Original Article

Ontogenetic variations in Salicylic acid and Jasmonic acid levels in the

galls of Garuga pinnata Roxb. induced by Phacopteron lentiginosum

Buckton

V.K. Bobika1, M. Nasser1, K.S. Shameer1,2

1Department of Zoology, University of Calicut, Thenhipalam, Malappuram, Kerala, India

2Department of Agricultural Sciences, University of Helsinki, Finland

Corresponding author: K.S. Shameer, Email: shameer.ks@helsinki.fi

Journal of Experimental Biology and Zoological Studies. 2(2): p 166-72, Jul-Dec 2026.

Received: 20/06/2026; Revised: 25/06/2026; Accepted: 26/06/2026; Published: 05/07/2026

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Abstract

Galls are distinct structures formed as a result of interactions between host plants and gall-inducing

insects. Gall inducers manipulate host plant physiology by altering the biochemical signalling

pathways of the plant. The present study aims to find the ontogenetic changes in phytohormones,

such as salicylic acid (SA) and jasmonic acid (JA), during gall development induced by

Phacopteron lentiginosum on the leaves of Garuga pinnata. Galls with 3rd, 4th and 5th instars of

the gall inducer were collected and the concentrations of SA and JA were analysed. Both

phytohormones varied significantly across the developmental stages of galls. The highest

concentrations of SA (147.67 ± 0.58 µg ml⁻¹) and JA (676.58 ± 25.48 µg ml⁻¹) were recorded in

galls containing 3rd instar nymphs, followed by a decline in 4th and 5th stages of galls. Elevated SA

and JA levels during early gall induction suggest a synergistic host plant defence mechanism,

whereas their subsequent decline may be associated with gall development and maintenance. This

study highlights the role of SA and JA in mediating insect–plant interactions.

Keywords: Cecidogenesis, gall inducer, Garuga pinnata, jasmonic acid, Phacopteron

lentiginosum, plant-insect interactions, salicylic acid.

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Introduction

Gall formation or cecidogenesis is one of the most specialized forms of plant-insect interactions.[1]

Galls are abnormal plant growths characterized by tissue proliferation, differentiation, and

structural reorganization induced by various organisms, including insects, mites, fungi, bacteria,

and nematodes.[2] The gall inducing stimuli can be oviposition fluids, salivary secretions or

mechanical stress imposed by gall-inducing organisms, triggering distinct morphological and

physiological alterations in host plant tissues, ultimately resulting in gall development.[3] These

elicitors bind to plant receptors, marking the earliest stage of gall initiation.[4,5]

Upon perception of gall-inducing stimuli, plants activate a range of defence responses. For

successful gall development, the gall inducers must suppress or modulate these defences and

redirect host tissues into specialized growth patterns.[6] The biotic stress induced by the elicitors

are defended by the wide range of inducible defence mechanisms activated by the plant during the

time of infestation.[6] Among these defence responses are the phytohormones jasmonic acid (JA)

and salicylic acid (SA), both of which function as key signalling molecules.[7] In plant tissues, JA

is synthesized from linolenic acid via octadecanoid pathway in response to wounding and

herbivore attack.[8] Similarly, SA, a derivative of benzoic acid, is another important plant hormone

regulating plant defence responses.[9] It functions as an endogenous growth regulator, influencing

various metabolic and physiological processes associated with plant defence, growth, and

development.[10] These compounds together coordinate complex signalling pathways that

ultimately trigger the activation of plant immune systems and associated protective responses.[11,12]

Previous studies have shown that hormonal signalling pathways are dynamically modulated, with

SA and JA levels rising during early gall induction but declining sharply during later stages,

suggesting a temporal modification of defence signalling that favours gall maintenance.[13]

The present study investigated the biochemical alterations in the levels of SA and JA, associated

with gall formation in Garuga pinnata Roxb. (Burseraceae) induced by Phacopteron lentiginosum

Buckton (Hempitera: Phacopteronidae) during different stages of gall development.

Materials and Methods

Gall system

The leaf galls induced by P. lentiginosum on G. pinnata are fruit-like, bilayered pouches (Figure

1) formed in response to the feeding activity of first-instar nymphs, with each gall harbouring 1–

14 individuals.

Gall formation is closely linked to nymphal development. Initial feeding by 1st instar nymphs

produces a localized swelling (first tier), within which nymphs moult to subsequent instars.

