Structural Complexity of Quercus virgiliana Galls Induced by Andricus quercustozae (Hymenoptera: Cynipidae)

Abstract

Cynipid gall wasps are known for their ability to manipulate host plant development, redirecting undifferentiated tissues into complex, highly specialised structures. In this study, we investigated how Andricus quercustozae larvae manipulate axillary bud tissues of Quercus virgiliana across four key stages of gall development: initiation, differentiation and growth, maturation, and lignification. Using detailed histological analyses, we characterised progressive tissue differentiation within galls, focusing on the organisation of nutritive, protective, and vascular tissues. Gall development was marked by sustained hyperplasia and hypertrophy, extensive vascular proliferation, and progressive cell wall lignification, resulting in a complex organ optimised for larval nutrition and protection. To complement these anatomical observations, we conducted a preliminary transcriptomic comparison between gall tissue and unmodified leaf tissue. Gene expression analyses revealed suppression of photosynthesis-related functions and coordinated modulation of developmental, regulatory, and metabolic pathways, consistent with a transition from assimilatory leaf tissue to a specialised nutrient sink. Integration of anatomical and transcriptomic evidence supports a model in which cynipid gall wasps intervene at key regulatory nodes of bud development, progressively reprogramming host tissues to form a functionally autonomous gall. These findings provide new insight into the extended phenotype and highlight the plasticity of plant developmental programmes under insect control.

1. Introduction

Cynipid wasps, among the most complex gall-bearing insects, are associated with some of the most intricate galls. More than 1300 species of wasps have been documented, alternating between sexual and parthenogenetic generations, each forming different types of galls. The genus Andricus Hartig, 1840 (Hymenoptera: Cynipidae: Cynipini) is a Holarctic genus with representatives in both Eurasia and North America. There are about 80 species in the western Palaearctic, particularly in Europe and Asia Minor. All are attracted to oak trees to form galls [1].

Cynipid wasps have developed a remarkable ability to control the growth of host plants, resulting in gall formation [2,3,4,5,6,7,8,9,10]. These gall structures provide a safe environment in which the larvae can feed and develop within the plant tissue. Cynipid wasps such as Andricus quercustozae (Bosc, 1792) have a complex life cycle with several developmental stages influenced by various factors. The four general stages of gall development are initiation, differentiation and growth, maturation, and finally lignification [5,8,11,12,13]. The mechanisms by which cynipid wasps induce gall formation are poorly characterised. One long-standing assumption is that chemicals released by the insects reprogramme plant development and trigger a cascade of events [14]. A key aspect of the gall wasp–plant interaction is that it is neither consensual nor cooperative on the part of the plant. The wasp actively manipulates the plant’s normal developmental and metabolic processes by injecting chemical effectors, essentially tricking the plant into producing the gall [15]. The question that arises is: which plant metabolic processes are fundamental for cynipid gall development?

Plant development follows the morphogenetic patterns determined in plant meristems. However, these patterns can be manipulated by gall-forming organisms, leading to over-differentiation or inhibition of certain plant features and the differentiation of distinct cell types. For instance, some galls maintain the protoderm, procambium, and ground meristem, capable of redifferentiating the three plant tissue systems [16,17]. Notably, Isaias et al. [17] highlight that in plant-gall interactions, galling organisms manipulate host promeristems to redirect development towards gall formation. Because apical and axillary meristems determine plant organogenesis, meristems must also be involved in gall morphogenesis. This involvement can occur through their overactivation or impairment at the induction site. When galls are induced on stem buds, they involve changes in existing apical or axillary meristems. In contrast, galls formed on other plant parts usually trigger parenchymatic cell responses. Furthermore, Meyer-Rochow [18] notes that the plant invests energy and resources to produce the gall, while the meristematic tissue of the plant cells proliferates and acts as a powerful “physiological sink.” By inducing the addition of new rows of vascular bundles, the larva ensures a dedicated “supply line” of nutrients that bypasses the plant’s normal resource allocation. As a result, starch, sugars, and many other chemicals are diverted from the phloem of the surrounding plant parts to benefit the developing insect. Supporting this, Ferreira et al. [19] investigated ontogenetic changes during gall formation in Schinus engleri F.A.Barkley (Anacardiaceae) lateral buds related to Eucecidoses minutanus Brèthes, 1917 (Lepidoptera) development and concluded that gall developmental stages correspond to specific gall-inducing stages, as gall chamber development progresses in line with the development of E. minutanus. The growth and development phase of galls is characterised by hyperplasia and hypertrophy of cells, and additional rows of vascular bundles form in young galls. During the maturation phase, centripetal lignification of the outer parenchymal cell layers, epidermal stratification, and activation of the cambium-like meristem are noted as main characteristics.

Cynipid galls also exhibit a high level of organisation, with transformed tissues that are clearly distinct from those of a typical plant organ. These tissues undergo extensive remodelling during gall development to serve both protective and nutritional functions [14,15,20]. The anatomical remodelling of plant tissue involves a sequence of developmental processes, including hyperplasia, cell hypertrophy, differentiation, and transformation of the tissue during the accumulation of primary and secondary metabolites [3,5,21,22,23,24,25,26,27,28]. These processes underpin the development of specialised microniches that meet the biosynthetic and nutritional needs of the developing larva. Histological observations have shown that tissue heterogeneity includes several important components, such as parenchymatous cells, collenchyma cells, vascular elements, sclerenchyma fibres, and epidermal layers. The most striking structural feature of A. quercustozae galls is the formation of concentric layers of different cells surrounding the larval chamber. The cambial zone, nutritive tissue, and lignified sheath constitute the inner gall, while the epidermis and parenchymatous cortical tissue form the outer gall [29].

