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Atmospheric gases, such as carbon dioxide (CO 2 ) and ozone (O 3 ), influence plant-insect interactions, with variable effects. The few studies that have investigated the direct effects of elevated CO 2 (eCO 2 ; 750-900 ppm) or elevated O 3 (eO 3 ; 60-200 ppb) on insects have shown mixed results. Instead, most research has focused on the indirect effects through changes in the host plant. In general, the lower nitrogen levels in C3 brassicaceous plants grown at eCO 2 negatively affect insects and may result in compensatory feeding. Phytohormones involved in plant resistance may be altered by eCO 2 or eO 3 . For example, stress-related jasmonate levels, which lead to induced resistance against chewing herbivores, are weakened at eCO 2 . In general, eCO 2 does not affect herbivore-induced plant volatiles, which remain attractive to natural enemies. However, floral volatiles and herbivore-induced plant volatiles may be degraded by O 3 , affecting pollination and foraging natural enemy behavior. Thus, eCO 2 and eO 3 alter plant-insect interactions; however, many aspects remain poorly understood.

Background: Integrated Pest Management (IPM) seeks to combine multiple management strategies for optimal pest control. One method that is successfully employed in IPM is the use of beneficial organisms. However, in severe circumstances when pest insects exceed threshold limits, insecticides may still need to be implemented. Thus, understanding the effects of insecticides on biocontrol agents, such as parasitoid wasps, is paramount to ensure sustainable agroecosystems. Sublethal effects of the bioinsecticide spinosyn, a mixture of the bacterial Saccharopolyspora spinosa (Mertz and Yao) fermentation products spinosyn A and D, on eggs of Trichoplusia ni (Hübner), a cruciferous crop pest, and its egg parasitoid Trichogramma brassicae (Bezdenko) was investigated., Results: The LC 50 for spinosyn A and D (dissolved in ethanol) on T. ni eggs is 54 ng mL -1 . Transcriptomics on caterpillars (1st and 3rd instars) that hatched from eggs treated with sublethal concentrations of spinosyn identified the upregulation of several genes encoding proteins that may be involved in insecticide resistance including detoxification enzymes, such as cytochrome P 450 s, glutathione S-transferases and esterases. Sublethal T. ni egg treatments did not affect parasitoid emergence, however, there was a marked increase in the size of T. brassicae hind tibia and wings that emerged from spinosyn-treated eggs., Conclusions: For the caterpillar, treatment of eggs with sublethal concentrations of spinosyn may induce insecticide resistance mechanisms. For the parasitoids, their increased size when reared in spinosyn-treated eggs suggests that the emerged wasps may have higher performance. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

In wounded Arabidopsis thaliana leaves, four 13 S -lipoxygenases (AtLOX2, AtLOX3, AtLOX4, AtLOX6) act in a hierarchical manner to contribute to the jasmonate burst. This leads to defense responses with LOX2 playing an important role in plant resistance against caterpillar herb-ivory. In this study, we sought to characterize the impact of AtLOX2 on wound-induced phytohormonal and transcriptional responses to foliar mechanical damage using wildtype (WT) and lox2 mutant plants. Compared with WT, the lox2 mutant had higher constitutive levels of the phytohormone salicylic acid (SA) and enhanced expression of SA-responsive genes. This suggests that AtLOX2 may be involved in the biosynthesis of jasmonates that are involved in the antagonism of SA biosynthesis. As expected, the jasmonate burst in response to wounding was dampened in lox2 plants. Generally, 1 h after wounding, genes linked to jasmonate biosynthesis, jasmonate signaling attenuation and abscisic acid-responsive genes, which are primarily involved in wound sealing and healing, were differentially regulated between WT and lox2 mutants. Twelve h after wounding, WT plants showed stronger expression of genes associated with plant protection against insect herbivory. This study highlights the dynamic nature of jasmonate-responsive gene expression and the contribution of AtLOX2 to this pathway and plant resistance against insects.