Continuous feeding by these nymphs leads to the formation of a second tier of galls appearing as

an inverted cup-shaped structure that serves as a protective shelter for the developing nymphs.[14]

The 5th instar represents the final nymphal stage. The adult emergence occurs through a

characteristic flower-like opening.

Figure 1: Galls induced on Garuga pinnata by Phacopteron lentignosum

Study area

Galls were collected from Pattambi, Kerala, India (10.8114°N, 76.1904°E) during 2019–2021. The

stages of nymphal instars were identified based on morphometric parameters (body length, body

width, head length, interocular distance and wing bud area), in accordance with Dyar’s law[15].

Galls were dissected to remove inducers and parasitoids, pooled by stage (n=20 per stage), and the

galls were subjected to LC-MS analysis. Ungalled leaves from uninfested plants served as controls.

Estimation of salicylic acid and jasmonic acid

The 3rd, 4th and 5th stages of galls containing the 3rd, 4th and 5th instars of the gall inducer

respectively and ungalled leaves (control) were collected and immediately frozen in liquid nitrogen

and stored at −80 °C until further analysis to prevent phytohormone degradation. Quantification

of endogenous SA and JA in gall tissues and ungalled leaves was carried out using the external

standard method. [16]

Preparation of SA and JA Standard Solutions: Stock solutions of SA and JA (0.1 mg ml⁻¹) were

prepared by dissolving each compound in methanol and stored at 4°C. Working standard solutions

were freshly prepared before analysis by diluting the stock solutions with the mobile phase to

obtain the required concentrations.

Generation of Standard Curves for SA and JA: Standard curves were generated using a series of

SA working solutions (10, 20, 30, 40, and 50 µg ml⁻¹) and JA working solutions (25, 50, 100, 150,

and 200 µg ml⁻¹). Each concentration was injected into the LC-MS system, and peak areas were

determined. Standard curves were constructed by plotting peak area against corresponding

concentrations, and linear regression analysis was performed to obtain calibration equations for

quantification.

Extraction of Endogenous SA and JA from different stages of galls and ungalled leaves:

Endogenous SA and JA were extracted using the following procedure.[17] Frozen samples of

different stages of gall tissues and ungalled leaves were separately ground to a fine powder in

liquid nitrogen. Powdered tissue was extracted using 10% methanol containing 1% acetic acid.

The samples were incubated on ice for 30 min, followed by centrifugation at 13,000 × g for 10

min at 4 °C. The supernatant was collected, and the pellet was re-extracted with additional

extraction solvent. After 30 min incubation on ice, extracts were centrifuged again, and

supernatants pooled. The extracts obtained from each maturation stage of the galls and from

ungalled leaves were filtered through a syringe filter prior to analysis.

Analysis and Quantification: Analysis was performed using an Agilent 6100 Series Quadrupole LC/MS

system. The mobile phase consisted of Solvent A: 100 % HPLC-grade water and Solvent B: 100 %

HPLC-grade acetonitrile. Injection volume was 10 µl for all samples. Detection of SA and JA was done

at 325 nm and 295 nm respectively. Peak areas obtained from sample chromatograms were compared

with standard calibration curves to calculate hormone concentrations, expressed as µg ml⁻¹.

Statistical analysis

All biochemical data were organized using Microsoft Excel prior to statistical processing. Statistical

analyses were conducted in R software (v4.3.3) using the R Studio user interface (2024-02-29 ucrt)[18]

using R Studio as the integrated development environment. Data were presented as mean ± standard

error (SE) unless stated otherwise.

Normality of data distribution was assessed using the Shapiro–Wilk test, while homogeneity of

variances was evaluated using Levene’s test. For comparisons between two groups (e.g., gall tissues

vs. ungalled leaves) and for comparisons of different gall developmental stages, one-way analysis of

variance (ANOVA) was used, followed by Tukey’s honestly significant difference (HSD) post hoc test

to identify pairwise differences.

Results

The present study showed that galls induced by P. lentiginosum on G. pinnata exhibit pronounced

variation in the concentration of SA and JA across different developmental stages of galls and these

profiles differ markedly from those of ungalled leaves (Table 1).

Table 1: Variation in biochemical parameters across different developmental stages of galls.