The galls of the asexual generation of A. quercustozae emerge from a bud on a two-year-old shoot when the female lays her eggs in specific tissue, usually meristematic [30]. The authors explain that females favour larger shoots as they provide better conditions for gall development and larval survival. The gall consists of host plant tissue but is structurally complex and unique to the wasp species. The larvae feed on the nutrient-rich tissue of the gall and pass through a series of stages. Climatic factors such as temperature and humidity influence the duration of the larval stage [31]. Once the larva is mature, it undergoes metamorphosis within the gall. The pupal stage is the crucial phase in which the larvae mature into an adult wasp. The duration of this stage also depends on environmental factors [7]. The adult wasps emerge from the gall and initiate the next generation by mating and laying eggs in suitable host tissue. The adult stage is very short and mainly dedicated to reproduction [32,33].

It remains unclear whether the modified tissue primarily serves the insect by providing food and shelter, or whether the oak traps the larvae in localised structures to limit further damage. Comparing the different stages of gall development and understanding the physiological and phenological patterns of gall formation could help resolve this question. This would also provide a valuable basis for future comparative proteomic and transcriptomic studies, which are of interest to the authors. Each gall-feeding wasp induces specific morphological changes in the host, reflecting precise molecular signalling [15,34]. By studying these interactions, researchers can uncover key biochemical pathways underlying gall formation and elucidate the dynamic interplay between plant defence mechanisms and insect manipulation strategies. These findings would deepen our understanding of plant–insect interactions at the molecular level.

This study aims to provide an integrated anatomical and physiological characterisation of A. quercustozae galls formed on virgilian oak (Quercus virgiliana (Ten.) Ten.; syn. Q. brachyphylloides Vuk.) across successive developmental stages. By combining detailed histological analyses with a preliminary transcriptomic comparison between gall tissue and unmodified leaf tissue, we address the hypothesis that cynipid gall wasps intervene at key regulatory nodes of bud and shoot development to redirect host developmental programmes. Specifically, we examine whether gall formation represents a transient wound- or repair-related response by the host plant, or a coordinated reprogramming process that progressively acquires functional autonomy, resulting in the formation of a specialised organ supporting larval nutrition and long-term survival.

2. Materials and Methods

2.1. Gall Sampling and Histological Analysis

Galls of various sizes containing larvae were sampled from the asexual generation of the gall wasp Andricus quercustozae on oak trees (Quercus virgiliana) in the Lepenica region of Central Dalmatia (43°37′31.5″ N 16°06′01.6″ E) between April 2023 and April 2025. Collections were conducted at a single site, sampling from five trees, each approximately five metres tall, where galls had been observed during an initial field survey. Based on preliminary findings, ten galls of similar dimensions were randomly removed from two-year-old shoots. Collection was performed at the end of each month during favourable weather conditions without precipitation. Galls were removed by hand or with manual or telescopic secateurs. Histological preparations were made from 5 to 6 galls collected each month. During the gall-appearing period in June and July, galls with a characteristic flower shape and a diameter of up to 1.0 cm were collected and referred to as “young galls”. Galls collected during August and September, which had already developed a round shape and a diameter of 1.0 to 2.5 cm, were referred to as “growing galls”. During October and November, when lignification began and gall growth stopped, galls with a diameter greater than 3.0 cm were collected and referred to as “mature galls”. During the winter dormancy period, the spring pupation period, and the period of emergence of adult insects, collection continued to study the anatomy and physiology of the insects themselves. During collection, some samples were immediately frozen in liquid nitrogen and stored at −80 °C for proteomic and transcriptomic studies. The timing of gall collection was recorded to improve understanding of the phenology of these interactions. Upon arrival at the laboratory, the galls’ diameter was measured, and the galls were carefully dissected with a razor blade and examined under a Leica TL 5000 stereomicroscope, with documentation provided by a Leica DMC 5400 camera (Leica, Wetzlar, Germany). For histological examination, the galls were fixed in formalin-acetic acid-alcohol (FAA), composed of 90 parts 70% alcohol, 5 parts 37% formalin, and 5 parts glacial acetic acid, following the method of O’Brien and McCully [35]. They were dehydrated through a graded ethanol series, cleared with xylene, embedded in Paraplast Plus^® (Surgipath®, Leica Biosystems, Richmond, IL, USA), and sectioned with a microtome (Leica, Wetzlar, Germany) at thicknesses between 5 and 10 μm (longitudinal sections). After removing the Paraplast Plus^® with xylene, the sections were placed on microscope slides and double-stained with Safranin-Alcian blue. Aqueous safranin (1%) (BioGnost Ltd., Zagreb, Croatia) was applied to the sections on the slide for about one minute, after which excess dye was washed off with distilled water. Counterstaining was performed with 1% Alcian blue (pH 2.5) (Alfa Aesar brand; Thermo Fisher (Kandel) GmbH, Karlsruhe, Germany) for three minutes, followed by thorough rinsing in distilled water to remove any excess stain. Sections were then differentiated using 96% ethanol until they were stained in a pale blue. They were washed with absolute ethanol for one minute, after which the paraffin was removed in two changes of xylene (one minute each). The dried sections were mounted with Biomount (BioGnost Ltd., Zagreb, Croatia). The staining produced purplish-red colours for lignified cell walls and tannins, while non-lignified cell walls and the cytoplasm of living cells appeared blue. The sections were analysed using a Leica TL 5000 microscope equipped with a Leica DMC 5400 for photodocumentation. Widefield fluorescence microscopy, which illuminates the entire specimen simultaneously with a broad beam of excitation light, was used to enhance interpretation of the results. Unstained sections for this purpose were analysed using a Zeiss Axio Imager.M1 microscope and photographed with an Axio-Cam MRm (Karl Zeiss, Vienna, Austria). Adobe Express was used for photo processing (order number: AE05029200455CHR).

