Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

Distinct nutritional and endocrine regulation of prothoracic gland activities underlies divergent life history strategies in Manduca sexta and Drosophila melanogaster.

Life history trade-offs lead to various strategies that maximize fitness, but the developmental mechanisms underlying these alternative strategies continue to be poorly understood. In insects, trade-offs exist between size and developmental time. Recent studies in the fruit fly Drosophila melanogaster have suggested that the steroidogenic prothoracic glands play a key role in determining the timing of metamorphosis. In this study, the nutrient-dependent growth and transcriptional activation of prothoracic glands were studied in D. melanogaster and the tobacco hornworm Manduca sexta. In both species, minimum viable weight (MVW) was associated with activation of ecdysteroid biosynthesis genes and growth of prothoracic gland cells. However, the timing of MVW attainment in M. sexta is delayed by the presence of the sesquiterpenoid hormone, juvenile hormone (JH), whereas in D. melanogaster it is not. Moreover, in D. melanogaster, the transcriptional regulation of ecdysteroidogenesis becomes nutrient-independent at the MVW/critical weight (CW) checkpoint. In contrast, in M. sexta, starvation consistently reduced transcriptional activation of ecdysteroid biosynthesis genes even after CW attainment, indicating that the nature of CW differs fundamentally between the two species. In D. melanogaster, the prothoracic glands dictate the timing of metamorphosis even in the absence of nutritional inputs, whereas in M. sexta, prothoracic gland activity is tightly coupled to the nutritional status of the body, thereby delaying the onset of metamorphosis before CW attainment. We propose that selection for survival under unpredictable nutritional availability leads to the evolution of increased modularity in both morphological and endocrine traits.

Contrasting Roles of Cannabidiol as an Insecticide and Rescuing Agent for Ethanol-induced Death in the Tobacco Hornworm Manduca sexta.

Cannabis sativa, also known as marijuana or hemp, produces a non-psychoactive compound cannabidiol (CBD). To investigate the defensive role of CBD, a feeding preference assay was performed with tobacco hornworm Manduca sexta. The larvae clearly show feeding preference towards the Cannabis tissue containing low CBD over high CBD. While the larva avoided the high CBD diet, we investigated detrimental effects of CBD in the insects' diet. Contrasted to the performance on low CBD-infused artificial diet (AD), larvae reared on the high CBD diet suffer significantly reduced growth and increased mortality. Through testing different carriers, we found that the increase of EtOH in the diet is negatively correlated with insect development and behaviors. Notably, CBD treatment significantly improved ethanol-intoxicated larval survival rate by 40% and also improved diet searching activity, resulting in increased diet consumption. Electrophysiology results revealed that the CBD-treated ganglia had delayed but much larger response with electric stimuli in comparison to the larvae reared on AD only and EtOH-added diet. Our results show CBDs' defensive role against pest insects, which suggests its possible use as an insecticide. We also provide evidence that CBD alleviates alcohol-induced stress; consequently, improving the performance and viability of M. sexta larvae.

An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco.

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining, we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(-)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions. We used these lines to test ovipositing moths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but the magnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity.

Cry1Ac production is costly for native plants attacked by non-Cry1Ac-targeted herbivores in the field.

Plants are the primary producers in most terrestrial ecosystems and have complex defense systems to protect their produce. Defense-deficient, high-yielding agricultural monocultures attract abundant nonhuman consumers, but are alternatively defended through pesticide application and genetic engineering to produce insecticidal proteins such as Cry1Ac (Bacillus thuringiensis). These approaches alter the balance between yield protection and maximization but have been poorly contextualized to known yield-defense trade-offs in wild plants. The native plant Nicotiana attenuata was used to compare yield benefits of plants transformed to be defenseless to those with a full suite of naturally evolved defenses, or additionally transformed to ectopically produce Cry1Ac. An insecticide treatment allowed us to examine yield under different herbivore loads in N. attenuata's native habitat. Cry1Ac, herbivore damage, and growth parameters were monitored throughout the season. Biomass and reproductive correlates were measured at season end. Non-Cry1Ac-targeted herbivores dominated on noninsecticide-treated plants, and increased the yield drag of Cry1Ac-producing plants in comparison with endogenously defended or undefended plants. Insecticide-sprayed Cry1Ac-producing plants lagged less in stalk height, shoot biomass, and flower production. In direct comparison with the endogenous defenses of a native plant, Cry1Ac production did not provide yield benefits for plants under observed herbivore loads in a field study.

