The ability of plants to recognize and adequately respond to herbivores attack is essential for their survival. Important hormones in such induced resistance are JA or salicylic acid, which trigger many defence mechanisms that reduce herbivore survival, reproductive capacity, or preference for a plant. It has been well documented that exogenous applications of JAs can protect plants against herbivores by inhibiting or disturbing their normal development and reproduction processes, e.g. reduction in egg numbers (Shahabinejad et al. 2014), larval and pupal average weight (Jiang and Yan 2018) or fecundity (Tianzi et al. 2018). Even with increasing MeJA concentration, the total weight of larvae and adults and their survival time decreased (Senthil-Nathan 2019). These results suggest that pre-treatment of plants with JAs could provide a greater opportunity for plant survival via the activation of JA-responsive genes (Selig et al. 2016).
E. americanus represent phloem feeding insect whose infestation of piercing-sucking mouthparts is manifest as silver dots, which later turn into necrotic lesions. In our work, numerous silver or brownish diffuse spots were observed which were slightly reduced in MeJA pre-treated plants. Similarly, a foliar application of JA caused a marked reduction in silver damage caused by western flower thrips which had dose-dependent trend (Mouden et al. 2020) or locally-dependent trend (Chen et al. 2020).
Insect feeding activity provokes a local wound, and consequently triggers various metabolic responses driven by ROS formation. Apoplastic ROS burst acts as a first barrier against the pathogens and herbivores attack. Their elevated levels are not only toxic to the invasive organisms, but also to the plants themselves. Oxidative damage to significant biomolecules can lead to programmed cell death, which is part of a hypersensitive reaction (War et al. 2012). Among ROS, highly stabile and freely diffusible hydrogen peroxide may also act as secondary messenger by triggering the expression of defence genes and synthesis of various secondary metabolites (Fürstenberg-Hägg et al. 2013). Moreover, hydrogen peroxide has been found to have direct toxicity against insects (Maffei et al. 2007). In our experiment, we noted significantly higher ROS level after thrips invasion, which was ameliorated via MeJA pre-treatment. Similar results were also recorded in white cabbage infected with larvae of Pieris brassicae or Phyllotreta nemorum (Kovalikova et al. 2019) or in sorghum plants attacked by the butterfly Chlio partellus (Hussain et al. 2014). The study of Tianzi et al. (2018) pointed out that MeJa-treated leaves of Populus x euramericana led to increased ROS generation, which was subsequently reflected in enhanced synthesis of antioxidant enzymes in Clostera anchoreta larvae fed on these leaves.
Overall, the characteristics of ROS accumulation can vary depending on the plant type and the invading insect. For example, in Arabidopsis, accumulation maximum of hydrogen peroxide after the oviposition of P. brassicae was after 72 hours (Little et al. 2006), while the attack of the phloem feeding aphid Brevicoryne brassicae showed changes after only 48 hours (Kunierczyk et al. 2008). In the case of Bemisia tabaci, no oxidative burst occurred even after 21 days of feeding (Kempema et al. 2006). In the aforementioned works, in parallel with the increasing concentration of ROS, an increased activity of not only antioxidant enzymes, specifically superoxide dismutase, catalase, peroxidase or polyphenoloxidase, but also other effective antioxidants, such as ascorbate and glutathione, was recorded. At the same time a number of corresponding genes associated with oxidative stress were reported to be upregulated (Kempema et al. 2006; Kunierczyk et al. 2008).
Malondialdehyde (MDA) represents a decomposition product of peroxidation of polyunsaturated fatty acid, and its concentration is usually correlated with the degree of membrane lipid degradation. Moreover, it has been suggested that elevated level of MDA may assist in the synthesis of more complex defence compounds and activate antioxidative enzymes. In addition, the emissions of leaf volatiles partially involved in indirect plant defence, are induced by lipid peroxidation after herbivore damage. Our results are in line with earlier reports, where MDA level was induced in cucumber plants infested with Bemisia tabaci (Zhang et al. 2008) or in rice plants attacked by Cnaphalocrocic medinalis (Chen et al. 2022). The rate of MDA formation can also depend on the insect type, e.g. scraping-sucking feeding activity is more stressful due to its concertation on large surface of plant tissues than the phloem-sucking insects which affect only vascular bundles (Kuster et al. 2022).