2.2. Transcriptome Sampling and RNA-Seq Analysis

Gall tissue and unmodified Q. virgiliana leaf tissue were sampled for transcriptomic analysis to characterise molecular reprogramming during gall development. Before RNA isolation, insect larvae were carefully removed from gall tissue by dissecting the galls under a magnifying glass and excising the larvae with a sterile scalpel. Throughout this procedure, tissues were kept immersed in RNAlater^® RNA Stabilization Solution (Sigma-Aldrich, St. Louis, MO, USA), which immediately inactivates RNases and preserves RNA integrity.

RNA was isolated from gall tissue at three developmental stages: early gall (Aqae_g_e; June, approximately 5.0 mm in diameter), mid gall (Aqae_g_m; July, approximately 1.0 cm), and late gall (Aqae_g_l; August, approximately 2.5 cm), as well as from unmodified leaf tissue collected from the same host plants (Aqae_l_e). For each gall sample, the entire gall tissue, from which the causative insect larvae had been removed, was used for RNA extraction to capture the full transcriptional profile of the gall structure.

Total RNA extraction, quality assessment, library preparation, and sequencing were performed according to standard Illumina-compatible protocols. Total RNA was extracted from lyophilised plant material using the Spectrum™ Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA), and RNA quantity and purity were assessed spectrophotometrically. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and only samples with RNA integrity values suitable for high-throughput sequencing were processed further. Poly-A mRNA was enriched, fragmented, reverse-transcribed, and used for library construction, followed by paired-end sequencing on an Illumina platform (Novogene Co., Ltd., Beijing, China) [36,37,38].

Raw sequencing reads were quality-filtered and aligned to the Quercus robur reference genome (assembly DHQuercusRobu3.1) using a splice-aware aligner optimised for eukaryotic transcriptomes [39]. Gene-level read counts were generated using featureCounts [40] and used for differential expression analysis with DESeq2 (R/Bioconductor package) [41,42]. Genes with an absolute log₂ fold change ≥ 1 and a Benjamini–Hochberg adjusted p-value ≤ 0.05 were considered significantly differentially expressed, following the DESeq2 statistical framework [41,42]. Normalised expression values were also used to construct an expression matrix for co-expression analyses across developmental stages. For gene-level heatmap visualisation (Supplementary Figures S6 and S7), log₂ fold-change values obtained from DESeq2 were capped at ±10 to prevent visual distortion from extreme fold-change estimates resulting from very low expression values in one condition. This transformation was applied solely for visualisation and did not affect differential expression testing, gene selection, or downstream functional enrichment analyses.

Functional enrichment analyses of differentially expressed genes were conducted using Gene Ontology (GO) and KEGG pathway frameworks, with GO enrichment accounting for RNA-seq gene length bias [43], and results were visualised using bar plots and bubble plots as described in the Results section. Full details of RNA isolation, sequencing procedures, quality control, differential expression analysis, and enrichment workflows are provided in the Supplementary Materials. Gall tissues representing three developmental stages were treated as biologically related manifestations of gall tissue and compared with unmodified leaf tissue to identify transcriptional signatures consistently associated with gall formation, as reflected in the gall-versus-leaf contrasts used for differential expression and functional enrichment analyses.

3. Results

Adult wasps lay their eggs between the bud scales on the adaxial side of axillary buds, on a second-year shoot. The adult wasp bores into the bud and lays eggs near the procambium (Figure 1a). The female Andricus quercustozae has a highly specialised, needle-like ovipositor used to puncture host oak tissue and lay eggs, along with chemicals from the venom gland and venom reservoir (Figure 1b). Ovipositors have three pairs of valvulae, as in most Hymenoptera. The first and second valvulae are intertwined and serve to pierce the plant tissue. The third valvula is equipped with sensory setae on the ovipositor sheaths to probe and assess potential host plant tissue.

In June, small green-coloured galls were visible (Figure 2a). From July to August, a sweet and sticky exudate appeared on the developing galls containing growing larvae (Figure 2b,c). This exudate attracted ants, as observed in other cynipid galls [44], and raises questions about the characteristics of interspecific relationships, which should be further investigated. By autumn, the galls were mature and lignification had begun (Figure 2d). The cynipid in the larval stage inhabits a gall chamber until the end of the maturation phase, that is, until winter.

During winter, inside the lignified gall, the larva within a lignified sheath was in diapause (Figure 3a,b), while the pupal stage was observed in March and April (Figure 3c). The adult wasp emerged from the galls in late spring (Figure 3d).

3.1. Histology

3.1.1. Young Galls Collected in June and July

In the early stages of growth, the main step is the formation of a chamber that protects the young larva. Even at this early stage of gall formation, two layers of the gall can be distinguished. The outer gall (OG) is formed by the epidermis, collenchyma, and homogeneous parenchyma with vascular bundles. The inner gall (IG) consists of nutritive parenchymal tissue, the crystal zone, a nutritive-like parenchymal tissue, and sclerenchyma plates. The larva has chewing mouthparts (Figure 4) that enable it to break open the cell walls of the nutritive tissue to suck up the contents of the nutrient cells.