Plant-mediated RNAi silences midgut-expressed genes in congeneric lepidopteran insects in nature.

BACKGROUND: Plant-mediated RNAi (PMRi) silencing of insect genes has enormous potential for crop protection, but whether it works robustly under field conditions, particularly with lepidopteran pests, remains controversial. Wild tobacco Nicotiana attenuata and cultivated tobacco (N. tabacum) (Solanaceae) is attacked by two closely related specialist herbivores Manduca sexta and M. quinquemaculata (Lepidoptera, Sphingidae). When M. sexta larvae attack transgenic N. attenuata plants expressing double-stranded RNA(dsRNA) targeting M. sexta's midgut-expressed genes, the nicotine-ingestion induced cytochrome P450 monooxygenase (invert repeat (ir)CYP6B46-plants) and the lyciumoside-IV-ingestion induced ß-glucosidase1 (irBG1-plants), these larval genes which are important for the larvae's response to ingested host toxins, are strongly silenced. RESULTS: Here we show that the PMRi procedure also silences the homologous genes in native M. quinquemaculata larvae feeding on irCYP6B46 and irBG1-transgenic N. attenuata plants in nature. The PMRi lines shared 98 and 96% sequence similarity with M. quinquemaculata homologous coding sequences, and CYP6B46 and BG1 transcripts were reduced by ca. 90 and 80%, without reducing the transcripts of the larvae's most similar, potential off-target genes. CONCLUSIONS: We conclude that the PMRi procedure can robustly and specifically silence genes in native congeneric insects that share sufficient sequence similarity and with the careful selection of targets, might protect crops from attack by congeneric-groups of insect pests.

ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth.

Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.

A Floral Fragrance, Methyl Benzoate, is An Efficient Green Pesticide.

Over-reliance on synthetic pesticides in insect pest control has caused widespread public and scientific concerns for human health and the environment, especially since many insect pests have already developed resistances to conventional pesticides and Bt products. For this reason, there is a considerable interest in development of alternative control methods for insect pest management. Based on laboratory studies, we report that methyl benzoate (MB), a naturally-occurring compound in many plants, may possess toxicity against various stages of a variety of insect pests, including the brown marmorated stinkbug, Halyomorpha halys, diamondback moth, Plutella xylostella, and tobacco hornworm, Manduca sexta, as well as the spotted wing drosophila, Drosophila suzukii. Based on our laboratory toxicity data, MB was at least 5 to 20 times more toxic than the conventional pyrethroid (ß-cyfluthrin), sulfur &pyrethrin mixture, and some organic commercial products available on the market against H. halys, P. xylostella, and M. sexta, eggs. Because MB is considered an environment-friendly, it has great potential to be used as an alternative tool to synthetic pesticide for insect pest management in crop production, thereby, reducing threats to natural ecosystems and human health caused by over-application of conventional synthetic pesticides.

EPR Studies of V-ATPase with Spin-Labeled Inhibitors DCC and Archazolid: Interaction Dynamics with Proton Translocating Subunit†c.

Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N'-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunit c of these medicinally important hetero-multimeric proton-translocating proteins. This study also demonstrates the usefulness of natural product derived spin labels as tools in medicinal chemistry.

A Nicotiana attenuata cell wall invertase inhibitor (NaCWII) reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants.

Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source-sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory.

Herbivore associated elicitor-induced defences are highly specific among closely related Nicotiana species.

