Figure 1

Immunolocalization of cyclotides in mite-damaged leaf tissues and in mites after ingestion. (a) Transverse sections of mite-infested leaves of V. uliginosa, and (b) V. odorata stained with immunohistochemistry. Cyclotides, found in the lower (le) and upper (ue) epidermis, palisade (pm) and spongy mesophyll (sm), are indicated in red; nuclei and chloroplasts are in blue. In the case of both Viola species, leaf injuries caused by mite feeding were localized in the palisade and spongy mesophyll (indicated with asterisks). (c) Simplified scheme of TSSM female anatomy. (d,e,f), Transections of different female mites fed on bean (control) (d1 and d2); and cyclotide-challenged females fed on V. odorata (e,e1,e2,f1,f2). Different tissues and structures and tissues can be distinguished: salivary gland (sg); nervous tissues or central nervous mass (nm); ovary (ov) with oocytes (OOCs) and egg (eg) adjacent to the ventriculus (v) and posterior midgut (pm); lumen of digestive tract filled with digestive cells (DCs) or floating cells originating mainly from the midgut epithelium and comprising phagocytes containing a bolus of food; generative cell (GCs) and lateral cell (LCs) cells located within caeca as well as from microvilli epithelial cells (MCs) located within the posterior midgut. DCs with undigested plant material containing cyclotides, to be finally extracted as fecal pellets are located in the rectum (r). Ventricular epithelium (VE) is located within the dorsal and ventral parts of the ventriculus, whereas the dorsal midgut is lined with dorsal epithelium (DE). Cyclotides, indicated with red fluorescence, are in close proximity to the epithelial cells (GCs, LCs, MCs), digestive cells (DCs) and ovary (ov). Abbreviations of mite anatomical structures adapted from Bensoussan et al.⁶⁰. Scale bars: a, b = 25 µm; d-f = 50 µm.