This study provides valuable insights into the morphology and composition of salivary glands in SLF adults. The presence of large salivary glands suggests the potential significance of these structures in SLF’s feeding interactions with host plants. Large salivary glands may ensure a constant supply of saliva to sustain long or frequent insect feeding activity. Wild-collected SLF adults, further confined to single diets of grapevines and TOH contain several phytohormones, including SA, ABA, JA, JA-ILE, OPDA, IAA, methyl IAA, cZR, and tZR in their salivary glands. Notably, SA was found in substantial quantities, followed by ABA, OPDA, and IAA. SLF individuals fed on grapevines had higher concentrations of ABA compared with those fed on TOH. Similarly, traces of JA-ILE were only found in the salivary glands of insects fed on grapevines, but the quantities of other hormones detected did not differ in insects fed on either host plant. Although this study doesn't directly demonstrate the secretion of these hormones into plants during insect feeding or their effect on plant immunity, the intriguing presence of these signaling molecules suggests they may be playing a significant role in regulating plant defenses as demonstrated in other insect and plant systems (Acevedo et al. 2019; Brütting et al. 2018).
SA has been identified in saliva, salivary glands, and eggs of different insect species (Acevedo et al. 2019; Saraiva et al. 2021; Tooker and De Moraes 2007). The role of SA in insect tissues has often been linked to the regulation of plant defenses and, more recently, to the suppression of insect immunity (Mollah et al. 2021). In plants, SA serves as a crucial signaling molecule, involved in responses against biotrophic pathogens, such as hypersensitive response (HR) and cell death (Ding and Ding 2020). SA accumulation in plants and downstream responses have also been reported after infestation with some sap-sucking insects (Deng et al. 2022). It has been demonstrated that some insect-derived effectors or molecules that suppress herbivore-induced defenses induce the SA pathway, which in turn suppresses JA-mediated herbivore resistance through an antagonistic interaction leading to enhanced herbivore performance (Cui et al. 2019; Ding and Ding 2020). SA-associated defenses are also induced by the exogenous application of SA in plants, which supports the hypothesis that SA in insect salivary glands can modulate plant defenses during insect feeding. The substantial quantities of SA in SLF, which remained consistent in insects fed on grape and TOH, suggest that the insect may rely on this signaling molecule to counter plant defenses in both host plants.
ABA is another hormone found in significant quantities in SLF salivary glands, especially when feeding on grapevines. ABA has been previously identified in the saliva of a Lepidoptera insect and nymphs (whole-body) of a Hemiptera species (Acevedo et al. 2019; Kai et al. 2017). In plants, this hormone is involved in plant growth and responses to abiotic and biotic stresses, including drought, cold stress, salinity, pathogens, and herbivore attacks (Erb et al. 2012; Li et al. 2022b; Singh et al. 2021). ABA is involved in the production and regulation of ROS (reactive oxygen species) molecules that play a critical role against pathogen infection and serve as signals in the activation of immune-related genes in plants (Li et al. 2022b). Increasing levels of ROS lead to higher Ca2+concentration in guard cells, controlling stomatal closure (Li et al. 2022b). The closure of stomata reduces leaf transpiration and prevents water loss which benefits plants under drought conditions and may also be advantageous for insect herbivores (Lin et al. 2022; Nilson and Assmann 2007). For example, aphid infestation triggers the accumulation of ABA leading to stomatal closure, which enhances phloem feeding time and increases aphid populations (Sun et al. 2015). ABA also works synergistically with JA and antagonistically with SA likely influencing hormone crosstalk and specific plant immune responses (Pieterse et al. 2012). The presence of ABA in SLF salivary glands and its presumptive deposition into plant tissues may prevent host dehydration through stomata closure and could modulate defense responses to insect feeding.
The oxylipin 12-oxo-phytodienoic acid, commonly known as OPDA was also detected in SLF salivary glands. To the author’s knowledge, this is the first time that OPDA has been detected in insect salivary glands. OPDA is commonly found in plants serving as a precursor of JA, and as a signaling molecule on its own (Jimenez Aleman et al. 2022). OPDA is accumulated in response to abiotic stress (flooding, heavy metals, salinity, drought) wounding, pathogen, and herbivore attacks (Archer et al. 2023; Kumar et al. 2019; Savchenko et al. 2014; Taki et al. 2005; Zhu et al. 2023). Similar to ABA, OPDA induces stomata closure (Savchenko et al. 2014) and is involved in the regulation of ROS (Taki-Nakano et al. 2014). OPDA is also involved in resistance to the phloem-feeding insects Nilaparvata lugens in rice and Myzus persicae in an Arabidopsis mutant (Archer et al. 2023; Guo et al. 2014). Although OPDA was detected in SLF fed on grape and TOH, it was more consistently detected in insects fed on TOH; speculatively, higher concentrations of this molecule in TOH may allow the insects to uptake and accumulate it in their salivary tissues.
