPO response and herbivore resistance in tea plants. Pretreatment with MeJA increased PPO activity by activating the transcripts of CsPPO2 and CsPPO4, whereas application of JA inhibitor DIECA suppressed PPO activity. GA3 strongly enhanced PPO activity, but ABA did not alter PPO activity. These findings strongly suggest that JA is a central player in PPO-mediated tea resistance against tea geometrids in a manner that prioritizes defense over growth.

Role of Saponins in Plant Defense Against Specialist Herbivores.

The diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae) is a very destructive crucifer-specialized pest that has resulted in significant crop losses worldwide. DBM is well attracted to glucosinolates (which act as fingerprints and essential for herbivores in host plant recognition) containing crucifers such as wintercress, Barbarea vulgaris (Brassicaceae) despite poor larval survival on it due to high-to-low concentration of saponins and generally to other plants in the genus Barbarea. B. vulgaris build up resistance against DBM and other herbivorous insects using glucosinulates which are used in plant defense. Aside glucosinolates, Barbarea genus also contains triterpenoid saponins, which are toxic to insects and act as feeding deterrents for plant specialist herbivores (such as DBM). Previous studies have found interesting relationship between the host plant and secondary metabolite contents, which indicate that attraction or resistance to specialist herbivore DBM, is due to higher concentrations of glucosinolates and saponins in younger leaves in contrast to the older leaves of Barbarea genus. As a response to this phenomenon, herbivores as DBM has developed a strategy of defense against these plant biochemicals. Because there is a lack of full knowledge in understanding bioactive molecules (such as saponins) role in plant defense against plant herbivores. Thus, in this review, we discuss the role of secondary plant metabolites in plant defense mechanisms against the specialist herbivores. In the future, trials by plant breeders could aim at transferring these bioactive molecules against herbivore to cash crops.

Gastropods and Insects Prefer Different Solanum dulcamara Chemotypes.

Solanum dulcamara (Bittersweet nightshade) shows significant intraspecific variation in glycoalkaloid (GA) composition and concentration. We previously showed that constitutive differences in overall GA levels are correlated with feeding preference of the grey field slug (GFS; Deroceras reticulatum). One particularly preferred accession, ZD11, contained low GA levels, but high levels of previously unknown structurally related uronic acid conjugated compounds (UACs). Here we test whether different slug species as well as insect herbivores show similar feeding preferences among six S. dulcamara accessions with different GA chemotypes. In addition, we investigate whether slug feeding can lead to induced changes in the chemical composition and affect later arriving herbivores. A leaf disc assay using greenhouse-grown plants showed that three slug species similarly preferred accessions with low GA levels. Untargeted metabolomic analyses showed that previous slug feeding consistently increased the levels of N-caffeoyl-putrescine and a structurally related metabolite, but not the levels of GAs and UACs. Slug-induced responses only affected slug preference in one accession. A common garden experiment using the same six accessions revealed that ZD11 received the highest natural gastropod feeding damage, but suffered the lowest damage by specialist flea beetles. The latter preferred to feed on accessions with high GA levels. Our study indicates that different selection pressures imposed by generalist gastropods and specialist insects may explain part of the observed chemical diversity in S. dulcamara.

Snails prefer it sweet: A multifactorial test of the metal defence hypothesis.

Metal defence against insect herbivory in hyperaccumulator plants is well documented. However, there are contradictory results regarding protection against snails. According to the joint effects hypothesis, inorganic and organic defences cooperate in plant protection. To test this hypothesis, we explored the relationships between snail (Cantareus aspersus) feeding and multiple inorganic and organic leaf components in the Cd hyperaccumulator plant Noccaea praecox. Plants grouped by rosette size growing in nutrient solution supplemented or not with 50 µM Cd were offered to the snails. After 3 days of snail feeding, the plants and snails were analysed. In addition to Cd concentrations, we analysed leaves for nutritional factors (sugar and protein), defence-related compounds (glucosinolates, phenolics, tannins, salicylic acid and jasmonate) and essential mineral nutrients. Cadmium concentrations in the snails and in snail excrements were also analysed. Snails preferentially fed on plants grown without Cd. Medium-sized plants exposed to Cd were the least consumed. Snail excrements from this trial weighed less and had higher Cd concentrations than those from other treatments. Cadmium increased salicylate and jasmonate production. A positive relationship between jasmonate levels and the number of attacked leaves was found. Principal component analysis revealed that leaf sugar concentration was the main factor positively affecting snails' leaf consumption, while leaf Cd had a negative but weaker influence. In conclusion, leaf sugar concentration mainly governs snails' feeding preferences. High leaf Cd concentrations do not deter herbivores from attacking leaves, but they do reduce leaf consumption. Our results clearly support the joint effects hypothesis.