Many plant species, particularly legumes, protect themselves with saponins. Previously, a correlation was observed between levels of oleanolic acid-derived saponins, such as hederagenin-derived compounds, in the legume Medicago truncatula and caterpillar deterrence. Using concentrations that reflect the foliar levels of hederagenin-type saponins, the sapogenin hederagenin was not toxic to 4th instar caterpillars of the cabbage looper Trichoplusia ni nor did it act as a feeding deterrent. Female caterpillars consumed more diet than males, presumably to obtain the additional nutrients required for oogenesis, and are, thus, exposed to higher hederagenin levels. When fed the hederagenin diet, male caterpillars expressed genes encoding trypsin-like proteins (LOC113500509, LOC113501951, LOC113501953, LOC113501966, LOC113501965, LOC113499659, LOC113501950, LOC113501948, LOC113501957, LOC113501962, LOC113497819, LOC113501946, LOC113503910) as well as stress-responsive (LOC113503484, LOC113505107) proteins and cytochrome P 450 6B2-like (LOC113493761) at higher levels than females. In comparison, female caterpillars expressed higher levels of cytochrome P 450 6B7-like (LOC113492289). Bioinformatic tools predict that cytochrome P 450 s could catalyze the oxygenation of hederagenin which would increase the hydrophilicity of the compound. Expression of a Major Facilitator Subfamily (MFS) transporter (LOC113492899) showed a hederagenin dose-dependent increase in gene expression suggesting that this transporter may be involved in sapogenin efflux. These sex-related differences in feeding and detoxification should be taken into consideration in insecticide evaluations to minimize pesticide resistance., (© 2024. The Author(s).)

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser 600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2 WT ). AtLOX2 WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser 600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2 S600D and Atlox2 S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2 S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser 600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser 600 is located at a critical position within the AtLOX2 structure and highlights how Ser 600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser 600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Before the end of the century, atmospheric carbon dioxide levels are predicted to increase to approximately 900 ppm. This will dramatically affect plant physiology and influence environmental interactions and, in particular, plant resistance to biotic stresses. This review is a broad survey of the current research on the effects of elevated CO 2 (eCO 2 ) on phytohormone-mediated resistance of C 3 agricultural crops and related model species to pathogens and insect herbivores. In general, while plants grown in eCO 2 often have increased constitutive and induced salicylic acid levels and suppressed induced jasmonate levels, there are exceptions that implicate other environmental factors, such as light and nitrogen fertilization in modulating these responses. Therefore, this review sets the stage for future studies to delve into understanding the mechanistic basis behind how eCO 2 will affect plant defensive phytohormone signaling pathways under future predicted environmental conditions that could threaten global food security to inform the best agricultural management practices.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

This article is part of the Top 10 Unanswered Questions in MPMI invited review series.The past few decades have seen major discoveries in the field of molecular plant-microbe interactions. As the result of technological and intellectual advances, we are now able to answer questions at a level of mechanistic detail that we could not have imagined possible 20 years ago. The MPMI Editorial Board felt it was time to take stock and reassess. What big questions remain unanswered? We knew that to identify the fundamental, overarching questions that drive our research, we needed to do this as a community. To reach a diverse audience of people with different backgrounds and perspectives, working in different areas of plant-microbe interactions, we queried the more than 1,400 participants at the 2019 International Congress on Molecular Plant-Microbe Interactions meeting in Glasgow. This group effort resulted in a list of ten, broad-reaching, fundamental questions that influence and inform our research. Here, we introduce these Top 10 unanswered questions, giving context and a brief description of the issues. Each of these questions will be the subject of a detailed review in the coming months. We hope that this process of reflecting on what is known and unknown and identifying the themes that underlie our research will provide a framework to use going forward, giving newcomers a sense of the mystery of the big questions and inspiring new avenues and novel insights.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The predicted future increase in tropospheric carbon dioxide (CO 2 ) levels will have major effects on C 3 plants and their interactions with other organisms in the biosphere. In response to attack by chewing arthropod herbivores or nectrotrophic pathogens, many plants mount a rapid and intense increase in jasmonate-related phytohormones that results in a robust defense response; however, previous studies have shown that C 3 plants grown at elevated CO 2 may have lower induced jasmonate levels, particularly in well nitrate-fertilized plants. Given the relationship between atmospheric CO 2 , photorespiration, cellular reductant and redox status, nitrogen assimilation and phytohormones, we compared wound-induced responses of the C 3 plant Arabidopsis thaliana . These plants were fertilized at two different rates (1 or 10 mM) with nitrate or ammonium and grown at ambient or elevated CO 2 . In response to artificial wounding, an increase in cellular oxidative status leads to a strong increase in jasmonate phytohormones. At ambient CO 2 , increased oxidative state of nitrate-fertilized plants leads to a robust 7- iso -jasmonyl-L-isoleucine increase; however, the strong fertilizer rate-associated increase is alleviated in plants grown at elevated CO 2 . As well, the changes in ascorbate in response to wounding and wound-induced salicylic acid levels may also contribute to the suppression of the jasmonate burst. Understanding the mechanism underlying the attenuation of the jasmonate burst at elevated CO 2 has important implications for fertilization strategies under future predicted climatic conditions., (Copyright © 2020 Martinez Henao, Demers, Grosser, Schedl, van Dam and Bede.)