Auxin (indole-3-acetic acid) or IAA and its nonpolar form methyl-IAA were detected in SLF salivary glands. IAA is a hormone commonly found in insects and other terrestrial arthropods, including those that don’t feed on plants (Tokuda et al. 2022). The large concentration of this hormone in insect bodies, in some cases higher than those detected in plants, led to the hypothesis that insects were able to synthesize IAA de novo. It is now well known that various insect species can synthesize IAA from tryptophan (Suzuki et al. 2014; Yamaguchi et al. 2012), and an aldehyde synthase enzyme seems to catalyze this reaction in sawflies (Miyata et al. 2021). In plants, auxins have a variety of critical functions for growth, development, and immunity (Luo et al. 2018). Elevated auxin levels have been associated with enhanced disease susceptibility from biotrophic and hemibiotrophic pathogens (Kunkel and Johnson 2021), promoting auxin production seems to benefit pathogen infection due in part to the antagonistic crosstalk between auxin and SA (Erb et al. 2012). The role of auxins in response to insect infestation is less understood, but the hormone seems to accumulate in response to feeding by gall-inducing insects and by some chewing herbivores (Machado et al. 2016; Tooker and De Moraes 2011). The amounts of IAA found in SLF salivary glands were low (2ng/g FW on average) compared with those found in other organisms (Tokuda et al. 2022), and its influence on grape and TOH plants remains elusive.
Low amounts of JA (0.6 ng/g FW on average) were detected in SLF salivary glands from insects fed on grapevines and TOH, but traces (0.037 ng/g FW on average) of the bioactive form, JA-isoleucine (JA-ILE) were only detected in insects fed on grapevines. High concentrations of JA have been found in eggs and neonate larvae from several insect species (Tooker and De Moraes 2005; Tooker and De Moraes 2007), as well as in insect tissues such as the gut and salivary glands (Tooker and De Moraes 2006), and insect saliva (Acevedo et al. 2019). JA and its derivates are well-known molecules that get activated in plants in response to insect herbivory and wounding (Erb et al. 2012). These key signaling molecules induce the expression of herbivore defense-related genes, the production of toxic secondary metabolites, and the emission of volatile organic compounds that are used by herbivores and natural enemies to locate their hosts (Li et al. 2022a). However, some herbivores use effector molecules or microbes to activate the SA pathway. This helps them thrive through the antagonistic interaction between SA and JA-regulated defense responses (Chung et al. 2013; Cui et al. 2019).
SLF salivary glands also contain low quantities of the cytokinin ribosides trans-zeatin (tZR, average = 0.6 ng/g FW), and cis-zeatin (cZR, average = 0.1 ng/g FW). CK ribosides have been previously identified in insect herbivores (Andreas et al. 2020), and larger amounts of this hormone are associated with insects that induce plant galls (Tokuda et al. 2022). Biosynthesis of CKs has not been demonstrated in insects, but bioinformatic analyses indicate that insects contain transcripts that encode proteins homologous to enzymes known to be involved in CK biosynthesis and metabolism (Mooi et al. 2024). CKs regulate nutrient allocation in plant tissues that can benefit some insect herbivores. For instance, CKs released in the saliva of the mirid bug Tupiocoris notatus alter the metabolism of tobacco plants to maintain their nutritional quality under herbivore attack. Feeding by T. notatus induces the accumulation of CKs in tobacco, which in turn promotes stable concentrations of glucose, fructose, and starch despite heavy herbivore damage (Brütting et al. 2018). Similarly, high concentrations of CKs were identified in green leaf sections (“green islands”) inhabited by mining insects in senescent apple leaves. The so-called “green islands” also had higher protein and sugar concentrations than the surrounding senescent tissues (Giron et al. 2007). CKs are also involved in the regulation of herbivore-induced defense responses in plants and interact with other signaling hormones, including auxins, ABA, JA, ethylene, etc (Schäfer et al. 2015).
Grapevine defense responses to SLF feeding have been previously investigated at the transcriptional level. Marquette grapevines infested with SLF had a high enrichment of genes associated with ABA, and auxin signaling, and equal induction of both JA and SA genes. There was also upregulation of genes associated with photosynthetic processes, biosynthesis of secondary metabolites, detoxification, and antioxidant activity, among others (Islam et al. 2022). The role that the identified phytohormones in SLF salivary glands may have on grapevine defense responses to SLF feeding remains elusive and deserves future investigation. It is also unknown if SLF or its associated symbionts synthesize plant hormones or if the insect accumulates them in their salivary glands after feeding on plants. The results of this study stimulate further investigation into the functional significance of phytohormones identified in SLF salivary glands on plant defense regulation.
In conclusion, this study provides significant insights into the morphology and composition of salivary glands in SLF adults. The presence of large salivary glands suggests their potential importance in SLF's feeding interactions with host plants. The detection of various phytohormones, including SA, ABA, JA, JA-ILE, OPDA, IAA, methyl IAA, cZR, and tZR in SLF salivary glands, raises intriguing questions about their role in regulating plant defenses. While this study does not directly demonstrate the secretion of these hormones into plants during feeding or their impact on plant immunity, it provides a foundation for further research in this area.