Interactive Responses of Solanum Dulcamara to Drought and Insect Feeding are Herbivore Species-Specific.

In nature, plants are frequently subjected to multiple biotic and abiotic stresses, resulting in a convergence of adaptive responses. We hypothesised that hormonal signalling regulating defences to different herbivores may interact with drought responses, causing distinct resistance phenotypes. To test this, we studied the hormonal and transcriptomic responses of Solanum dulcamara subjected to drought and herbivory by the generalist Spodoptera exigua (beet armyworm; BAW) or the specialist Leptinotarsa decemlineata (Colorado potato beetle; CPB). Bioassays showed that the performance of BAW, but not CPB, decreased on plants under drought compared to controls. While drought did not alter BAW-induced hormonal responses, it enhanced the CPB-induced accumulation of jasmonic acid and salicylic acid (SA), and suppressed ethylene (ET) emission. Microarray analyses showed that under drought, BAW herbivory enhanced several herbivore-induced responses, including cell-wall remodelling and the metabolism of carbohydrates, lipids, and secondary metabolites. In contrast, CPB herbivory enhanced several photosynthesis-related and pathogen responses in drought-stressed plants. This may divert resources away from defence production and increase leaf nutritive value. In conclusion, while BAW suffers from the drought-enhanced defences, CPB may benefit from the effects of enhanced SA and reduced ET signalling. This suggests that the fine-tuned interaction between the plant and its specialist herbivore is sustained under drought.

Oviposition by Spodoptera exigua on Solanum dulcamara Alters the Plant's Response to Herbivory and Impairs Larval Performance.

Plant resistance traits against insect herbivores are extremely plastic. Plants respond not only to the herbivory itself, but also to oviposition by herbivorous insects. How prior oviposition affects plant responses to larval herbivory is largely unknown. Combining bioassays and defense protein activity assays with microarray analyses and metabolite profiling, we investigated the impact of preceding oviposition on the interaction of Solanum dulcamara with the generalist lepidopteran herbivore Spodoptera exigua at the levels of the plant's resistance, transcriptome and metabolome. We found that oviposition increased plant resistance to the subsequent feeding larvae. While constitutive and feeding-induced levels of defensive protease inhibitor activity remained unaffected, pre-exposure to eggs altered S. dulcamara's transcriptional and metabolic response to larval feeding in leaves local and systemic to oviposition. In particular, genes involved in phenylpropanoid metabolism were more strongly expressed in previously oviposited plants, which was reflected by reciprocal changes of primary metabolites upstream and within these pathways. Our data highlight that plants integrate signals from non-threatening life stages of their natural enemies to optimize their response when they become actually attacked. The observed transcriptional and metabolic reshaping of S. dulcamara's response to S. exigua herbivory suggests a role of phenylpropanoids in oviposition-primed plant resistance.

New Antifeedant Grayanane Diterpenoids from the Flowers of Pieris formosa.

Three new grayanane diterpenoids, pierisoids C‒E (1-3), as well as 10 known ones (4-13), were evaluated from the flowers of Pieris formosa, which is used as an insecticide in rural areas of China. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analyses. Significant antifeedant activity of 1, 3 and 10 against the beet armyworm (Spodoptera exigua) was found, indicating that these diterpenoids might also be involved in the plant defense against insect herbivores.

Pervasive interactions between foliar microbes and soil nutrients mediate leaf production and herbivore damage in a tropical forest.

Producing and retaining leaves underlie the performance and survivorship of seedlings in deeply shaded tropical forests. These habitats are characterized by conditions ideal for foliar bacteria, which can be potent plant pathogens. Leaf production, retention and susceptibility to enemies may ultimately depend upon interactions among soil nutrients and foliar microbes, yet this has never been tested. We experimentally evaluated the degree that foliar bacteria and soil resource supply mediate leaf dynamics for five common tree species (five different families) in a Panamanian forest. We reduced foliar bacteria with antibiotics for 29 months and measured leaf production, retention and damage for seedlings nested within a replicated 15-yr factorial nutrient enrichment experiment (nitrogen, N; phosphorus, P; potassium, K). Our results demonstrate that when we applied antibiotics, soil nutrients - particularly N - always regulated seedling leaf production (and to a lesser extent herbivore damage) for all five tree species. In addition, it was common for two macronutrients together to negate or completely reverse the impact of applying either one alone. Our findings of frequent plant-microbe-nutrient interactions are novel and suggest that these interactions may reinforce plant species-environment associations, thereby creating a fairly cryptic and fine-scale dimension of niche differentiation for coexisting tree species.