Stages of galls

SA (µg ml⁻¹)

JA (µg ml⁻¹)

0.26 ± 0.01

190.39 ± 10.26

147.67 ± 0.58

676.58 ± 25.48

91.10 ± 1.11

450.05 ± 8.07

31.99 ± 0.54

140.75 ± 5.63

Values represent mean ± standard error (SE). L=ungalled leaf tissue; G3=Galls containing third instar nymphs of

the gall inducer; G4=Galls containing fourth instar nymphs of the gall inducer; G5=Galls containing fifth instar

nymphs of the gall inducer; JA=Jasmonic acid; SA=Salicylic acid; No. of observations=20.

Salicylic acid content in different stages of galls and ungalled leaves

SA content differed significantly among gall developmental stages (3rd to 5th instars) and ungalled

leaves (one-way ANOVA: F= 4048, p < 0.001). SA levels were the highest in 3rd stage galls (with

3rd instar nymphs) and declined progressively with gall maturation (Table 1). Ungalled leaves

exhibited the lowest SA concentration and differed significantly from all gall stages (all p < 0.001)

(Figure 2).

Jasmonic acid content in different stages of galls and ungalled leaves

JA content differed significantly among gall developmental stages (third to fifth instars) and

ungalled leaves (one-way ANOVA: F = 137.1, p < 0.001). JA levels were the highest in galls

containing 3rd nymphal instars compared to galls containing 4th and 5th instars and ungalled leaves

(Table 1). JA levels were not significantly different in galls with 5th nymphal instar and ungalled

leaves, both of which were significantly lower than 3rd and 4th developmental stage of galls (Figure

3).

Figure 2: Variation in salicylic acid across different developmental stages of galls and ungalled leaves.

(A) Forest plot showing Tukey-adjusted pairwise differences between groups. Horizontal lines represent

95% confidence intervals. (B) Boxplot showing SA concentration across different developmental stages

of galls and ungalled leaves. G3, G4 and G5=galls with 3rd, 4th and 5th instar nymphs of the gall inducer

respectively; L=ungalled leaves (control).

Figure 3: Variation in jasmonic acid across different developmental stages of galls and ungalled leaves.

(A) Forest plot showing Tukey-adjusted pairwise differences between groups. Horizontal lines represent

95% confidence intervals. (B) Boxplot showing JA concentration across different developmental stages of

galls and ungalled leaves. G3, G4 and G5=galls with 3rd, 4th and 5th instar nymphs of the gall inducer

respectively; L=ungalled leaves (control).

Discussion

In the present study, we investigated the levels of salicylic acid and jasmonic acid across different

developmental stages of galls induced by P. lentiginosum on G. pinnata in comparison with

ungalled leaves. The results clearly demonstrate significant alterations in these phytohormone

levels during gall development. Earlier reports have already established that the concentration of

phytohormones varies across the developmental stages of the gall and the gall inducers.[19-21] These

metabolic imbalances are likely the consequences of elicitors secreted by the gall inducer, which

reprogram host plant metabolism and redirect normal organogenesis into the formation of a

specialized structure.[22,23]

We observed a markedly higher concentration of SA and JA in galls with 3rd instar of the gall

inducer, followed by a consistent decline as galls matured with the 4th and 5th nymphal instars. This

suggests an activation of SA mediated defence response to counteract the tissue manipulation by

the gall inducer. This pattern is consistent with the role of SA in activating systemic acquired

resistance (SAR) against biotic stresses.[24] The significantly lower SA levels in ungalled leaves

further indicates that gall induction enhances SA signalling as an immediate response to the elicitor

released by the gall inducer.[25] JA is also associated with defence against herbivory[26], the

suppression of which during the 5th stage could be due to the decline in elicitor levels required for

the successful manipulation of gall formation and maintenance. In oak leaf galls induced by

Cynipid wasps, gall tissues showed alterations in JA biosynthesis pathways compared to

surrounding leaf tissue, consistent with our observations of JA levels in galled and ungalled

leaves.[27]

As observed in our study, the concurrent decline of both SA and JA across gall maturation stages

defines a coordinated plant defence signalling. The crosstalk between the two inducible hormones

indicate that herbivory is associated with activation of SA defences and pathogenesis with

activation of JA pathway.[28] However, phloem feeding is considered as a special case, where the

sap-sucking insects act as pathogen-like agents in plants and activate both SA and JA signalling

pathways simultaneously in plants.[29] The synergistic effect of SA and JA is reported in the gene

expression of Squash plants in response to Silverleaf whitefly feeding.[30] However, the root galls

of Vitis spp. induced by Daktulosphaira vitifoliae showed an initial hike in JA concentration

followed by a sharp decline due to subsequent SA induction; this simultaneous SA-JA crosstalk

suppress JA mediated host responses.[31]