In addition to secondary metabolites, which are the main components of chemical defence, proteins also play an important role. Proteins are the main nutrient for insect growth and changes in their content can affect the overall physiological state of herbivores by reducing their growth rate, adult size or survival rate. In this study, a sharp increase in protein content was noted. Hussain et al. (2014) reported similar results, where their content increased even more after the addition of JA. Increased levels of proteins may reflect the plants' current need for antioxidant enzymes or other protein-based defensive compounds. For example, MeJA spraying on Larix olgensis seedlings significantly increased the bio-activities of superoxide dismutase, polyphenol oxidase and two proteinase inhibitors, which resulted in strongly reduced larval/pupal weights and survivals, as well as the fecundity of Lamantria dispar (Jiang and Yan 2018).
Among the secondary metabolites, plant phenols constitute one of the most common and widespread group of defensive compounds, which play a major role in plant resistance against herbivores, including insects. In plants, polyphenols are synthesized through the phenylpropanoid pathway in which L-phenylalanine is converted to cinnamic acid via PAL enzyme. Further, different enzymes at different levels modulate the structure of the compounds formed at different levels of the pathway (Singh et al. 2021). PAL together with polyphenol oxidase have been considered as key enzymes in phenol metabolism, and their activity has been correlated to plant protection against insect feeding. In our study, the accumulation of phenols and flavonoids corresponded with the enzymatic activity of PAL with the maximum in plants attacked by thrips. Pre-treatment with MeJA slightly reduced PAL activity together with phenol levels. Similar results were also found in sorghum plants (Hussain et al. 2014), but on the contrary, an increased level of phenols and flavonoids was recorded in lettuce under the influence of MeJA and pill bugs (Tierranegra-García et al. 2011). Moreover, the level of activity may also depend on the type of plant cultivar. Aphid induced stronger enzyme elevation during the early period after infestation in resistant cultivars of chrysanthemum than to susceptible (He et al. 2011). Changes were also noted at the level of gene expression. For example, in pepper plants, the relative expression of PAL gene increased with prolonged invasion of western flower thrips (Zheng et al. 2019). Surprisingly, Selig et al. (2016) pointed out that the expression level can also be influenced by the type of insect itself, when phloem-feeder thrips upregulated and cell-content feeder aphid downregulated soybean PAL2 expression.
Among the phenolic acids, chlorogenic acid is the most abundant among the caffeoylquinic acid isomers in nature. It is considered as a defence metabolite in plants that provides protection against different herbivores via its oxidation to chlorogenoquinone which binds to free amino acids and proteins and reduced their digestibility. Higher accumulation of chlorogenic acid has been described in several plant species regardless of the feeding method, i.e. stem boring (Lee et al. 2017), piercing–sucking (Leiss et al. 2009) and chewing (Kundu et al. 2018). In these studies, anti-herbivore effect of chlorogenic acid was also confirmed by dietary tests, when a growth retardation, survival rate or larval weight reduction was detected. In our work, we noted an increase not only in chlorogenic acid, but also in 1,5-dicaffeoylquinic acid and 5-ferruloylquinic acid, both due to MeJA and thrips. Overall, the levels were higher in the case of the diploid cultivar, which, according to the conclusions of the study by Leiss et al. (2009), indicated that cultivars with higher values of the mentioned metabolites showed higher resistance. In addition to chlorogenic acid, other phenolic acids can also play an important role in protection. For example, exogenous application of cinnamic and vanillic acid negatively affected the oviposition and feeding preference of the larvae of Achaea janata and Spodoptera litura on castor plants (Usha Rani and Pratyusha 2014).
Among other phenolic metabolites we focused on coumarins, which content was significantly influenced by thrips infestation. Almost in all cases, pre-treatment with MeJA reduced their levels. A significant elevation not only due to the effect of thrips, but also due to the effect of MeJA itself was monitored in the work Repčák and Suvák (2013), so we can conclude that concentration (0.4 mM) used here could already have a stressful rather than acclimatizing effect. Increased levels of coumarins in chamomile plants were also observed under the influence of various abiotic, e.g. heavy metals (Kováčik et al. 2012), nitrogen nutrition (Sajko et al. 2018) or UV radiation (Petruľová et al. 2014), and biotic stimuli, e.g. Erypside cichoracearum infection (Repčák et al. 2001).
Many phenolic metabolites, including coumarins, are toxic not only to herbivores, but also to the plants themselves. This negative effect is reduced by their sequestration into an inactive form in vacuoles, mostly by conjugation with glucose. With an increased need for active aglycones, the bond is broken by a specific glucosyltransferase (Singh et al. 2021). Occurrence of cellular membrane disruption, monitored here as an elevated level of MDA, should lead to a decrease in glycosidic form, GMCA and skimmin, and an increase in aglycones, herniarin and umbelliferone. Surprisingly, continuous increment in all mentioned coumarins was monitored here. Similar results were found in chamomile plants treated with salicylic acid (Ducaiova et al. 2013) or MeJA (Ducaiova et al. 2016).