Solanum dulcamara's response to eggs of an insect herbivore comprises ovicidal hydrogen peroxide production.

Plants can respond to insect oviposition, but little is known about which responses directly target the insect eggs and how. Here, we reveal a mechanism by which the bittersweet nightshade Solanum dulcamara kills the eggs of a generalist noctuid herbivore. The plant responded at the site of oviposition by Spodoptera exigua with formation of neoplasms and chlorotic tissue, accumulation of reactive oxygen species and induction of defence genes and proteins. Transcriptome analysis revealed that these responses were reflected in the transcriptional reprogramming of the egg-laden leaf. The plant-mediated egg mortality on S. dulcamara was not present on a genotype lacking chlorotic leaf tissue at the oviposition sites on which the eggs are exposed to less hydrogen peroxide. As exposure to hydrogen peroxide increased egg mortality, while catalase supplementation prevented the plants from killing the eggs, our results suggest that reactive oxygen species formation directly acts as an ovicidal plant response of S. dulcamara.

Influence of Host-Plant Surface Chemicals on the Oviposition of the Cereal Stemborer Busseola Fusca.

The chemical composition of plant surfaces plays a role in selection of host plants by herbivorous insects. Once the insect reaches the plant, these cues determine host acceptance. Laboratory studies have shown that the stem borer Busseola fusca (Lepidoptera: Noctuidae), an important pest of sorghum and maize in sub-Saharan Africa, is able to differentiate between host and non-host plant species. However, no information is available on the cues used by this insect to seek and accept the host plant. Thus, the role of surface phytochemical stimuli on host selection and oviposition by B. fusca was studied in the laboratory using two host plants, sorghum, Sorghum bicolor, and maize, Zea mays, and one non-host plant, Napier grass, Pennisetum purpureum. The numbers of eggs and egg masses deposited on the three plant species were compared first under no-choice and choice conditions. In both cases, more eggs and egg masses were laid on maize and sorghum than on the non-host. Artificial surrogate stems treated with a water or chloroform surface extract of each plant were then compared with surrogate stems treated with, respectively, water or chloroform as controls, under similar conditions. Surrogate stems treated with plant water extracts did not show an increase in oviposition when compared to controls, indicating that the major compounds in these extracts, i.e., simple sugars and free amino acids, are not significantly responsible for the oviposition preference. By contrast, a chloroform extract of sorghum enhanced oviposition on the surrogate stems compared to the control, while those of maize and Napier grass showed no significant effects. Analysis of the chloroform extract of sorghum showed higher amounts of α-amyrin, ß-amyrin, and n-nonacosane compared to those of maize and Napier grass. A blend of the three chemicals significantly increased oviposition compared to the chloroform-treated control, indicating that these compounds are part of the surface chemical signature of the plant responsible for host recognition and oviposition by B. fusca.

A specialist herbivore pest adaptation to xenobiotics through up-regulation of multiple Cytochrome P450s.

The adaptation of herbivorous insects to their host plants is hypothesized to be intimately associated with their ubiquitous development of resistance to synthetic pesticides. However, not much is known about the mechanisms underlying the relationship between detoxification of plant toxins and synthetic pesticides. To address this knowledge gap, we used specialist pest Colorado potato beetle (CPB) and its host plant, potato, as a model system. Next-generation sequencing (454 pyrosequencing) was performed to reveal the CPB transcriptome. Differential expression patterns of cytochrome P450 complement (CYPome) were analyzed between the susceptible (S) and imidacloprid resistant (R) beetles. We also evaluated the global transcriptome repertoire of CPB CYPome in response to the challenge by potato leaf allelochemicals and imidacloprid. The results showed that more than half (51.2%) of the CBP cytochrome P450 monooxygenases (P450s) that are up-regulated in the R strain are also induced by both host plant toxins and pesticide in a tissue-specific manner. These data suggest that xenobiotic adaptation in this specialist herbivore is through up-regulation of multiple P450s that are potentially involved in detoxifying both pesticide and plant allelochemicals.