SA–JA crosstalk is often represented as a mutual antagonism.[32] The present study implies that

gall inducers can modulate both pathways simultaneously to suppress host defences, thereby

facilitating sustained gall growth, development and maturation. In contrast to the earlier mentioned

antagonistic interaction between SA and JA, our findings suggest a synergistic interplay between

the SA and JA pathways in protecting plants against biotic stress. When both pathways are

activated together, plant resistance can become stronger and more effective as observed in

Nicotiana glutinosa, where SA and JA work together to enhance disease resistance.[33] Thus, the

present study provides insight into the mechanisms by which gall inducing insects overcome host

defences and highlights the potential need to study whether synergism can lead to simultaneous

resistance to insect attack.

Conclusion

Galls represent an extended phenotype where an inducing insect manipulates host plant gene

expression to create a protective, nutrient-rich microenvironment. This study investigates the

interaction between the host plant Garuga pinnata and the gall-inducer Phacopteron lentiginosum,

focusing on the roles of SA and JA. SA is known to interact with auxins and cytokinins to drive

tissue hypertrophy and hyperplasia, needed to physically build the gall. JA modulates stress

responses and resource allocation. Its signalling is hijacked to modulate metabolic pathways and

establish a nutrient sink for developing larvae. Contrary to the typical antagonism between these

pathways, our findings reveal that the inducer elicits a synergistic, highly localized temporal wave

of both SA and JA. This precise hormonal balancing may suppress the plant's lethal hypersensitive

response while successfully directing the development of the gall structure.

References

1. Raman A. Gall-inducing insects and plants. Curr Sci 2021; 120(1): 66–78.

2. Raman A, Schaefer CW, Withers TM. Galls and gall-inducing arthropods: an overview of their biology, ecology, and

evolution, In: Raman A, Schaefer CW, Withers TM, editors. Biology, ecology and evolution of gall inducing arthropods:

Science Publishers Inc., Enfield, New Hampshire, USA; 2005. pp. 1–33.

3. Ferreira BG, Álvarez R, Avritzer SC, Isaias RMS. Revisiting the histological patterns of storage tissues: beyond the limits of

gall-inducing taxa. Botany 2017; 95(2):173–84.

4. Yoshikawa M, Yamada Y, Masago H. Elicitors: Their significance and primary modes of action in the induction of plant

defense reactions. Plant Cell Physiol 1993; 34(8):1163–73.

5. Mishra P, Saini P, Patni V. Biochemical dynamics during development of insect-induced plant galls: a review. J Plant Dis Prot

2024;131(6):1803–18.

6. Harris MO, Pitzschke A. Plants make galls to accommodate foreigners: some are friends, most are foes. New Phytol 2020;

225:1852–72.

7. Lazebnik J, Frago E, Dicke M, Van Loon JJ. Phytohormone mediation of interactions between herbivores and plant pathogens.

J Chem Ecol 2014; 40(7):730–741.

8. Zhang SZ, Hua BZ, Zhang F. Induction of the activities of antioxidative enzymes and the levels of malondialdehyde in

cucumber seedlings as a consequence of Bemisia tabaci (Hemiptera: Aleyrodidae) infestation. Arthropod Plant-Interact 2008;

2(4):209–13.

9. War AR, Paulraj MG, Ahmad T, Buhroo AA, Hussain B, Ignacimuthu S, et al. Mechanisms of plant defense against insect

herbivores. Plant Signal Behav 2012;7(10): 1306–20.

10. Rivas-San VM, Plasencia J. Salicylic acid beyond defence: its role in plant growth and development. J Exp Bot

2011;62(10):3321–38.

11. Wasternack C. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and

development. Ann Bot 2007;100(4):681–97.

12. Smith JL, De Moraes CM, Mescher MC. Jasmonate and salicylate mediated plant defense responses to insect herbivores,

pathogens and parasitic plants. Pest Manag Sci 2009; 65(5): 497–503.

13. Martinuz A, Sikora RA, Vilchez S, Schouten A. The role of salicylic and jasmonic acid in the induction of resistance in

Arabidopsis thaliana against Meloidogyne incognita. Physiol Mol Plant Pathol 2025;140:102943.