BACKGROUND: Herbivore-induced defence responses are often specific - different herbivores induce different defence responses in plants - and their specificity is largely mediated by chemical cues (herbivore-associated elicitors: HAEs) in insect oral or oviposition secretions. However, the specificity and the mechanisms of HAE-induced defence have not been investigated in the context of the evolutionary relationships among plant species. Here we compare the responses of six closely related Nicotiana species to a synthetic elicitor, N-linolenoyl-glutamic acid (C18:3-Glu) and HAE of two insect herbivores (the Solanaceae specialist Manduca sexta and generalist Spodoptera littoralis). RESULTS: HAE-induced defences are highly specific among closely related Nicotiana species at three perspectives. 1) A single Nicotiana species can elicit distinct responses to different HAEs. N. pauciflora elicited increased levels of JA and trypsin proteinase inhibitors (TPI) in response to C18:3-Glu and the oral secretions of M. sexta (OS Ms ) but not to oral secretions of S. littoralis (OS Sl ). In contrast, N. miersii only responded to OS Sl but not to the other two HAEs. The specific responses to different HAEs in Nicotiana species are likely due to the perception by the plant of each specific component of the HAE. 2) One HAE can induce different defence responses among closely related Nicotiana species. OS Ms and C18:3-Glu induced JA and TPI accumulations in N. linearis, N. attenuata, N. acuminata and N. pauciflora, but not in N. miersii and N. obtusifolia. 3) The effect of HAE-induced defences differ for the Solanaceae specialist M. sexta and the generalist S. littoralis. Among the four tested Nicotiana species, while the growth rate of M. sexta was only reduced by the induced defences elicited by C18:3-Glu; the growth rate of S. littoralis can be reduced by the induced defences elicited by all three HAEs. This is likely due to differences in the susceptibility of the specialist M. sexta and generalist S. littoralis to induced defences of their host. CONCLUSIONS: Closely related Nicotiana species elicit highly specific defence responses to herbivore associated elicitors and provide an ideal framework for investigating the molecular mechanisms and evolutionary divergence of induced resistance in plants.

Use of molecular and in silico bioinformatic tools to investigate pesticide binding to insect (Lepidoptera) phenoloxidases (PO): insights to toxicological aspects.

In the present work, the promising bioinformatic tools, based on structure-affinity analysis, allowed to screen several pesticides supposed to bind to the insect immune Phenoloxidases (PO). First, the binding of aminoparathion, a reference compound, to the PO was structurally (3D) validated in accordance with previous reports. Second, using the same docking conditions, a range of pesticides was checked for their binding ability to the crystal 3D structure (PDB 3HSS) of the insect Manduca sexta (Lepidoptera) PO. The obtained data showed that many of the tested pesticides were able to bind, in silico, to M. sexta PO. The combination of in vitro (chemical and biochemical) and in silico (automated docking) approaches was found advantageous to elucidate the behavior of phenolic pesticides as substrate-analogues when binding to the active site of insect POs. Our findings emphasize new ecotoxicological aspects of pesticide residues in the agro-chemical and environmental circles.

RuBPCase activase (RCA) mediates growth-defense trade-offs: silencing RCA redirects jasmonic acid (JA) flux from JA-isoleucine to methyl jasmonate (MeJA) to attenuate induced defense responses in Nicotiana attenuata.

• RuBPCase activase (RCA), an abundant photosynthetic protein, is strongly down-regulated in response to Manduca sexta's oral secretion (OS) in Nicotiana attenuata. RCA-silenced plants are impaired not only in photosynthetic capacity and growth, but also in jasmonic acid-isoleucine (JA-Ile) signaling, and herbivore resistance mediated by JA-Ile-dependent defense traits. These responses are consistent with a resource-based growth-defense trade-off. • As JA + Ile supplementation of OS restored wild-type (WT) levels of JA-Ile, defenses and resistance to M. sexta, but OS supplemented individually with JA or Ile did not, the JA-Ile deficiency of RCA-silenced plants could not be attributed to lower JA or Ile pools or JAR4/6 conjugating activity. Similar levels of JA-Ile derivatives after OS elicitation indicated unaltered JA-Ile turnover, and lower levels of other JA conjugates ruled out competition from other conjugation reactions. • RCA-silenced plants accumulated more methyl jasmonate (MeJA) after OS elicitation, which corresponded to increased jasmonate methyltransferase (JMT) activity. RCA silencing phenocopies JMT overexpression, wherein elevated JMT activity redirects OS-elicited JA flux towards inactive MeJA, creating a JA sink which depletes JA-Ile and its associated defense responses. • Hence, RCA plays an additional non-photosynthetic role in attenuating JA-mediated defenses and their associated costs, potentially allowing plants to anticipate resource-based constraints on growth before they actually occur.

Toxicity and mode of action of insecticidal Cry1A proteins from Bacillus thuringiensis in an insect cell line, CF-1.