The diameters of the six analysed galls collected in June ranged from 1.5 to 3.3 mm. The young gall with a diameter of 2 mm (Figure 2a, Figure 5 and Figure 6), contains a centrally located single larval chamber (diameter of ≈0.5 mm) with one larva.

The epidermis (EP) of the galls is homogeneous and covered by a thin cuticle (cu) (Figure 5 and Figure 6a). Bilaterally to the larval chamber, the epidermal cells contain large, intensely coloured vacuoles, indicating the presence of tannin compounds (Figure 5 and Figure 6a). The lamellar collenchyma (CO) (2–3 layers) is located beneath the well-developed epidermis (Figure 5 and Figure 6a). At the top of the gall, above and below the larval chamber, there are two sclerenchyma plates (SCp) formed by about six layers of cells (Figure 5 and Figure 6b). Homogeneous parenchyma (PA) is situated beneath the collenchyma and consists of thin-walled, densely packed, non-lignified cells arranged in parallel rows, extending laterally into the two sclerenchyma plates and surrounding the larval chamber (Figure 5 and Figure 6b,c). Vascular bundles (vb) are present between the parenchyma cells (Figure 6c). Drusen and other types of crystals are highly abundant in the parenchyma. The nutritive-like parenchyma (NLT) of the inner gall or cambial zone forms a thin, compact tissue that fills the corners between the two sclerenchyma plates (Figure 5 and Figure 6b). A thin layer of nutritive tissue (NT) lines the larval chamber (Figure 5 and Figure 6b). Between the nutritive tissue and the nutritive-like tissue is a zone of crystals, i.e., CaOx idioblasts (CZ) (Figure 5 and Figure 6b). In the basal part of the gall is the stalk (st), which connects the gall to the bud (bu). The vascular bundles of the stalk (vb) are narrow and have spiral thickenings typical of the primary xylem (Figure 5 and Figure 6d)

The diameters of the six analysed galls collected in July ranged from 3.5 to 7.0 mm.

When the galls reached approximately 5 mm in diameter (Figure 2b and Figure 7), some structural changes were observed. The lateral homogeneous parenchyma (PA) is much thicker than in younger galls, and the tissue is more compact, indicating that many additional cell divisions had occurred (hyperplasia) (Figure 8a). Dense cytoplasm and food reserves (proteins, lipids, or soluble sugars) were observed in the nutritive tissue. The spaces between the nutritive cells allow gas exchange and facilitate the movement of water and nutrients (Figure 8b). No accumulation of starch was observed in the nutritive-like tissue. The larval chamber (LC) during the early differentiation and growth stage reaches a diameter of approximately 1 mm (Figure 7).

3.1.2. Growing Galls Collected in August and September

Of the galls collected in August, five with diameters ranging from 1.3 to 1.9 cm were histologically analysed. When the gall reaches a diameter of approximately 1.5 cm (Figure 2c and Figure 9a), the lateral homogeneous parenchyma (PA) is observed to close the upper side of the gall by covering the upper sclerenchyma plate (SCp) (Figure 9a). The nutrient-like tissue (NLT) is significantly thicker, with cells that are rectangular or square in shape and loosely packed. No accumulation of starch has yet been observed (Figure 10a). The diameters of the five analysed galls collected in September ranged from 2.0 to 2.4 cm. Starch begins to accumulate in the nutrient-like tissue, indicating cell hypertrophy (Figure 10b). The larval chamber still measures approximately 1 mm.

3.1.3. The Mature Gall, Collected in October

For this period of gall development, five galls with a diameter of 3.0 to 3.7 cm were histologically processed. When the gall exceeds 3 cm in diameter and lignification begins (Figure 2d, Figure 11 and Figure 12), a sclerenchyma sheath (scs) forms around the nutritive-like tissue. This sheath consists of about five layers of sclereids, which later form a protective capsule for the diapausing larva. It is about 5 mm in diameter, corresponding to the maximum size of a larva, which will feed on the remaining nutrients within the sheath until it grows and enters diapause (Figure 3a,b).

Inside the sclerenchyma sheath, the nutrient-like tissue, as thick cambial storage parenchyma (CSP), surrounds the inner parts of the gall: crystal zone, nutritive tissue, and larval chamber (Figure 12a,c). In the storage parenchyma, a large amount of starch and large vacuoles are observed (Figure 12b). In the inner gall, the larval chamber still measures approximately 1 mm and is lined with the remaining parts of nutrient cells (NT), above which a crystal zone remains visible (Figure 12b). Meanwhile, the parenchymatous outer gall tissue becomes lignified (Figure 11b and Figure 12d) to provide a place for the larva to overwinter (Figure 3).

3.1.4. The Lignified Gall, Collected in January

During winter, the gall tissue becomes completely lignified, providing the larva with a place to overwinter (Figure 3a). Inside the gall, the larva enters diapause (Figure 3b), surrounded by a lignified sclerenchyma sheath that is dorsally attached to the lignified upper sclerenchyma plate. Cracks formed in the parenchyma around the sheath allow the imago to move until it breaks through and leaves the gall (Figure 3d).

3.2. Transcriptomic Reprogramming During Gall Development

To investigate the molecular basis underlying the pronounced anatomical differentiation observed during gall development, we compared the transcriptomes of A. quercustozae gall tissue and unmodified Q. virgiliana leaf tissue using RNA sequencing. Differential expression analysis revealed extensive transcriptional reprogramming associated with gall formation, reflecting coordinated modulation of host developmental, metabolic, and regulatory pathways.

