Jacqueline C. Bede

Caterpillar saliva interferes with induced Arabidopsis thaliana defence responses via the systemic acquired resistance pathway

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.

The role of neuropeptides in caterpillar nutritional ecology.

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.

Nutrient utilization by caterpillars of the generalist beet armyworm, Spodoptera exigua

Abstract Beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), caterpillars are destructive crop pests responsible for considerable annual economic losses. These generalist herbivores are challenged with a diversity of dietary choices that can impact their survival, development and fecundity. In the present study, dietary choices of S. exigua caterpillars, based on the protein to digestible carbohydrate (P : C) ratio of the food, and the impact of nutritionally unbalanced foods on caterpillar performance are assessed. In choice experiments, individual third‐instar caterpillars are offered simultaneously a P‐biased and a C‐biased food until pupation. Caterpillars feed nonrandomly and select a slightly P‐biased diet (22P : 20C). In no‐choice experiments, second instar caterpillars are reared until pupation on diets ranging in P : C ratio from extremely P‐ to extremely C‐biased. High mortality and delayed development are observed on the C‐rich, P‐poor diets, highlighting the potential deleterious effects of excess carbohydrates and the importance of protein for growth and development. Diet‐dependent differences in pupal weight or pupal lipid reserves are not observed. This contrasts with closely‐related Spodoptera species where pupal mass and lipid stores increase on C‐rich, P‐poor diets. On the extremely P‐biased diet, performance is similar to that of individuals reared on the self‐selected diet, suggesting that these caterpillars may efficiently be deaminating excess amino acids to generate carbon skeletons, which are shunted into lipid biosynthesis. Spodoptera exigua caterpillars exhibit flexible and efficient pre‐ingestive nutrient intake regulation and post‐ingestive utilization, allowing these generalist feeders to cope with the heterogeneous diets they may encounter.

Caterpillar- and salivary-specific modification of plant proteins.

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.

Della proteins modulate arabidopsis defences induced in response to caterpillar herbivory

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.

Diet-specific salivary gene expression and glucose oxidase activity in Spodoptera exigua (Lepidoptera: Noctuidae) larvae

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.

Insights into the insect salivary gland proteome: diet-associated changes in caterpillar labial salivary proteins.

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.

Arabidopsis redox status in response to caterpillar herbivory

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.

Ethylene Signaling Modulates Herbivore-Induced Defense Responses in the Model Legume Medicago truncatula

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.

Two genera of Aulacoscelinae beetles reflexively bleed azoxyglycosides found in their host cycads.

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.