Our understanding of microRNA (miRNA) regulation of gene expression and protein translation, as a critical area of cellular regulation, has blossomed in the last two decades. Recently, it has become apparent that in plant-insect interactions, both plants and insects use miRNAs to regulate their biological processes, as well as co-opting each others' miRNA systems. In this review article, we discuss the current paradigms of miRNA-mediated cellular regulation and provide examples of plant-insect interactions that utilize this regulation. Lastly, we discuss the potential biotechnological applications of utilizing miRNAs in agriculture.

Plant resistance mechanisms to insect herbivory can potentially be bred into crops as an important strategy for integrated pest management. Medicago truncatula ecotypes inoculated with the rhizobium Ensifer medicae (Sinorhizobium medica) WSM419 were screened for resistance to herbivory by caterpillars of the beet armyworm, Spodoptera exigua, through leaf and whole plant choice studies; TN1.11 and F83005.5 are identified as the least and most deterrent ecotypes, respectively. In response to caterpillar herbivory, both ecotypes mount a robust burst of plant defensive jasmonate phytohormones. Restriction of caterpillars to either of these ecotypes does not adversely affect pest performance. This argues for an antixenosis (deterrence) resistance mechanism associated with the F83005.5 ecotype. Unbiased metabolomic profiling identified strong ecotype-specific differences in metabolite profile, particularly in the content of oleanolic-derived saponins that may act as antifeedants. Compared to the more susceptible ecotype, F83005.5 has higher levels of oleanolic-type zanhic acid- and medicagenic acid-derived compounds. Together, these data support saponin-mediated deterrence as a resistance mechanism of the F83005.5 ecotype and implicates these compounds as potential antifeedants that could be used in agricultural sustainable pest management strategies.

Plants have multiple strategies, including phytochemicals that protect their vulnerable tissues against pathogens and herbivores. Dioon edule, like all cycads, possess unique azoxy-type compounds, azoxyglycosides (AZGs) as a chemical defense; however, the ontogenetic variability of these compounds in this long-lived cycad is unknown. Here, we investigated the effects of plant age, sex, genotype and individual heterozygosity on AZG levels in mature leaves of wild D. edule populations from eastern Mexico. Individuals were divided into three ontogenetic stages: seedlings, juveniles and adults. We established overall leaf quality by quantifying pigments associated with photosynthesis; chlorophyll a , chlorophyll b and lutein. Leaf chlorophyll a levels were higher in seedlings compared to adult cycads. Plants were genotyped using 11 microsatellite markers and foliar AZG levels were quantified by HPLC. AZG levels do not correlate with plant genotype or the individual's heterozygosity. Genetic analysis identified a distinction between lowland and highland individuals; foliar AZG levels were higher in lowland adult cycads compared to highland individuals. In both populations, the highest AZG levels were found in seedlings compared to adult cycads. These young cycads are highly reliant on their few leaves since seedlings bear one or two leaves for the first years of their life and, thus, are unlikely to recover from defoliation. The results suggest that cycad leaves with a greater nutritive content and a higher value for long-term survival are better protected with higher AZG levels. Female adult cycads have higher AZG levels compared to males, suggesting that the benefits of defense could also be linked to reproductive costs.