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.

Testing Dose-Dependent Effects of the Nectar Alkaloid Anabasine on Trypanosome Parasite Loads in Adult Bumble Bees.

The impact of consuming biologically active compounds is often dose-dependent, where small quantities can be medicinal while larger doses are toxic. The consumption of plant secondary compounds can be toxic to herbivores in large doses, but can also improve survival in parasitized herbivores. In addition, recent studies have found that consuming nectar secondary compounds may decrease parasite loads in pollinators. However, the effect of compound dose on bee survival and parasite loads has not been assessed. To determine how secondary compound consumption affects survival and pathogen load in Bombus impatiens, we manipulated the presence of a common gut parasite, Crithidia bombi, and dietary concentration of anabasine, a nectar alkaloid produced by Nicotiana spp. using four concentrations naturally observed in floral nectar. We hypothesized that increased consumption of secondary compounds at concentrations found in nature would decrease survival of uninfected bees, but improve survival and ameliorate parasite loads in infected bees. We found medicinal effects of anabasine in infected bees; the high-anabasine diet decreased parasite loads and increased the probability of clearing the infection entirely. However, survival time was not affected by any level of anabasine concentration, or by interactive effects of anabasine concentration and infection. Crithidia infection reduced survival time by more than two days, but this effect was not significant. Our results support a medicinal role for anabasine at the highest concentration; moreover, we found no evidence for a survival-related cost of anabasine consumption across the concentration range found in nectar. Our results suggest that consuming anabasine at the higher levels of the natural range could reduce or clear pathogen loads without incurring costs for healthy bees.

Meta-transcriptomics indicates biotic cross-tolerance in willow trees cultivated on petroleum hydrocarbon contaminated soil.

BACKGROUND: High concentrations of petroleum hydrocarbon (PHC) pollution can be hazardous to human health and leave soils incapable of supporting agricultural crops. A cheap solution, which can help restore biodiversity and bring land back to productivity, is cultivation of high biomass yielding willow trees. However, the genetic mechanisms which allow these fast-growing trees to tolerate PHCs are as yet unclear. METHODS: Salix purpurea 'Fish Creek' trees were pot-grown in soil from a former petroleum refinery, either lacking or enriched with C10-C50 PHCs. De novo assembled transcriptomes were compared between tree organs and impartially annotated without a priori constraint to any organism. RESULTS: Over 45% of differentially expressed genes originated from foreign organisms, the majority from the two-spotted spidermite, Tetranychus urticae. Over 99% of T. urticae transcripts were differentially expressed with greater abundance in non-contaminated trees. Plant transcripts involved in the polypropanoid pathway, including phenylalanine ammonia-lyase (PAL), had greater expression in contaminated trees whereas most resistance genes showed higher expression in non-contaminated trees. CONCLUSIONS: The impartial approach to annotation of the de novo transcriptomes, allowing for the possibility for multiple species identification, was essential for interpretation of the crop's response treatment. The meta-transcriptomic pattern of expression suggests a cross-tolerance mechanism whereby abiotic stress resistance systems provide improved biotic resistance. These findings highlight a valuable but complex biotic and abiotic stress response to real-world, multidimensional contamination which could, in part, help explain why crops such as willow can produce uniquely high biomass yields on challenging marginal land.

Effect of Cyantraniliprole on Probing Behavior of the Potato Psyllid (Hemiptera: Triozidae) as Measured by the Electrical Penetration Graph Technique.

Zebra chip (ZC), an economically important disease of potato, is caused by 'Candidatus Liberibacter solanacearum' (Lso) transmitted by the potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae). Currently, using insecticides against potato psyllid is the only means to manage ZC. However, the ability of the potato psyllid to rapidly transmit Lso represents a substantial challenge in preventing the spread of ZC. Cyantraniliprole, a novel second-generation anthranilic diamide insecticide has been shown to deter insect feeding and reduce disease transmission. During this study, the effect of cyantraniliprole on potato psyllid probing behavior was assessed using electrical penetration graph technology and compared with abamectin, a commonly used insecticide to control potato psyllid. Results showed that both cyantraniliprole and abamectin significantly deterred probing behavior of the potato psyllid. Average duration of intercellular stylet penetration on cyantraniliprole- and abamectin-treated and untreated control plants was 2.36, 1.80, and 9.15 h, respectively. It took psyllids 1.82, 1.10, and 2.42 h to reach the xylem of cyantraniliprole- and abamectin-treated and untreated plants, respectively. Xylem sap ingestion duration averaged 0.53, 0.57, and 3.66 h on cyantraniliprole- and abamectin-treated and untreated controls, respectively. None of the psyllids exposed to insecticide-treated plants reached the phloem tissue, except one that bypassed the xylem. The insects completely ceased probing after 4.44 and 3.64 h on cyantraniliprole- and abamectin-treated plants, respectively, in contrast with those on untreated plants that probed throughout the entire 24-h experiment duration. These results indicate that cyantraniliprole is as effective as abamectin in deterring potato psyllid feeding and could significantly reduce transmission of Lso and the spread of ZC.