3.2.1. Global Functional Reorganisation of Gall Transcriptomes

Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) revealed strong and systematic functional shifts between gall and leaf tissue (Figure 13). Enriched Biological Process (BP) categories were dominated by terms associated with photosynthesis, chloroplast organisation, and primary metabolic processes, alongside categories related to cytoskeletal organisation, signalling, and cellular transport. Cellular Component (CC) enrichment highlighted chloroplast- and thylakoid-associated structures as well as cytoskeletal and membrane-related components, while Molecular Function (MF) categories included binding and catalytic activities linked to metabolic and regulatory functions.

Together, these enrichment patterns indicate that gall development is accompanied by large-scale restructuring of core cellular functions, with suppression of canonical leaf-associated processes and concurrent modulation of pathways linked to tissue differentiation and cellular reorganisation. The statistical breadth and robustness of GO enrichment across BP, CC, and MF categories are further illustrated in Supplementary Figure S4, which visualises gene ratios, gene counts, and adjusted p-values for enriched terms, supporting the conclusions drawn from the bar plot in Figure 13.

3.2.2. Directionality of Transcriptional Changes

To determine whether enriched functional categories were predominantly activated or suppressed in gall tissue, we examined the directionality of differential expression within selected GO categories (Supplementary Figure S1). This analysis revealed that photosynthesis-related categories were overwhelmingly composed of down-regulated genes in gall tissue compared with unmodified leaves. In contrast, categories related to signalling, cytoskeleton organisation, and cellular transport showed a more balanced distribution of up- and down-regulated genes. These patterns indicate that gall development involves targeted repression of leaf-specific physiological programmes rather than uniform transcriptional downscaling, accompanied by selective modulation of regulatory and structural pathways.

3.2.3. Developmental Continuity and Gall-Core Transcriptional Programmes

To assess whether gall development represents a modification of the host leaf transcriptional framework or the establishment of a novel expression programme, gene expression overlap across developmental stages was examined using a four-way co-expression Venn diagram (Supplementary Figure S2). A large core set of genes was shared among early, mid, and late gall stages, indicating the persistence of a conserved gall-associated transcriptional programme throughout development.

Functional annotation of this gall-core gene set revealed enrichment for processes related to cell wall modification, oxidative and redox-associated processes, and cell expansion, consistent with sustained tissue growth and structural differentiation across gall development. These genes include regulators and enzymes associated with cell wall remodelling (e.g., cell wall-modifying enzymes and structural proteins), oxidative metabolism, and growth-related cellular processes, suggesting that gall tissues maintain an active, developmentally plastic state from early through late stages.

In contrast, genes uniquely expressed in unmodified leaf tissue were predominantly associated with photosynthesis, light harvesting, and carbon fixation, reflecting the maintenance of canonical leaf physiological functions. Early gall-specific gene sets were enriched for jasmonate/oxylipin signalling, wounding responses, and terpenoid biosynthesis, indicating an initial stress- and defence-associated transcriptional response following oviposition. Mid-stage gall-specific genes were associated with signal transduction, carbohydrate metabolism, and brassinosteroid signalling, consistent with active growth and metabolic reorganisation. Late-stage gall-specific genes showed enrichment for cell wall organisation, defence response, and lignin metabolism, paralleling the histologically observed transition towards tissue lignification and structural reinforcement (Figure 11 and Figure 12).

Together, these patterns demonstrate that gall development proceeds through stage-specific modulation of a conserved gall-core transcriptional framework, rather than wholesale replacement of leaf gene expression programmes. The persistence of shared regulatory and structural gene sets across gall stages, combined with progressive activation of stage-specific functional modules, supports a model of gradual transcriptomic reprogramming tightly linked to anatomical differentiation.

3.2.4. Pathway-Level Metabolic Reorganisation

Pathway-level analysis using KEGG enrichment further substantiated the global transcriptional shifts observed in GO analyses (Supplementary Figure S3). Enriched pathways included photosynthesis, carbon metabolism, plant–pathogen interaction, and signalling-related pathways, indicating coordinated metabolic and regulatory reorganisation during gall development.

The breadth and statistical strength of KEGG pathway enrichment are visualised in Supplementary Figure S5, which highlights the proportion of DEGs contributing to each pathway and reinforces the conclusion that gall formation involves extensive physiological restructuring rather than isolated pathway perturbations.

3.2.5. Gene-Level Modulation of Secondary Metabolism and Hormone-Related Regulators

To complement global enrichment analyses and resolve gene-level regulatory patterns, we examined representative sets of secondary metabolism-related and hormone-associated genes extracted directly from the DESeq2 results.

A heatmap of secondary metabolism-related genes revealed significant expression shifts between gall and leaf tissue (Supplementary Figure S6). In particular, genes associated with flavonoid and phenolic biosynthesis were predominantly down-regulated in gall tissue, whereas phenylpropanoid- and lignin-related enzymes were preferentially up-regulated. These patterns reflect strong directional changes in gene expression rather than absolute fold-change magnitude, and are consistent with the anatomical differentiation of lignified and protective tissues observed during later stages of gall development (Figure 11 and Figure 12).

Similarly, a heatmap of hormone-related genes showed targeted modulation of regulators associated with auxin, ethylene, gibberellin, abscisic acid, jasmonic acid, and cytokinin signalling pathways (Supplementary Figure S7). Instead of uniform activation of entire hormone pathways, gall development was characterised by selective up- and down-regulation of specific hormone-responsive genes, indicating fine-scale hormonal reprogramming independent of extreme fold-change values underlying tissue differentiation and growth regulation.