14. Bobika VK, Nasser M, Shameer KS. Decoding chemical profiles in insect-induced gall development: Insights

from Phacopteron lentiginosum–Garuga pinnata interactions. Entomol Sci 2026 (In Press).

15. Saleem UKA. Diversity of gall associated insects in north Kerala [dissertation]. India: University of Calicut; 2015.

16. Kakade S, Anil V. Detection and estimation of endogenous jasmonic acid and salicylic acid in potato genotypes imposed with

defense modulators using high performance liquid chromatography. Plant Cell Biotechnol Mol Biol 2023; 24(7–8): 51–9.

17. Islam J, Phukan S, Chattopadhyay P. Development of a validated RP-HPLC/DAD method for the quantitative determination

of methyl jasmonate in an insect repellent semi-solid formulation. Heliyon 2019; 5(5):e01775.

18. R Core Team. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna,

Austria 2024. Available from: https://www.R-project.org/ [Last accessed on 2025 Jun 05].

19. De Bruyn L, Vandevyvere I, Jaminé D, Prinsen E. The effects of gall formation by Lipara lucens (Diptera, Chloropidae) on

its host Phragmites australis (Poaceae). In: Csóka G, Mattson WJ Jr, Stone GN, Price PW, editors. The Biology of Gall-

Inducing Arthropods. General Technical Report NC-199. St. Paul, Minnesota, USA: U.S Department of Agriculture, Forest

Service, North Central Forest Experiment Station; 1998. pp. 173–87.

20. Yamaguchi H, Tanaka H, Hasegawa M, Tokuda M, Asami T, Suzuki Y. Phytohormones and willow gall induction by a gall–

inducing sawfly. New Phytol 2012;196:586–95.

21. Wang H, Liu J, Cui K, Chen H, Yang Z, Wu H, et al. Gibberellic acid is selectively downregulated in response to aphid-

induced gall formation. Acta Physiol Plant 2016;38:214.

22. Mani MS. Ecology of Plant Galls. Monographiae Biologicae, Vol. 12. The Hague, The Netherlands: Dr. W. Junk Publishers;

1964. pp. 156.

23. Snoeck S, Guayazán-Palacios N, Steinbrenner AD. Molecular tug-of-war: plant immune recognition of herbivory. Plant Cell

2022;34:1497–513.

24. Peng J, Deng X, Huang J, Jia S, Miao X, Huang Y. Role of salicylic acid in tomato defense against cotton bollworm,

Helicoverpa armigera Hübner. Z Naturforsch C J Biosci 2004;59(11–12):856–62.

25. Tooker JF, Helms AM. Phytohormone dynamics associated with gall insects, and their potential role in the evolution of the

gall-inducing habit. J Chem Ecol 2014;40(7):742–53.

26. Shivaji R, Camas A, Ankala A, Engelberth J, Tumlinson JH, Williams WP, et al. Plants on constant alert: elevated levels of

jasmonic acid and jasmonate-induced transcripts in caterpillar-resistant maize. J Chem Ecol 2010;36(2):179–91.

27. Wang Y, Xue C, Wu S, Zhang Y, Li R, Li Y, et al. Transcriptome and phytohormone analysis reveal mechanism of gall

formation by Trichagalma acutissimae larvae on oak leaves. Front Plant Sci 2025;16:1646230.

28. Thaler JS, Humphrey PT, Whiteman NK. Evolution of jasmonate and salicylate signal crosstalk. Trends Plant Sci

2012;17(5):260–70.

29. Walling LL. The myriad plant responses to herbivores. J Plant Growth Regul 2000;19(2): 195–216.

30. van de Ven WT, LeVesque CS, Perring TM, Walling LL. Local and systemic changes in squash gene expression in response

to silverleaf whitefly feeding. Plant Cell 2000;12(8): 1409–23.

31. Eitle MW, Griesser M, Vankova R, Dobrev P, Aberer S, Forneck A. Grape phylloxera (D. vitifoliae) manipulates SA/JA

concentrations and signalling pathways in root galls of Vitis spp. Plant Physiol Biochem 2019;144:85–91.

32. Mur LA, Kenton P, Atzorn R, Miersch O, Wasternack C. The outcomes of concentration–specific interactions between

salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol

2006;140(1):249–62.

33. Shang J, Xi DH, Xu F, Wang SD, Cao S, Xu MY, et al. A broad–spectrum, efficient and nontransgenic approach to control

plant viruses by application of salicylic acid and jasmonic acid. Planta 2011;233(2):299–308.

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