The involvement of coumarins in defence processes suggests both their elevated concentrations in stressful conditions and also their antioxidant potential linked with the presence of conjugated system, ester bonds and with the number of bound hydroxyl groups. According to Lin et al. (2008), coumarins with higher amount of hydroxyl groups express better scavenging properties than unhydroxylated coumarins. From hydroxylated coumarins, we observed strong increment in the content of umbelliferone (7-hydroxycoumarin), but surprisingly, daphnetin (7,8-dihydroxycoumarin), was found only in trace amounts, but its glucosidic precursor daphnin (daphnetin-7-O-glucoside) significantly rose in MJT and T variants. The work of Petruľová-Poracká et al. (2013), points out that there are significantly higher concentrations of daphnetin in the anthodia than in the leaves.
Umbelliferone plays an important role, both as a stress coumarin and as a precursor for the synthesis of other coumarins (herniarin, esculetin or skimmin) (Chu et al. 2017). In this way, low amounts of umbelliferone under normal physiological conditions and elevated levels under stressful conditions were expected. Both in our experiments and in abovementioned works, a significant increase in umbelliferone was noted.
Polyacetylenes of spiroketal enol ether type are known secondary metabolites synthesized by Asteraceae plants. Together with other substances such as monoterpenes or sesquiterpenes, they are part of the volatile substances that create the typical aroma. In addition, they play an important role in plant defence thanks to their toxic properties, such as growth and reproductive inhibitors, repellents, and oviposition-deterrent (Minto and Blacklock 2008). Several studies showed that essential oils or substances alone showed strong larvicidal potential against mosquito (Ali et al. 2018; Govindarajan and Benelli 2016). The literature data about the changes of dicycloethers in chamomile plants during the stress conditions are expressed in two way, as their sum or as free form, so it is difficult to compare to our results. Similar to our results, an increasing trend was observed after biotic stress (Repčák et al. 2001). However, MeJA application alone resulted in different accumulation trends, an increase in the (Z)- isoforms and a decrease in the (E)-isoforms (Dučaiová et al., 2016). In the case of other abiotic stressors, a predominantly declining trend was observed (Repčák et al. 2001, Sajko et al. 2018).
An increased ploidy level not only leads to a higher accumulation of pharmaceutically important metabolites, but can also help better adaptation to environmental stimuli. Yang et al 2021 showed that plants with higher ploidy level produced more phenolic metabolites with allelopathic effects, which allowed seedlings to survive better and colonize new habitats faster. The effect of ploidy level on the rate of attack by herbivores is not yet completely clear. Authors report both a lower (Nuismer et Thompson 2001) and a higher attack rate (Arvanitisa et al. 2010) in polyploid plants. Furthermore, it is known that polyploidy can cause several changes in physiology, morphology, and gene expression that can impact resistance to herbivores. For example, in Brassicales, genome duplication led to an increased rate of glucosinolate synthesis, which play a key role in defence against invading butterflies (Edger et al. 2015). Qualitative changes of terpenes in Solidago gigantea, namely an increase in sesquiterpenes in tetraploids and, conversely, an increase in monoterpenes and diterpenes in hexaploids, led to changes in the survival and growth of invading herbivores (Hull-Sanders et al. 2009a, b).
Due to the fact that in the case of our experiment diploid and tetraploid plants were separated, it is not possible to clearly determine the preference of invading thrips. Macroscopically observable manifestations of feeding in the form of silver and brown spots did not show significant differences in number between cultivars. Activation of defence mechanisms was manifested by increased accumulation of total proteins (possibly increased synthesis of antioxidant enzymes) and increased accumulation of coumarins and phenolic acids with potential antioxidant properties (chlorogenic acid, 5-dicaffeoylquinic acid, and 5-feruloylquinic acid). A higher content of phenolic metabolites in diploid Centaurea phrygia plants compared to tetraploid plants was also noted by Münzbergová et al. (2015), which led to increased resistance to insects. However, the total content of hydrogen peroxide and the rate of its increase was significantly higher in the case of diploid plants, which indicates a higher level of stress. Overall, we can say that diploid plants responded to stress to a higher extent, but the use of MeJA as a protective agent alleviated the subsequent insect-induced stress more significantly than tetraploid plants.