Background: Increased atmospheric carbon dioxide (CO2) levels predicted to occur before the end of the century will impact plant metabolism. In addition, nitrate availability will affect metabolism and levels of nitrogen-containing defense compounds, such as glucosinolates (GSLs). We compared Arabidopsis foliar metabolic profile in plants grown under two CO2 regimes (440 vs 880 ppm), nitrate fertilization (1 mM vs 10 mM) and in response to mechanical damage of rosette leaves., Results: Constitutive foliar metabolites in nitrate-limited plants show distinct global patterns depending on atmospheric CO2 levels; in contrast, plants grown under higher nitrate fertilization under elevated atmospheric CO2 conditions have a unique metabolite signature. Nitrate fertilization dampens the jasmonate burst in response to wounding in plants grown at elevated CO2 levels. Leaf GSL profile mirrors the jasmonate burst; in particular, indole GSLs increase in response to damage in plants grown at ambient CO2 but only in nitrate-limited plants grown under elevated CO2 conditions., Conclusions: This may reflect a reduced capacity of C3 plants grown under enriched CO2 and nitrate levels to signal changes in oxidative stress and has implications for future agricultural management practices.

Premise of the Study: Dioon (Zamiaceae) is an endangered North American cycad genus of evolutionary and ornamental value. We designed and validated a set of microsatellite markers from D. edule that can be used for population-level and conservation studies, and that transferred successfully to D. angustifolium, D. spinulosum, and D. holmgrenii., Methods and Results: We tested 50 primers from 80 microsatellite candidate loci in the OneKP D. edule transcriptome. Genotypes from 21 loci in 20 D. edule individuals revealed up to 14 alleles per locus and observed heterozygosity from 0.15 to 0.92; one locus was monomorphic. Seven of those 21 loci were polymorphic in D. angustifolium, D. spinulosum, and D. holmgrenii, with up to seven alleles, and an observed heterozygosity up to 0.89., Conclusions: The transcriptome-derived microsatellites generated here will serve as tools to advance population genetic studies and inform conservation strategies of Dioon, including the identification and origin of illegal plants in the cycad trade.

One or more effectors in the labial saliva (LS) of generalist Noctuid caterpillars activate plant signaling pathways to modulate jasmonate (JA)-dependent defense responses; however, the exact mechanisms involved have yet to be elucidated. A potential candidate in this phytohormone interplay is the ethylene (ET) signaling pathway. We compared the biochemical and molecular responses of the model legume Medicago truncatula and the ET-insensitive skl mutant to herbivory by fourth instar Spodoptera exigua (Hübner) caterpillars with intact or impaired LS secretions. Cellular oxidative stress increases rapidly after herbivory, as evidenced by changes in oxidized-to-reduced ascorbate (ASC) and glutathione (GSH) ratios. The caterpillar-specific increase in GSH ratios and the LS-specific increase in ASC ratios are alleviated in the skl mutant, indicating that ET signaling is required. Ten hours postherbivory, markers of the JA and JA/ET pathways are differentially expressed; MtVSP is induced and MtHEL is repressed in a caterpillar LS- and ET-independent manner. In contrast, expression of the classic marker of the systemic acquired resistance pathway, MtPR1, is caterpillar LS-dependent and requires ET signaling. Caterpillar LS further suppresses the induction of JA-related trypsin inhibitor activity in an ET-dependent manner. Findings suggest that ET is involved in the caterpillar LS-dependent, salicylic acid/NPR1-mediated attenuation of JA-dependent induced responses.

Premise of the Study: Slow-growing understory cycads invest heavily in defenses to protect the few leaves they produce annually. The Neotropical cycad Zamia stevensonii has chemical and mechanical barriers against insect herbivores. Mechanical barriers, such as leaf toughness, can be established only after the leaf has expanded. Therefore, chemical defenses may be important during leaf expansion. How changes in leaf traits affect the feeding activity of cycad specialist insects is unknown. We investigated leaf defenses and incidence of specialist herbivores on Z. stevensonii during the first year after leaf flush., Methods: Herbivore incidence, leaf production, and leaf traits that might affect herbivory-including leaf age, lamina thickness, resistance-to-fracture, work-to-fracture, trichome density, and chlorophyll, water, and toxic azoxyglycoside (AZG) content-were measured throughout leaf development. Principal component analysis and generalized linear models identified characteristics that may explain herbivore incidence., Key Results: Synchronized leaf development in Z. stevensonii is characterized by quick leaf expansion and delayed greening. Specialist herbivores feed on leaves between 10 and 100 d after flush and damage ∼37% of all leaflets produced. Young leaves are protected by AZGs, but these defenses rapidly decrease as leaves expand. Leaves older than 100 d are protected by toughness., Conclusions: Because AZG concentrations drop before leaves become sufficiently tough, there is a vulnerable period during which leaves are susceptible to herbivory by specialist insects. This slow-growing gymnosperm invests heavily in constitutive defenses against highly specialized herbivores, underlining the convergence in defensive syndromes by major plant lineages.