Effects of Oral Exposure to Fungicides on Honey Bee Nutrition and Virus Levels.

Sublethal exposure to fungicides can affect honey bees (Apis mellifera L.) in ways that resemble malnutrition. These include reduced brood rearing, queen loss, and increased pathogen levels. We examined the effects of oral exposure to the fungicides boscalid and pyraclostrobin on factors affecting colony nutrition and immune function including pollen consumption, protein digestion, hemolymph protein titers, and changes in virus levels. Because the fungicides are respiratory inhibitors, we also measured ATP concentrations in flight muscle. The effects were evaluated in 3- and 7-d-old worker bees at high fungicide concentrations in cage studies, and at field-relevant concentrations in colony studies. Though fungicide levels differed greatly between the cage and colony studies, similar effects were observed. Hemolymph protein concentrations were comparable between bees feeding on pollen with and without added fungicides. However, in both cage and colony studies, bees consumed less pollen containing fungicides and digested less of the protein. Bees fed fungicide-treated pollen also had lower ATP concentrations and higher virus titers. The combination of effects we detected could produce symptoms that are similar to those from poor nutrition and weaken colonies making them more vulnerable to loss from additional stressors such as parasites and pathogens.

Spatial and Temporal Potato Intensification Drives Insecticide Resistance in the Specialist Herbivore, Leptinotarsa decemlineata.

Landscape-scale intensification of individual crops and pesticide use that is associated with this intensification is an emerging, environmental problem that is expected to have unequal effects on pests with different lifecycles, host ranges, and dispersal abilities. We investigate if intensification of a single crop in an agroecosystem has a direct effect on insecticide resistance in a specialist insect herbivore. Using a major potato pest, Leptinotarsa decemlineata, we measured imidacloprid (neonicotinoid) resistance in populations across a spatiotemporal crop production gradient where potato production has increased in Michigan and Wisconsin, USA. We found that concurrent estimates of area and temporal frequency of potato production better described patterns of imidacloprid resistance among L. decemlineata populations than general measures of agricultural production (% cropland, landscape diversity). This study defines the effects individual crop rotation patterns can have on specialist herbivore insecticide resistance in an agroecosystem context, and how impacts of intensive production can be estimated with general estimates of insecticide use. Our results provide empirical evidence that variation in the intensity of neonicotinoid-treated potato in an agricultural landscape can have unequal impacts on L. decemlineata insecticide insensitivity, a process that can lead to resistance and locally intensive insecticide use. Our study provides a novel approach applicable in other agricultural systems to estimate impacts of crop rotation, increased pesticide dependence, insecticide resistance, and external costs of pest management practices on ecosystem health.

Identification of plant compounds that disrupt the insect juvenile hormone receptor complex.

Insects impact human health through vector-borne diseases and cause major economic losses by damaging crops and stored agricultural products. Insect-specific growth regulators represent attractive control agents because of their safety to the environment and humans. We identified plant compounds that serve as juvenile hormone antagonists (PJHANs). Using the yeast two-hybrid system transformed with the mosquito JH receptor as a reporter system, we demonstrate that PJHANs affect the JH receptor, methoprene-tolerant (Met), by disrupting its complex with CYCLE or FISC, formation of which is required for mediating JH action. We isolated five diterpene secondary metabolites with JH antagonist activity from two plants: Lindera erythrocarpa and Solidago serotina. They are effective in causing mortality of mosquito larvae at relatively low LD50 values. Topical application of two diterpenes caused reduction in the expression of Met target genes and retardation of follicle development in mosquito ovaries. Hence, the newly discovered PJHANs may lead to development of a new class of safe and effective pesticides.