Plant-Herbivore Interaction: Dissection of the Cellular Pattern of Tetranychus urticae Feeding on the Host Plant

Abstract

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores feeding on cell contents of over 1100 plant species including more than 150 crops. It is being established as a model for chelicerate herbivores with tools that enable tracking of reciprocal responses in plant-spider mite interactions. However, despite their important pest status and a growing understanding of the molecular basis of interactions with plant hosts, knowledge of the way mites interface with the plant while feeding and the plant damage directly inflicted by mites is lacking. Here, utilizing histology and microscopy methods, we uncovered several key features of T. urticae feeding. By following the stylet path within the plant tissue, we determined that the stylet penetrates the leaf either in between epidermal pavement cells or through a stomatal opening, without damaging the epidermal cellular layer. Our recordings of mite feeding established that duration of the feeding event ranges from several minutes to more than half an hour, during which time mites consume a single mesophyll cell in a pattern that is common to both bean and Arabidopsis plant hosts. In addition, this study determined that leaf chlorotic spots, a common symptom of mite herbivory, do not form as an immediate consequence of mite feeding. Our results establish a cellular context for the plant-spider mite interaction that will support our understanding of the molecular mechanisms and cell signaling associated with spider mite feeding.

Keywords: Arabidopsis; Tetranychus urticae; bean; chlorosis; microscopy; plant-pest interaction; stylet; two-spotted spider mite.

Figure 1

T. urticae feeding: duration and plant damage. (A), Duration of individual mite feeding events in minutes (n = 27). (B), T. urticae mouth parts. i–iii, SEM imaging and v–vii, brightfield of mite gnathosoma. i and v, dorsal view with inset close up view of the rostral gutter (Rg); ii and vi, ventral view with inset close up view of the inferior oral commissure (Or); iii and vii lateral view; iv: phalloidin staining of actin filaments and viii: schematic representation of muscles associated with the mouth parts. Sty, stylophore; Bc, buccal cavity; Es, Esophagus; Ph, Pharynx; Pr, Propharynx; scale bars: in inset i and ii, 10μm; in iv (i–iv and vi–viii), 50μm; in v, 50μm. (C–F), Mite feeding and plant damage. (C), Mite at the beginning of the feeding event. (D), Mite at the end of the feeding event. (E,F), Feeding site. F inset, a typical chlorotic spot (arrow head). Scale bars: 500μm in (C–E); 250μm in (F) (main panel and inset).

Figure 2

Plant damage associated with spider mite feeding. (A), Longitudinal cross sections of bean and Arabidopsis leaves stained with toluidine blue (on the top) and Arabidopsis optical sections (at the bottom) visualized using confocal (upper row) and brightfield (lower row) microscopy. ad, adaxial epidermis; pm, palisade mesophyll; sm, spongy mesophyll; ab, abaxial epidermis. (B–E), Representative images of damaged cells within trypan blue stained bean and Arabidopsis leaves after spider mite feeding for 10 min. Damaged cells appear blue and are marked with arrowheads in optical and cross sections. Scale bars: 50μm in (A) through (E).

Figure 3

Interface between T. urticae and plant tissues during feeding. (A), A longitudinal cross section of mite feeding from the abaxial side of the Arabidopsis leaf. (B,C), Serial sections (1μm apart) of stylet penetration through epidermal cellular layers of Arabidopsis leaves while mites fed from the abaxial side (in B) and adaxial side (in C). Guard cells were labeled with arrows in (B). (D), T. urticae stylet penetration while feeding from the abaxial side of bean leaf. (E), Plant cell damage associated with spider mite feeding. Longitudinal cross sections of Arabidopsis (i and ii) and bean (iii and iv) leaves showing cells (marked with asterisks) that were in direct contact with mite stylet. Stylet is marked with arrowhead throughout. Scale bars: 100μm in (A), 25μm in (B–E).

Figure 4

Model of the interactions between the plant and the cell-content feeding herbivores—the two-spotted spider mite and the aphid. (A), Schematic of leaf cross-section and feeding herbivores. Both TSSM and aphid use stylets (red) to penetrate the leaf without disturbing epidermal cells. TSSM feeds from the immediate cell in the mesophyll layer that stylet encounters, while aphid navigates its stylets through mesophyll apoplast to reach sieve element cell (SE). The schematic is drawn to scale; scale bar: 50μm. (B), Close-up diagrams of TSSM and aphid feeding sites. On the left, TSSM feeding results in a cell whose content has been removed. Stylet hole (marked with the arrowhead) is disturbing the unity of the plasma membrane and the cell wall. Damage- and Herbivore-Associated Molecular Patterns (DAMPs and HAMPs) are shown as blue and black dots, while mite effector molecules are shown in red. These molecules are expected within the damaged feeding cell and in the apoplast surrounding it, where they may diffuse. Cells that directly respond to DAMPs and HAMPs trigger local responses and are shown in pink. Model predicts that some TSSM effectors (red dots) targeting the modulation of plant transcriptional response are internalized by these cells. Cells surrounding the feeding site and not directly exposed to DAMPs and HAMPs (light green) mount the systemic response. On the right, aphid stylet (red) is surrounded with a salivary sheet (yellow). It penetrates the sieve element cell (SE) where effectors are delivered. Effectors that modulate plant transcriptional reprogramming diffuse into adjacent companion (CC) and bundle sheath (BSC) cells that are symplastically connected with the enucleated sieve element cell. DAMPs and HAMPs also accumulate within the SE and diffuse into CC and BSC cells. PM, palisade mesophyll; SM, spongy mesophyll; pd, plasmodesmata. Schematic in (B) is not drawn to scale.