Upon insect herbivory, many plant species change the direction of metabolic flux from growth into defence. Two key pathways modulating these processes are the gibberellin (GA)/DELLA pathway and the jasmonate pathway. In this study, the effect of caterpillar herbivory on plant-induced responses was compared between wild-type Arabidopsis thaliana (L.) Heynh. and quad-della mutants that have constitutively elevated GA responses. The labial saliva (LS) of caterpillars of the beet armyworm, Spodoptera exigua, is known to influence induced plant defence responses. To determine the role of this herbivore cue in determining metabolic shifts, plants were subject to herbivory by caterpillars with intact or impaired LS secretions. In both wild-type and quad-della plants, a jasmonate burst is an early response to caterpillar herbivory. Negative growth regulator DELLA proteins are required for the LS-mediated suppression of hormone levels. Jasmonate-dependent marker genes are induced in response to herbivory independently of LS, with the exception of AtPDF1.2 that showed LS-dependent expression in the quad-della mutant. Early expression of the salicylic acid (SA)-marker gene, AtPR1, was not affected by herbivory which also reflected SA hormone levels; however, this gene showed LS-dependent expression in the quad-della mutant. DELLA proteins may positively regulate glucosinolate levels and suppress laccase-like multicopper oxidase activity in response to herbivory. The present results show a link between DELLA proteins and early, induced plant defences in response to insect herbivory; in particular, these proteins are necessary for caterpillar LS-associated attenuation of defence hormones.

Plant responses to insect herbivory are regulated through complex, hormone-mediated interactions. Some caterpillar species have evolved strategies to manipulate this system by inducing specific pathways that suppress plant defense responses. Effectors in the labial saliva (LS) secretions of Spodoptera exigua caterpillars are believed to induce the salicylic acid (SA) pathway to interfere with the jasmonic acid (JA) defense pathway; however, the mechanism underlying this subversion is unknown. Since noctuid caterpillar LS contains enzymes that may affect cellular redox balance, this study investigated rapid changes in cellular redox metabolites within 45 min after herbivory. Caterpillar LS is involved in suppressing the increase in oxidative stress that was observed in plants fed upon by caterpillars with impaired LS secretions. To further understand the link between cellular redox balance and plant defense responses, marker genes of SA, JA and ethylene (ET) pathways were compared in wildtype, the glutathione-compromised pad2-1 mutant and the tga2/5/6 triple mutant plants. AtPR1 and AtPDF1.2 showed LS-dependent expression that was alleviated in the pad2-1 and tga2/5/6 triple mutants. In comparison, the ET-dependent genes ERF1 expression showed LS-associated changes in both wildtype and pad2-1 mutant plants and the ORA 59 marker AtHEL had increased expression in response to herbivory, but a LS-dependent difference was not noted. These data support the model that there are SA/NPR1-, glutathione-dependent and ET-, glutathione-independent mechanisms leading to LS-associated suppression of plant induced defenses.