3.2.6. Integration of Anatomical and Transcriptomic Evidence

Taken together, global enrichment analyses, pathway-level reorganisation, developmental continuity of expression programmes, and gene-level modulation of regulatory and metabolic modules demonstrate that gall development involves extensive transcriptomic reprogramming of host leaf tissue. These molecular changes parallel the progressive anatomical differentiation described above, linking suppression of leaf-specific functions with activation and modulation of pathways supporting gall growth, structural reinforcement, and larval accommodation.

4. Discussion

Insect galls are plant structures composed of host tissue, but are developed and controlled by the insect to provide food and shelter for the developing larvae. Gall wasps, for example, manipulate plant hormones and use secretions to induce the plant to create unique structures around their eggs and larvae, essentially programming the plant to build a tailored environment [34,45]. The persistent dilemma is whether gall initiation begins as the creation of ectopic food storage organs for the larva that hatches from the egg, or a complex plant wound response. Detailed research on the developmental anatomy of oak galls and galling physiology, induced by different species of Cynipidae, and their comparison could help answer this question.

To date, detailed research on the developmental anatomy and physiology of oak galls has been conducted for the cynipid Amphibolips michoacaensis Reinhard, 1865 [46], Biorhiza pallida Linnaeus, 1758 [47,48], Cynips longiventris Hartig, 1840 [49], Cynips quercusfolii Linnaeus, 1758 [49,50], Neuroterus numismalis Geoffroy in Fourcroy, 1785 [49], and Neuroterus quercusbaccarum (Linnaeus, 1758) [29,51,52]. This research provides the first such detailed description of Andricus quercustozae galls. All the research mentioned above has shown that there are distinct tissue types in galls, with the innermost tissue responsible for providing nutrition to the wasp larva and the outer tissue protecting through structural and chemical defences [11,23,53]. Gall formation begins when the female lays eggs in a specific plant organ or tissue, usually a meristematic one. The egg deposition site and secretions deposited at the oviposition site play a crucial role in gall initiation [54,55,56,57]. The female A. quercustozae possesses a highly specialised, needle-like ovipositor for puncturing host oak tissue and laying eggs (Figure 1). Before laying an egg, the female wasp uses the third valvula, with sensory setae on her ovipositor sheaths [58], to probe and assess potential host plant tissue. Once a suitable site is found, the wasp uses the interlocking first and second valvulae to pierce the plant tissue. The female wasp simultaneously injects a cocktail of chemical compounds, including venom and potentially other substances [59,60]. Oak gall wasps of the tribe Cynipini have exceptionally large venom glands and reservoirs that occupy a large part of their metasome [61,62]. Accessory glands, also called oviductal or colleterial glands, are lipid-rich organs that, among other functions, can secrete lubricant to facilitate the transport of oocytes through the ovipositor [63]. The anatomy and mechanics of the ovipositor and the content of the venom and accessory glands are likely adapted to the diversity of hosts and host-associated substrates in or on which insects lay their eggs, and this may have contributed to the great diversity of species in Hymenoptera [63,64].

Although injury to plant tissue caused by oviposition and venom secretion can trigger defence responses in the plant and induce several internal signals from the wounded tissues, we assume that the plant wound response is not a major factor in gall initiation. Generally, plants have evolved various defences against inducers, such as the production of bioactive specialised compounds or secondary metabolites, inducible defence proteins, reallocation of resources from the wounding site to more distant tissues, and morphological features such as epidermal outgrowths [65,66,67]. According to the authors, insects that use plants for growth would, by investing energy in avoiding or responding to these defence mechanisms, slow their growth and development; however, this is not the case with Cynipidae. The A. quercustozae larva begins to grow and develop immediately at the onset of gall development, when the diameter is less than 2.0 mm, and no slowdown or cessation of growth is observed in subsequent stages (Figure 2, Figure 4 and Figure 5). Additionally, by reallocating resources, plants can strategically divert nutrients away from inducers at the feeding site, whereas in the gall, resources are diverted directly to the cynipid larvae and used entirely for their own needs. Even the morphological features of galls actually produced by the plant, such as the cuticle and epidermal outgrowths, are not designed to protect the plant from inducers, but to protect the larva within the larval chamber.

It has been observed that these structures develop from the primary meristem. At the very beginning of gall formation, the insect, by laying eggs, appears to take over the mechanisms that activate the dormant bud. In fact, at the initial stage of gall formation, all three primary meristems are used or activated: the protoderm, the procambium, and the ground meristem, as the plant would normally trigger their activation during bud growth [68]. Examination of a dozen young galls, each about 2.0 mm in diameter, revealed that galls always appear at the base of the adaxial side of the axillary bud, which is an embryonic shoot protected by layers of scales (Figure 2a). The egg is laid between the scales and comes into contact with the procambium, the precursor of the gall vascular system (Figure 1a). Egg laying is followed by cell lysis, which creates a chamber for the larva. The bud growth mechanism is triggered, and the meristematic potential of the primary meristems is activated. The gall epidermis develops from the protoderm, while homogeneous parenchyma develops from the ground meristem. Before this, differentiation of the procambium provides a vascular connection between the tiny larval chamber formed around the newly hatched larva and the primary vascular tissue of the stem. The procambium in a developing gall differentiates acropetally, as in a healthy bud [69], and from the beginning the gall grows uniformly upwards (Figure 5 and Figure 7). Acropetal growth elevates the larval chamber, the sclereids already arranged in sclerenchyma plates below and above the larval chamber (Figure 7 (scl)), as well as the rest of the formed tissue of the inner and outer gall. The upper sclerenchyma plate in these early stages of development (Figure 2a, Figure 5 and Figure 7) can be seen as a bright cone rising from the middle of the gall. In the axillary bud of oak, the appearance of sclereids is not uncommon. Indeed, they have been recorded within healthy oak buds as part of the structural reinforcement that provides mechanical protection to the sensitive meristematic tissues within [70]. Creation of the larval chamber is quickly followed by the formation of the protective calcium oxalate (CaOx) crystal layer and nutrient-rich parenchymatous layers. It appears that the position of the crystal layer is not accidental, but very specific, strategically placed to protect the nutrient tissue and the developing larva.

