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Key MessageThis review provides an overview, analysis, and reflection on insect elicitors and effectors (particularly from oral secretions) in the context of the ‘arms race’ with host plants.Following injury by an insect herbivore, plants rapidly activate induced defenses that may directly or indirectly affect the insect. Such defense pathways are influenced by a multitude of factors; however, cues from the insect’s oral secretions are perhaps the most well studied mediators of such plant responses. The relationship between plants and their insect herbivores is often termed an ‘evolutionary arms race’ of strategies for each organism to either overcome defenses or to avoid attack. However, these compounds that can elicit a plant defense response that is detrimental to the insect may also benefit the physiology or metabolism of an insect species. Indeed, several insect elicitors of plant defenses (such as the fatty acid-amino acid conjugate, volicitin) are known to enhance an insect’s ability to obtain nutritionally important compounds from plant tissue. Here we re-examine the well-known elicitors and effectors from chewing insects to demonstrate not only our incomplete understanding of the specific biochemical and molecular cascades involved in these interactions but also to consider the role of these compounds for the insect species itself. Finally, this overview discusses opportunities for research in the field of plant-insect interactions by utilizing tools such as genomics and proteomics to integrate the future study of these interactions through ecological, physiological, and evolutionary disciplines.

In response to herbivore attack, plants release large amounts of volatiles that can serve as attractants for the natural enemies of the attacking herbivores. Such responses are typically triggered by damage- and insect-associated factors. Cotton plants are somewhat peculiar because they release specific blends of volatiles in two waves in response to caterpillar attack. They first emit constitutively stored volatile compounds, and after about 24 h a second wave that includes various de novo synthesized compounds. The relative importance of damage-associated and insect associated-factors in this induction of cotton volatile emissions is not yet fully clear. We evaluated how cotton plants respond to mechanical damage and to the application of the oral secretion from the generalist lepidopteran pest Spodoptera exigua , by measuring the local and systemic emissions of volatile compounds from their leaves. Our results confirm that cotton plants respond to damage-associated molecular patterns (DAMPs) as well as to herbivore-associated molecular patterns (HAMPs) present in the caterpillars’ oral secretion. Interestingly, a stronger response was observed for cotton plants that were treated with oral secretion from cotton-fed caterpillars than those fed on maize. We tested the possibility that volicitin, a common fatty acid-derived elicitor in caterpillar regurgitant plays a role in this difference. Volicitin and volicitin-like compounds were detected in equal amounts in the oral secretion of S. exigua fed on either cotton or maize leaves. We conclude that other elicitors must be involved. The identification of these eliciting cues is expected to contribute to the development of novel strategies to enhance the resistance of cotton plants to insect pests.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.

Global sorghum production has been significantly reduced due to the occurrence of sorghum root rot caused by the fungus Fusarium graminearum. The utilization of biocontrol microorganisms has emerged as an effective strategy. However, the underlying mechanisms remain unclear. Therefore, the aim of this study was to investigate the effectiveness of biocontrol bacteria in inducing sorghum resistance against sorghum root rot and explore the potential induced resistance mechanisms through metabolomics analysis. The results revealed that the biocontrol bacteria Lnkb100, identified as Serratia marcescens (GenBank: PP152264), significantly enhanced the resistance of sorghum against sorghum root rot and promoted its growth, leading to increased seed weight. Targeted metabolomics analysis demonstrated that the highest concentration of the hormone IAA (indole-3-acetic acid) was detected in the metabolites of Lnkb100. Treatment with IAA enhanced the activity of disease-related enzymes such as SOD, CAT, POD and PPO in sorghum, thereby improving its resistance against sorghum root rot. Further untargeted metabolomic analysis revealed that IAA treatment resulted in higher concentrations of metabolites involved in the resistance against F. graminearum, such as geniposidic acid, 5-L-Glutamyl-taurine, formononetin 7-O-glucoside-6″-O-malonate, as well as higher concentrations of the defense-related molecules volicitin and JA. Additionally, “secondary bile acid biosynthesis” and “glycerophospholipid metabolism” pathways were found to play significant roles in the defense response of sorghum against fungal infection. These findings provide a reliable theoretical basis for utilizing biocontrol microorganisms to control sorghum root rot.

Fatty acid amino acid conjugates (FACs) have been found in noctuid as well as sphingid caterpillar oral secretions; in particular, volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and its biochemical precursor, N-linolenoyl-L-glutamine, are known elicitors of induced volatile emissions in corn plants. These induced volatiles, in turn, attract natural enemies of the caterpillars. In a previous study, we showed that N-linolenoyl-L-glutamine in larval Spodoptera litura plays an important role in nitrogen assimilation which might be an explanation for caterpillars synthesizing FACs despite an increased risk of attracting natural enemies. However, the presence of FACs in lepidopteran species outside these families of agricultural interest is not well known. We conducted FAC screening of 29 lepidopteran species, and found them in 19 of these species. Thus, FACs are commonly synthesized through a broad range of lepidopteran caterpillars. Since all FAC-containing species had N-linolenoyl-L-glutamine and/or N-linoleoyl-L-glutamine in common, and the evolutionarily earliest species among them had only these two FACs, these glutamine conjugates might be the evolutionarily older FACs. Furthermore, some species had glutamic acid conjugates, and some had hydroxylated FACs. Comparing the diversity of FACs with lepidopteran phylogeny indicates that glutamic acid conjugates can be synthesized by relatively primitive species, while hydroxylation of fatty acids is limited mostly to larger and more developed macrolepidopteran species.