The primary function of salivary glands is fluid and protein secretion during feeding. Compared to mammalian systems, little is known about salivary protein secretion processes and the effect of diet on the salivary proteome in insect models. Therefore, the effect of diet nutritional quality on caterpillar labial salivary gland proteins was investigated using an unbiased global proteomic approach by nanoLC/ESI/tandem MS. Caterpillars of the beet armyworm, Spodoptera exigua Hübner, were fed one of three diets: an artificial diet containing their self-selected protein to carbohydrate (p:c) ratio (22p:20c), an artificial diet containing a higher nutritional content but the same p:c ratio (33p:30c) or the plant Medicago truncatula Gaertn. As expected, most identified proteins were associated with secretory processes and not influenced by diet. However, some diet-specific differences were observed. Nutrient stress-associated proteins, such as peptidyl-propyl cis-trans isomerase and glucose-regulated protein94/endoplasmin, and glyceraldehyde 3-phosphate dehydrogenase were identified in the labial salivary glands of caterpillars fed nutritionally poor diets, suggesting a link between nutritional status and vesicular exocytosis. Heat shock proteins and proteins involved in endoplasmic reticulum-associated protein degradation were also abundant in the labial salivary glands of these caterpillars. In comparison, proteins associated with development, such as arylphorin, were found in labial salivary glands of caterpillars fed 33p:30c. These results suggest that caterpillars fed balanced or nutritionally-poor diets have accelerated secretion pathways compared to those fed a protein-rich diet., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aulacoscelinae beetles have an ancient relationship with cycads (Cycadophyta: Zamiaceae), which contain highly toxic azoxyglycoside (AZG) compounds. How these "primitive" leaf beetles deal with such host-derived compounds remains largely unknown. Collections were made of adult Aulacoscelis appendiculata from Zamia cf. elegantissima in Panama, A. vogti from Dioon edule in Mexico, and Janbechynea paradoxa from Zamia boliviana in Bolivia. Total AZG levels were quantified in both cycad leaves and adult beetles by high performance liquid chromatography (HPLC). On average, cycad leaves contained between 0.5-0.8% AZG (frozen weight, FW), while adult beetles feeding on the same leaves contained even higher levels of the compounds (average 0.9-1.5% FW). High AZG levels were isolated from reflex bleeding secreted at the leg joints when beetles were disturbed. Nuclear magnetic resonance and mass spectroscopy identified two AZGs, cycasin and macrozamin, in the reflex bleeding; this is the first account of potentially plant-derived compounds in secretions of the Aulacoscelinae. These data as well as the basal phylogenetic position of the Aulacoscelinae suggest that sequestration of plant secondary metabolites appeared early in leaf beetle evolution.

Saliva secreted during caterpillar feeding contains enzymes to initiate digestion or detoxify noxious plant compounds. Activity of some salivary enzymes is diet-dependent and may be transcriptionally regulated. In this study, cDNA-amplified fragment length polymorphism was used to identify beet armyworm, Spodoptera exigua Hübner, labial salivary genes that are differentially expressed in response to diet. In addition, SeGOX was sequenced based on homology and characterized to confirm that the transcript encodes a functional enzyme. Three labial salivary transcripts, encoding glucose oxidase (GOX) and two proteins of unknown function (Se1H and Se2J), were expressed in a diet-specific manner. Since diet, particularly the protein to digestible carbohydrate levels and ratio, may affect labial salivary enzyme activity, the influence of nutritional quality on gene expression was determined. Transcript levels of the labial salivary genes Se1H, Se2J and SeGOX increased with dietary carbohydrate levels, regardless of protein concentrations. In contrast GOX enzymatic activity increased with increasing dietary carbohydrates when caterpillars were fed protein-rich diets, but not when caterpillars were fed protein-poor diets. Our results suggest that dietary carbohydrates affect SeGOX, Se1H and Se2J transcription, but dietary protein or amino acid levels affect translational and/or post-translational regulation of the enzyme GOX., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Though there is overlap, plant responses to caterpillar herbivory show distinct variations from mechanical wounding. In particular, effectors in caterpillar oral secretions modify wound-associated plant responses. Previous studies have focused on transcriptional and protein abundance differences in response to caterpillar herbivory. This study investigated Spodoptera exigua caterpillar-specific post-translational modification of Arabidopsis thaliana soluble leaf proteins by liquid chromatography/electrospray ionization/mass spectroscopy/mass spectroscopy (LC/ESI/MS/MS). Given that caterpillar labial saliva contains oxidoreductases, such as glucose oxidase, particular attention was paid to redox-associated modifications, such as the oxidation of protein cysteine residues. Caterpillar- and saliva-specific protein modifications were observed. Differential phosphorylation of the jasmonic acid biosynthetic enzyme, lipoxygenase 2, and a chaperonin protein is seen in plants fed upon by caterpillars with intact salivary secretions compared to herbivory by larvae with impaired labial salivary secretions. Often a systemic suppression of photosynthesis is associated with caterpillar herbivory. Of the five proteins modified in a caterpillar-specific manner (a transcription repressor, a DNA-repair enzyme, PS I P700, Rubisco and Rubisco activase), three are associated with photosynthesis. Oxidative modifications are observed, such as caterpillar-specific denitrosylation of Rubisco activase and chaperonin, cysteine oxidation of Rubisco, DNA-repair enzyme, and chaperonin and caterpillar-specific 4-oxo-2-nonenal modification of the DNA-repair enzyme.