In the second phase (differentiation and growth), the larva continues to grow alongside the enlargement of the chamber and the ongoing development of the nutrient-rich parenchymal tissue of the inner gall. The outer gall, which includes the epidermis, collenchyma, and homogeneous parenchyma, differentiates together with the vascular tissue and forms concentric layers in the gall that function as protective tissue. The vascular bundles within the outer gall parenchyma are usually distorted or enlarged due to selective hyperplasia and neovascularisation [8,57], leading to the formation of a continuous ring of vascular cambium. This new cambium ring, formed by the fusion of fascicular and interfascicular cambium, is responsible for the radial growth of the gall and its increase in volume, while ensuring a constant supply of water and nutrients (Figure 6c). Nutritive tissue (Figure 6b and Figure 8b) generally consists of thin-walled cells with a well-defined nucleus and active metabolism [2,3,4,9]. These parenchyma cells are located either in patches along the edge of the larval chamber or in a region [8,57] and accumulate higher amounts of sugars, proteins, amino acids, lipids, and minerals [6,10]. As the larva’s hunger increases, the nutritive tissue grows more regularly and surrounds the entire larval chamber. During the growth and differentiation phase, the nutrient-like tissue (cambial zone) surrounding the nutritive tissue and crystal layer thickens to increase the size of the gall and replace the nutritive layers that the larva mechanically consumes during feeding (Figure 10a). The cambial zone, which is not directly fed by the inducers, becomes enriched with starch (Figure 10b), followed by a maturation phase in which the sclerenchyma sheath is formed and the gall begins to lignify (Figure 11 and Figure 12). Plant cell differentiation stops, and the larva feeds on the remaining adjacent nutrients within the sclerenchyma sheath until it enters diapause. In parallel, the gall tissue becomes increasingly lignified to provide the larva with a place to overwinter.

Complementing the developmental sequence documented here, our transcriptomic comparison of A. quercustozae gall tissue and unmodified leaf tissue provides molecular support for long-standing hypotheses about cynipid manipulation of host developmental pathways. Studies in several Cynipidae species—including B. pallida, Dryocosmus quercuspalustris (Osten Sacken, 1861) and Amphibolips michoacaensis Nieves-Aldrey & Pascual, 2012. on Quercus robur L. and Q. petraea L. [10,15,34,71,72,73]—have consistently shown that early gall development shares transcriptional signatures with the activation of dormant or developing buds. These signatures typically include upregulation of genes associated with cell cycle progression, phytohormone signalling, cytoskeletal dynamics, and members of the early nodulin (ENOD) gene family.

The enriched GO categories identified in our dataset (Figure 13) indicate similar functional shifts. Biological processes related to intracellular transport, microtubule organisation, and signalling were among the most strongly represented, reflecting activation of pathways closely associated with early meristematic activity and the cytoskeletal remodelling. These transcriptomic patterns correspond with the histological evidence presented here, which documents the coordinated activation of all three primary meristems—protoderm, procambium, and ground meristem—during the earliest stages of gall formation. Directional gene-level patterns underlying these enrichments, including the relative proportions of up- and down-regulated genes within these functional categories, are shown in Supplementary Figure S1, while the breadth and statistical strength of these GO enrichments are further illustrated in Supplementary Figure S4.

In addition to developmental signatures, several enriched categories correspond to plant–pathogen interaction pathways, including components of calcium signalling, receptor-like kinases, and early stress-response signalling modules (Supplementary Figures S3 and S5). Such pathways are commonly activated during herbivory and microbial infection; however, in cynipid systems they appear to be modulated rather than fully activated, with some defence systems suppressed or redirected as part of gall development rather than broadly induced as in classic stress responses [34,74,75]. This interpretation is consistent with our anatomical observations, which show uninterrupted larval development and the absence of visible hypersensitive or necrotic responses, even during the early gall stages when the gall diameter remains below 2 mm. The transcriptomic signal therefore likely reflects a controlled, localised activation of signalling pathways normally associated with wound or pathogen perception, which are co-opted as developmental triggers rather than defence endpoint.

Hearn et al. [15] showed that genes highly expressed in young galls overlap substantially with those active in ungalled developing buds, reinforcing the idea that gall induction begins by commandeering endogenous developmental trajectories. As galls development progresses, transcriptomic profiles diverge markedly from canonical leaf development and instead reflect the emergence of a strong nutrient-sink physiology. This divergence is evident in our enrichment results (Figure 13), where photosynthesis-related biological processes and cellular components—including “photosystem”, “thylakoid”, and “photosynthetic membrane”—show some of the highest enrichment scores, indicating broad transcriptional suppression of photosynthetic machinery. The co-expression analysis (Supplementary Figure S2) further supports this segregation by identifying a large set of leaf-specific genes absent from all gall stages. These patterns align closely with the extensive vascular redirection towards the larval chamber and the expansion of nutritive parenchyma documented in our anatomical series, and mirror observations reported in other cynipid-oak systems [34,72].