Bacillus thuringiensis Cry toxins are insecticidal proteins used to control insect pests. The interaction of Cry toxins with the midgut of susceptible insects is a dynamic process involving activation of the toxin, binding to midgut receptors in the apical epithelium and conformational changes in the toxin molecule, leading to pore formation and cell lysis. An understanding of the molecular events underlying toxin mode of action is essential for the continued use of Cry toxins. In this work, we examined the mechanism of action of Cry1A toxins in the lepidopteran cell line CF-1, using native Cry1Ab and mutant forms of this protein that interfer with different steps in the mechanism of action, specifically, receptor binding, oligomerization or pore formation. These mutants lost activity against both Manduca sexta larvae and CF-1 cells. We also analyzed a mutation created in domain I of Cry1Ab, in which helix α-1 and part of helix α-2 were deleted (Cry1AbMod). Cry1AbMod is able to oligomerize in the absence of toxin receptors, and although it shows reduced activity against some susceptible insects, it kills insect pests that have developed resistance to native Cry1Ab. Cry1AbMod showed enhanced toxicity to CF-1, suggesting that oligomerization of native Cry1Ab may be a limiting step in its activity against CF-1 cells. The toxicity of Cry1Ac and Cry1AcMod were also analyzed. Our results suggest that some of the steps in the mode of action of Cry1A toxins are conserved in vivo in insect midgut cells and in vitro in an established cell line, CF-1.

A characterization of the Manduca sexta serotonin receptors in the context of olfactory neuromodulation.

Neuromodulation, the alteration of individual neuron response properties, has dramatic consequences for neural network function and is a phenomenon observed across all brain regions and taxa. However, the mechanisms underlying neuromodulation are made complex by the diversity of neuromodulatory receptors expressed within a neural network. In this study we begin to examine the receptor basis for serotonergic neuromodulation in the antennal lobe of Manduca sexta. To this end we cloned all four known insect serotonin receptor types from Manduca (the Ms5HTRs). We used phylogenetic analyses to classify the Ms5HTRs and to establish their relationships to other insect serotonin receptors, other insect amine receptors and the vertebrate serotonin receptors. Pharmacological assays demonstrated that each Ms5HTR was selective for serotonin over other endogenous amines and that serotonin had a similar potency at all four Ms5HTRs. The pharmacological assays also identified several agonists and antagonists of the different Ms5HTRs. Finally, we found that the Ms5HT1A receptor was expressed in a subpopulation of GABAergic local interneurons suggesting that the Ms5HTRs are likely expressed heterogeneously within the antennal lobe based on functional neuronal subtype.

Inbreeding in horsenettle (Solanum carolinense) alters night-time volatile emissions that guide oviposition by Manduca sexta moths.

Plant volatiles serve as key foraging and oviposition cues for insect herbivores as well as their natural enemies, but little is known about how genetic variation within plant populations influences volatile-mediated interactions among plants and insects. Here, we explore how inbred and outbred plants from three maternal families of the native weed horsenettle (Solanum carolinense) vary in the emission of volatile organic compounds during the dark phase of the photoperiod, and the effects of this variation on the oviposition preferences of Manduca sexta moths, whose larvae are specialist herbivores of Solanaceae. Compared with inbred plants, outbred plants consistently released more total volatiles at night and more individual compounds-including some previously reported to repel moths and attract predators. Female moths overwhelmingly chose to lay eggs on inbred (versus outbred) plants, and this preference persisted when olfactory cues were presented in the absence of visual and contact cues. These results are consistent with our previous findings that inbred plants recruit more herbivores and suffer greater herbivory under field conditions. Furthermore, they suggest that constitutive volatiles released during the dark portion of the photoperiod can convey accurate information about plant defence status (and/or other aspects of host plant quality) to foraging herbivores.

Identification and profiling of miRNAs during herbivory reveals jasmonate-dependent and -independent patterns of accumulation in Nicotiana attenuata.

BACKGROUND: Plant microRNAs (miRNAs) play key roles in the transcriptional responses to environmental stresses. However, the role of miRNAs in responses to insect herbivory has not been thoroughly explored. To identify herbivory-responsive miRNAs, we identified conserved miRNAs in the ecological model plant Nicotiana attenuata whose interactions with herbivores have been well-characterized in both laboratory and field studies. RESULTS: We identified 59 miRNAs from 36 families, and two endogenous trans-acting small interfering RNAs (tasiRNA) targeted by miRNAs. We characterized the response of the precursor and mature miRNAs to simulated attack from the specialist herbivore Manduca sexta by quantitative PCR analysis and used ir-aoc RNAi transformants, deficient in jasmonate biosynthesis, to identify jasmonate-dependent and -independent miRNA regulation. Expression analysis revealed that groups of miRNAs and tasiRNAs were specifically regulated by either mechanical wounding or wounding plus oral secretions from M. sexta larvae, and these small RNAs were accumulated in jasmonate-dependent or -independent manners. Moreover, cDNA microarray analysis indicated that the expression patterns of the corresponding target genes were correlated with the accumulation of miRNAs and tasiRNAs. CONCLUSIONS: We show that a group of miRNAs and tasiRNAs orchestrates the expression of target genes involved in N. attenuata's responses to herbivore attack.