Caterpillars are faced with nutritional challenges when feeding on plants. In addition to harmful secondary metabolites and protein- and water-limitations, tissues may be carbohydrate-rich which may attenuate optimal caterpillar performance. Therefore, caterpillars have multiple strategies to cope with surplus carbohydrates. In this study, we raise the possibility of a pre-ingestive mechanism to metabolically deal with excess dietary sugars. Many Noctuid caterpillars secrete the labial salivary enzyme glucose oxidase (GOX), which oxidizes glucose to hydrogen peroxide and gluconate, a nutritionally unavailable carbohydrate to the insect. Beet armyworm, Spodoptera exigua, larvae were restricted to diets varying in protein to digestible carbohydrate (P:C) ratio (42p:21c; 33p:30c; 21p:42c) and total nutrient concentration (42% and 63%). High mortality and longer developmental time were observed when caterpillars were reared on the C-biased, P-poor diet (21p:42c). As the carbohydrate content of the diet increased, caterpillars egested excess glucose and a diet-dependent difference in assimilated carbohydrates and pupal biomass was not observed, even though caterpillars restricted to the C-biased diet (21p:42c) accumulated greater pupal lipid reserves. Larval labial salivary GOX activity was also diet-dependent and gluconate, the product of GOX activity, was detected in the frass. Unexpectedly, GOX activity was strongly and positively correlated with dietary protein content.

Arabidopsis thaliana (L.) Heynh. genotypes limited in their ability to mount either octadecanoid-dependent induced resistance (IR(-)) or systemic acquired resistance (SAR(-)) were used to characterize the roles of these pathways in plant-herbivore interactions. Molecular and biochemical markers of IR were analysed in plants subject to herbivory by caterpillars of the beet armyworm, Spodoptera exigua Hübner, which had either intact or impaired salivary secretions since salivary enzymes, such as glucose oxidase, have been implicated in the ability of caterpillars to circumvent induced plant defences. Transcript expression of genes encoding laccase-like multicopper oxidase [AtLMCO4 (polyphenol oxidase)] and defensin (AtPDF1.2) showed salivary-specific patterns which were disrupted in the SAR(-) mutant plants. The activity of octadecanoid-associated anti-nutritive proteins, such as LMCO and trypsin inhibitor, showed similar patterns. Gene and protein changes parallel plant hormone levels where elevated jasmonic acid was observed in wild-type plants fed upon by caterpillars with impaired salivary secretions compared with plants subject to herbivory by normal caterpillars. This salivary-specific difference in jasmonic acid levels was alleviated in SAR(-) mutants. These results support the model that caterpillar saliva interferes with jasmonate-dependent plant defences by activating the SAR pathway.

Plant diet strongly impacts the fitness of insect herbivores. Immediately, we think of plant defensive compounds that may act as feeding deterrents or toxins. We are, probably, less aware that plants also influence insect growth and fecundity through their nutritional quality. However, most herbivores respond to their environment and select the diet which optimizes their growth and development. This regulation of nutritional balance may occur on many levels: through selecting and ingesting appropriate plant tissue and nutrient digestion, absorption and utilization. Here, we review evidence of how nutritional requirements, particularly leaf protein to digestible carbohydrate ratios, affect caterpillar herbivores. We propose a model where midgut endocrine cells assess and integrate hemolymph nutritional status and gut content and release peptides which influence digestive processes. Understanding the effects of diet on the insect herbivore is essential for the rational design and implementation of sustainable pest management practices.

In response to caterpillar herbivory, alfalfa and related plant species defend themselves through the induction of saponin and volatile terpenoid biosynthesis. Both these types of defensive compounds are derived from the metabolic intermediate, isopentenyl diphosphate (IPP). In plants, two distinct biosynthetic pathways can generate IPP; the cytosolic mevalonate pathway and the plastid-associated 2C-methyl erythritol 4-phosphate (MEP) pathway. In Medicago truncatula, transcript levels of key regulatory genes active in the early steps of these biosynthetic pathways were measured in response to larval herbivory by the beet army worm, Spodoptera exigua. Transcripts encoding enzymes at early steps of both terpenoid pathways were lower in caterpillar-damaged leaves. Higher degrees of herbivore damage accentuated the decrease in transcript levels; however, transcript amounts were not affected by insect larval stage. Insect larvae, manipulated to reduce labial gland salivary secretions, were used to examine the role of the salivary elicitors in modulating gene expression. Results suggest that an insect salivary factor, possibly glucose oxidase (GOX), may be involved in reduction of transcript levels following herbivory. Addition of GOX or hydrogen peroxide to mechanically wounded leaves confirm these findings. In comparison, transcript levels of a gene encoding a putative terpene synthase are induced in mechanically- or insect-damaged leaves. These data show that insect salivary factors can act to suppress transcript levels of genes involved in plant defense pathways. Findings also suggest that in response to stress such as insect herbivory, regulation occurs at the early steps of the MEP pathway.