Co-expression analysis across early, mid, and late gall stages and unmodified leaf tissue (Supplementary Figure S2) further indicates that gall development occurs through progressive modification of a conserved host transcriptional framework, rather than abrupt replacement of leaf gene expression programmes [68,74]. A substantial set of genes is shared among all gall stages, consistent with the retention of basic leaf-derived cellular functions throughout gall ontogeny. In contrast, genes co-expressed across all gall stages but absent from unmodified leaf tissue define a consistent gall-associated expression pattern enriched for processes related to cell wall organisation and oxidative remodelling, including peroxidase- and laccase-associated activities. Leaf-specific genes, dominated by photosynthesis- and chloroplast-associated processes, are consistently excluded from gall tissues. Together, these patterns support the histological evidence for gradual tissue remodelling during gall development, whereby protective and nutritive structures arise through coordinated, stage-dependent reprogramming of host tissues [34].

At the gene level, targeted analyses of secondary metabolism-related genes (Supplementary Figure S6) provide further detail on these patterns. Gall tissues show marked directional shifts, including predominant downregulation of flavonoid and phenolic biosynthetic genes and preferential upregulation of phenylpropanoid- and lignin-associated enzymes, consistent with the observed transition towards lignified protective tissues during later developmental stages. These transcriptomic trends closely correspond to the anatomical differentiation of sclerenchyma plates and lignified parenchyma documented here (Figure 11 and Figure 12), reinforcing the link between metabolic reprogramming and structural reinforcement of the gall.

Similarly, gene-level analysis of hormone-associated regulators (Supplementary Figure S7) reveals selective modulation of auxin-, ethylene-, gibberellin-, abscisic acid-, jasmonic acid-, and cytokinin-responsive genes. Rather than uniform activation of entire hormone signalling pathways, gall development is characterised by fine-scale up- and down-regulation of specific regulatory nodes, supporting a model in which cynipid wasps manipulate endogenous hormonal networks to steer host developmental outcomes. Such targeted hormonal modulation has been implicated in other cynipid systems and is thought to underlie coordinated control of cell proliferation, differentiation, and nutrient allocation within galls [15,68,74].

Transcriptomic studies across Cynipidae have also reported enrichment of nutrient catabolism, ion-binding functions, and stress-response pathways in mature galls [15,75]. Our findings are consistent with this broader pattern: enriched molecular function categories such as microtubule motor activity, ion binding, and tetrapyrrole binding (Figure 13; Supplementary Figure S4) correspond to the metabolically active inner gall tissues and dynamic cambial zone, which continually regenerates nutritive layers during larval feeding. These molecular signatures support our anatomical evidence of coordinated differentiation of protective sclerenchyma, nutritionally rich parenchyma, and an acropetally developing vascular system that ensures sustained water and nutrients supply (Figure 5, Figure 6, Figure 7 and Figure 8).

Taken together, the integration of detailed anatomical observations with transcriptomic profiling supports a model in which cynipid gall wasps intervene at key regulatory nodes of bud and shoot development to initiate a developmental programme that progressively acquires functional autonomy. The combined structural and molecular patterns observed in A. quercustozae on Q. virgiliana closely resemble those reported from other oak systems, such as Biorhiza pallida galls on Quercus robur, where transcriptome and genome analyses have revealed developmental parallels with normal bud programmes and extensive host remodelling [15], and in Dryocosmus quercuspalustris-induced galls, where large-scale reprogramming of oak host gene networks has been documented [74]. These parallels provide a robust conceptual framework and generate clear hypotheses for future transcriptomic studies to resolve the universality and mechanistic basis of gall morphogenesis in Q. virgiliana.

5. Conclusions

This study presents a detailed anatomical and histological characterisation of gall development induced by Andricus quercustozae on Quercus virgiliana, revealing a highly coordinated process of tissue differentiation driven by larval activity. Gall formation is initiated by oviposition and maintained by larval feeding, which stimulates continuous production of nutritive cells, progressive hyperplasia and hypertrophy of parenchymal tissues, and the addition of new vascular bundles that establish a dedicated nutrient supply to the developing larva. As development progresses, gall tissues undergo marked structural specialisation, including the formation of a sclerenchymatous sheath and extensive cell wall lignification, ultimately producing a complex organ that combines nutritive, protective, and storage functions.

In addition to these anatomical observations, preliminary transcriptomic analyses indicate that gall development is accompanied by extensive reprogramming of host gene expression. Rather than establishing a novel organ identity, gall tissues retain a conserved transcriptional framework derived from leaf tissue that is progressively modified across developmental stages. Global enrichment and pathway-level analyses highlight suppression of photosynthesis-related functions alongside coordinated modulation of developmental, regulatory, and metabolic pathways, consistent with the functional transition of galls from assimilatory leaf structures to metabolically specialised nutrient sinks. Gene-level analyses further suggest fine-scale adjustment of phytohormone-associated regulators and secondary metabolic enzymes, providing molecular context for the observed patterns of tissue growth, differentiation, and structural reinforcement.

Taken together, the integration of anatomical and transcriptomic evidence supports a model in which cynipid gall wasps manipulate key host developmental and physiological processes to redirect normal bud and leaf programmes towards the formation of a structurally complex, nutritionally optimised gall. While the transcriptomic component of this study is exploratory, it provides a coherent molecular framework that complements the detailed anatomical data and lays the groundwork for future, stage-resolved and replicated transcriptomic analyses aimed at dissecting the mechanisms underlying gall induction and development in Q. virgiliana.