Bioaccumulation of gold nanomaterials by Manduca sexta through dietary uptake of surface contaminated plant tissue.

We investigated the potential for bioaccumulation of engineered nanomaterials (ENMs) by tobacco hornworm (Manduca sexta) caterpillars resulting from the ingestion of plant tissue surface contaminated with ENMs. Caterpillars were fed tomato leaf tissue that had been surface contaminated with 12 nm tannate coated Au ENMs. After dosing was complete, bulk Au concentrations in individual caterpillars were measured after 0, 1, 4, and 7 days of elimination. Growth, mortality, and ingestion rate were monitored. This experiment revealed (1) no evidence that caterpillars were affected by ingestion of ENM contaminated plant tissue; (2) low bioaccumulation factors (BAF = 0.16) compared to a previous study where hornworm caterpillars were fed plants that had previously bioaccumulated Au ENMs (BAF = 6.2-11.6); (3) inefficient elimination of accumulated Au ENMs not associated with hornworm gut contents; and (4) regional differences in translocation of Au ENMs into tissues surrounding the hornworm gut, possibly the result of the interaction between ENM surface chemistry and regional differences in hornworm gut chemistry. These data, along with previous findings, indicate that although ENMs resuspended from soil onto plant surfaces by wind, water, biota, and/or mechanical disturbances are bioavailable to terrestrial consumers, bioaccumulation efficiency may be much lower via this pathway than through direct trophic exposure.

Nicotiana attenuata α-DIOXYGENASE1 through its production of 2-hydroxylinolenic acid is required for intact plant defense expression against attack from Manduca sexta larvae.

Nicotiana attenuata α-DIOXYGENASE1 (α-DOX1) is an oxylipin-forming gene elicited during herbivory by fatty acid amino acid conjugates (FACs) contained in oral secretions of Manduca sexta. To understand the roles of Naα-DOX1 and its major product, 2-hydroxylinolenic acid (2-hydroxylinolenic acid), in N. attenuata's anti-herbivore defenses, we used a transgenic line specifically silenced in Naα-DOX1 expression (ir-α-dox1) and monitored 2-HOT production in M. sexta-damaged tissues and its role in influencing the production of direct defense compounds and resistance to this insect. Attack by M. sexta larvae amplified 2-HOT formation at the feeding sites; a reaction probably facilitated by Naα-DOX1's high pH optimum which allows 2-HOT formation to occur in the more alkaline conditions at the feeding sites or potentially in the insect mouth parts after the leaf tissue is ingested. Manduca sexta larvae performed better on ir-α-dox1 plants than on wild-type (WT) plants as a result of attenuated herbivory-specific JA and 2-HOT bursts as well as JA-inducible well-established defenses (nicotine, caffeoylputrescine and trypsin proteinase inhibitors). Repeated applications of 2-HOT to wounds before insect feeding partly amplified JA-controlled defenses and restored the resistance of ir-α-dox1 plants. We conclude that 2-HOT, produced by attack-activated α-DOX1 activity, participates in defense activation during insect feeding.

Nitric oxide has differential effects on currents in different subsets of Manduca sexta antennal lobe neurons.

Nitric oxide has been shown to regulate many biological systems including olfaction. In the moth olfactory system nitric oxide is produced in the antennal lobe in response to odor stimulation and has complex effects on the activity of both projection neurons and local interneurons. To examine the cell autonomous effects of nitric oxide on these cells, we used patch-clamp recording in conjunction with pharmacological manipulation of nitric oxide to test the hypothesis that nitric oxide differentially regulates the channel properties of these different antennal lobe neuron subsets. We found that nitric oxide caused increasing inward currents in a subset of projection neurons while the effects on local neurons were variable but consistent within identifiable morphological subtypes.