Many researchers investigating plant-insect interactions maintain their insect colonies on artificial diet and assume that salivary enzymes and elicitors remain representative of natural situations. These salivary elicitors, such as the enzyme glucose oxidase (GOX), play important roles in influencing plant defense responses. In fact, GOX has been implicated in suppressing induced nicotine-production in tobacco plants (Musser et al., 2002). In this study, we investigated the effect of artificial or plant diet on the GOX activity in caterpillars of the beet armyworm, Spodoptera exigua. In the later developmental stages, whole body GOX of S. exigua caterpillars reared on a wheat germ-based artificial diet is over ten times higher than when insects were fed plants of the legume, Medicago trunctula. Labial salivary GOX accounted for most of this whole body activity in 4th instar caterpillars (57.8%), with the remaining activity present in the carcass. Hemolymph GOX levels were below the detection limits of the o-dianisidine-peroxidase assay used to measure activity. Labial salivary GOX activity was significantly higher in 4th larval instars reared on artificial diet compared with plant-fed caterpillars (U/mg per pair labial salivary gland, p = 0.0062), suggesting that diet effects GOX activity. When 4th instar plant-fed caterpillars were transferred to artificial diet, increased labial salivary GOX activity is closely correlated with the amount of time spent feeding on artificial diet. This study shows that the labial salivary GOX activity of S. exigua caterpillars is dependent on diet and developmental stage and that caution must be exercised in the design of plant-insect experiments.

Terpenes are an important class of defense compounds that accumulate in plants after pathogen infection or arthropod injury. Sequences predicted to encode terpene synthases were selected from an expressed sequence tag (EST) database of Medicago truncatula. Four putative terpene synthase clones (MtTps1-MtTps4), originating from a chewing insect-damaged M. truncatula leaf cDNA library, were isolated. Transcript levels of each gene examined increased in response to artificial wounding, Spodoptera exigua herbivory, and treatment with volatile methyl jasmonate (meJA). Addition of S. exigua regurgitant to wound sites triggered transcript accumulation of MtTps1 and levels increased with higher concentrations of regurgitant. Furthermore, induction of MtTps1 occurred after application of N-linolenoyl-glutamate or N-linoleoyl-glutamate, factors found in lepidopteran regurgitant. Genomic DNA blots indicate that each of the putative proteins is encoded by a single-copy gene or a small gene family. Proteins encoded by MtTps3 and MtTps4 are imported into the soluble fraction of chloroplasts in in vitro assays, whereas proteins encoded by MtTps1 and MtTps2 are not imported into chloroplasts. Combined with sequence comparisons of multiple plant terpene synthases, the import data indicate that MtTps1 and MtTps2 likely encode sesquiterpene synthases and that MtTps3 and MtTps4 encode mono- or di-terpene synthases. In addition to serving as a valuable model legume species for genomic studies, M. truncatula should prove a valuable source of novel terpene-producing enzymes. Induction of wound-responsive genes by insect oral factors suggests that M. truncatula senses biotic damage through the presence of elicitors originating in the herbivore., ((c) 2005 Wiley-Liss, Inc.)

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase.

Juvenile hormones (JHs) are sesquiterpenoids that regulate metamorphosis and reproduction in most insect species. There has been one report of an insect JH in plants: JH III, methyl-10R,11-epoxy-3,7,11-trimethyl 2E, 6E-dodecadienoate, has been identified in two sedge species, Cyperus iria L. and C. aromaticus (Ridley) Mattf and Kük. This is the first report of callus and cell suspension cultures derived from C. iria. Farnesol and methyl farnesoate, two biosynthetic intermediates of JH III in insects, as well as JH III have been identified in suspension culture cell extracts by gas chromatography-mass spectroscopy. These cultures thus provide a useful in vitro model to investigate the biosynthesis of JH III in the sedge, C. iria.