Since the first fatty acid amino acid conjugate (FAC) was isolated from regurgitant of Spodoptera exigua larvae in 1997 [volicitin: N-(17-hydroxylinolenoyl)-L-glutamine], their role as elicitors of induced responses in plants has been well documented. However, studies of the biosyntheses and the physiological role of FACs in the insect have been minimal. By using ¹⁴C-labeled glutamine, glutamic acid, and linolenic acid in feeding studies of Spodoptera litura larvae, combined with tissue analyses, we found glutamine in the midgut cells to be a major source for biosynthesis of FACs. Furthermore, 20% of the glutamine moiety of FACs was derived from glutamic acid and ammonia through enzymatic reaction of glutamine synthetase (GS). To determine whether FACs improve GS productivity, we studied nitrogen assimilation efficiency of S. litura larvae fed on artificial diets containing ¹⁵NH₄Cl and glutamic acid. When the diet was enriched with linolenic acid, the nitrogen assimilation efficiency improved from 40% to >60%. In the lumen, the biosynthesized FACs are hydrolyzed to fatty acids and glutamine, which are reabsorbed into tissues and hemolymph. These results strongly suggested that FACs play an active role in nitrogen assimilation in Lepidoptera larva and that glutamine containing FACs in the gut lumen may function as a form of storage of glutamine, a key compound of nitrogen metabolism.

Induced plant responses to insect attack include the release of volatile chemicals. These volatiles are used as host-location signals by foraging parasitoids, which are natural enemies of insect herbivores. A plant's response to herbivory can be influenced by factors present in insect oral secretions. Volicitin (N-(17-hydroxylinolenoyl)-L-glutamine), identified in beet armyworm (Spodoptera exigua) oral secretions, stimulates volatile release in corn (Zea mays L.) seedlings in a manner similar to beet armyworm herbivory. Volicitin is hypothesized to trigger release of induced volatiles, at least in part, by modulating levels of the wound hormone, jasmonic acid (JA). We compare the sesquiterpene volatile release of damaged leaves treated with aqueous buffer only or with the same buffer containing volicitin or JA. Leaves were damaged by scratching with a razor and test solutions were applied to the scratched area. The leaves were either excised from the plant or left intact shortly after this treatment. Plants were treated at three different times (designated as Evening, Midnight, and Morning) and volatiles were collected in the subsequent photoperiod. JA and volicitin treatments stimulated the release of volatile sesquiterpenes, namely β-caryophyllene, (E)-α-bergamotene, and (E)-β-farnesene. In all cases, JA stimulated significant sesquiterpene release above mechanical damage alone. Volicitin induced an increase in sesquiterpene volatiles for all excised-leaf bioassays and the Midnight intact plants. Volicitin treatments in the Evening and Morning intact plants produced more sesquiterpenes than the untreated controls, while mechanical damage alone produced an intermediate response that did not differ from either treatment group. Excised leaves produced a 2.5- to 8.0-fold greater volatile response than similarly treated intact plants. Excision also altered the ratio of JA-and volicitin-induced sesquiterpene release by preferentially increasing (E)-β-farnesene levels relative to β-caryophyllene. The inducibility of volatile release varied with time of treatment. On average, sesquiterpene release was highest in the Midnight excised leaves and lowest in the Morning intact plants. The duration of induced volatile release also differed between treatments. On average, JA produced a sustained release of sesquiterpenes over time, with over 20% of the combined sesquiterpenes released in the third and final volatile collection period. In contrast, less than 8% of the combined sesquiterpenes induced by volicitin were emitted during this period. The large quantitative differences between intact plants and detached leaves suggest that the results of assays using excised tissues should be cautiously interpreted when considering intact-plant models.

Plants attacked by insect herbivores release a blend of volatile organic compounds (VOCs) that serve as chemical cues for host location by parasitic wasps, natural enemies of the herbivores. Volicitin, N-(17-hydroxylinolenoyl)-L-glutamine, is one of the most active VOC elicitors found in herbivore regurgitants. Our previous study revealed that hydroxylation on the 17th position of the linolenic acid moiety of N-linolenoyl-L-glutamine increases by more than three times the elicitor activity in corn plants. Here, we identified N-(18-hydroxylinolenoyl)-L-glutamine (18OH-volicitin) from larval gut contents of tobacco hornworm (THW), Manduca sexta. Eggplant and tobacco, two solanaceous host plants of THW larvae, and corn, a non-host plant, responded differently to this new elicitor. Eggplant and tobacco seedlings emitted twice the amount of VOCs when 18OH-volicitin was applied to damaged leaf surfaces compared to N-linolenoyl-L-glutamine, while both these fatty acid amino acid conjugates (FACs) elicited a similar response in corn seedlings. In both solanaceous plants, there was no significant difference in the elicitor activity of 17OH- and 18OH-volicitin. Interestingly, other lepidopteran species that have 17OH-type volicitin also attack solanaceous plants. These data suggest that plants have developed herbivory-detection systems customized to their herbivorous enemies.

Biotic elicitors produced by plant pathogens or herbivore pests rapidly activate a range of plant chemical defenses when translocated to plant tissue. The fatty acid conjugate volicitin has proven to be a robust elicitor model for studying herbivore-induced plant defense responses. Here we review the role of insect-derived volicitin (N-[17-hydroxylinolenoyl]-L-glutamine) as an authentic elicitor of defense responses, specifically as an activator of signal volatiles that attract natural enemies of herbivore pests. Comparisons are drawn between volicitin as an elicitor of plant defenses and two other classes of signaling molecules, C(6) green-leaf volatiles and C(4) bacterial volatiles that appear to prime plant defenses thereby enhancing the capacity to mobilize cellular defense responses when a plant is faced with herbivore or pathogen attack.