Formation of Stylet Sheaths in āere (in air) from eight species of phytophagous hemipterans from six families (Suborders: Auchenorrhyncha and Sternorrhyncha)

Abstract

Stylet sheath formation is a common feature among phytophagous hemipterans. These sheaths are considered essential to promote a successful feeding event. Stylet sheath compositions are largely unknown and their mode of solidification remains to be elucidated. This report demonstrates the formation and solidification of in āere (in air) produced stylet sheaths by six hemipteran families: Diaphorina citri (Psyllidae, Asian citrus psyllid), Aphis nerii (Aphididae, oleander/milkweed aphid), Toxoptera citricida (Aphididae, brown citrus aphid), Aphis gossypii (Aphididae, cotton melon aphid), Bemisia tabaci biotype B (Aleyrodidae, whitefly), Homalodisca vitripennis (Cicadellidae, glassy-winged sharpshooter), Ferrisia virgata (Pseudococcidae, striped mealybug), and Protopulvinaria pyriformis (Coccidae, pyriform scale). Examination of in āere produced stylet sheaths by confocal and scanning electron microscopy shows a common morphology of an initial flange laid down on the surface of the membrane followed by continuous hollow core structures with sequentially stacked hardened bulbous droplets. Single and multi-branched sheaths were common, whereas mealybug and scale insects typically produced multi-branched sheaths. Micrographs of the in āere formed flanges indicate flange sealing upon stylet bundle extraction in D. citri and the aphids, while the B. tabaci whitefly and H. vitripennis glassy-winged sharpshooter flanges remain unsealed. Structural similarity of in āere sheaths are apparent in stylet sheaths formed in planta, in artificial diets, or in water. The use of 'Solvy', a dissolvable membrane, for intact stylet sheath isolation is reported. These observations illustrate for the first time this mode of stylet sheath synthesis adding to the understanding of stylet sheath formation in phytophagous hemipterans and providing tools for future use in structural and compositional analysis.

Figure 1. Scanning electron micrographs of D.citri in a¯ere stylet sheaths.

Panel A, is an image of two sheaths, the first (left sheath) is single-canal and the second (right) is a multi-branched sheath. Panel B is a non-gold sputtered image of Panel A with the white arrows indicating internal hollow canal tracks for the D. citri stylets. Panels C and D indicate the hollow canal of the D. citri stylets track with Panel D being an enlargement Panel C stylets canal opening. The opening is ∼3 µm in diameter. Panels E and F, indicate angular stylets probing (in āere) with panel E having a ∼93° bend within a single-canal sheath. Panels G and H are typical of linear sheaths formed in āere with panel G (white arrow indicating a closure of the stylet canal) suggesting secretion of sheath material as the stylet was retracted from the sheath.

Figure 2. Micrograph images of D.citri in a¯ere formed stylet sheath and flange, and D. citri labrum and stylet bundle.

Panels A – C are individual confocal slices, with Panel D being a merged composite image of Panels A – C confocal slice images. Panel D, white arrows, indicate the visible internal stylet canal traversing the central core of the stylet sheath extending from the base to the terminal end of the sheath. Panel E is a SEM micrograph of a D. citri labium, stylets, and labial tip sensilla (ss). Panel F is a single slice confocal DIC micrograph of a D. citri third instar nymph exuvial stylet bundle with a detached flange circumambient the stylets. Panel G is a TEM micrograph cross-section of an adult D. citri stylet bundle indicating interlocking maxillary (mx) with mandibular (md) stylets, salivary (sc), food (fc), and dendrite (dn) canals. Panel H is a SEM micrograph of an adult D. citri flange formed in a mock feeding chamber. White arrows designate the location of indentations in the flange are sensilla cavities (ssc) with the central protrusion (black arrow) indicating a mound of retraction secreted sheath (rss) material formed upon withdrawal of the stylets from the sheath.

Figure 3. P.pyriformis and F. virgata in āere formed stylet sheaths.

Panels A – C and D – F are P. pyriformis and stripped F. virgata associated micrographs, respectively. Panels A and D are stereoscopic views of a P. pyriformis and F. virgata (respectively) mock feeding across a clear plastic membrane, with red arrows indicating multi-branched air formed sheaths. Panels B and E are SEM micrographs of P. pyriformis and F. virgata multi-branched sheaths, respectively. Panels C and F are magnified SEM micrographs of selected terminal sheath branch tips from Panels B and E, respectively.

Figure 4. H.vitripennis, B. tabaci, and A. nerii in āere formed sheaths and flanges.

Panels A – D, E – H, and I – L are sheath and flanges from H. vitripennis, B. tabaci, and A. nerii, respectively. Panels A, B, E, F, I, and J are sheath and Panels C, D, G, H, K, and L are flange SEMs, respectively. For each, the sheaths are short and lack the branching as seen previously for D. citri, P. pyriformis, and F. virgata (for comparison see Figure 1 - Panels A and B, Figure 2 - Panel D composite, and Figure 4 - Panels B and E). Flanges for both the H. vitripennis (Panel C) and B. tabaci (Panel H) indicate an open access point into the central canal (designated by the black arrows) for the stylets. Panel D is an increased magnification SEM of the H. vitripennis flange opening for the stylets indicating the hollow canal for the stylets. Panels K and L indicate that the A. nerii flange are sealed closed by retraction secreted sheath material (green arrows). White arrows indicate sensilla cavities in the flange surface (Panels C, H and L), yellow arrows indicate the labial groove imprint on the H. vitripennis flange surface (Panel C), the red arrow indicating the coalescence of secreted whitefly ‘waxy’ cuticular lipid hydrocarbons shed and covering the flange (Panel G), and the black arrows (Panels B and H) indicate the open cavity of the stylet canal for both the H. vitripennis and B. tabaci flanges.

Figure 5. D.citri in a¯ere formed stylet sheaths from water-soluble Solvy™ stabilizer membranes.

Panel A image is DIC and panels B – D are SEM micrographs of D. citri Solvy™ isolated stylet sheaths. Structural features of these intact (attached sheath with flange) stylet sheaths are indicated including: ‘neck’ segments that correspond to the membrane traversing span connecting the flange and sheath segments (Panels A – D), the membrane sheath interface (msi) on the sheath face side (Panels A – D), continuous bulbous secretion formations and fork locations in the sheath segment (Panels C and D), or the flange (Panel D) segment.

Figure 6. Naturally formed D.citri stylet sheaths within leaf midrib of Duncan grapefruit (Citrus paradisi Macfadyen).

Panel A indicates a stylet sheath segment having bulbous stylet sheath formations (bssf) within the ground tissue region of the midrib that originated from a D. citri probe on the lower surface of the midrib. Propidium iodide fluorescence staining is used to provide contrasting resolution between the tissue types and the stylet sheath structure. Panel B is a magnified view of Panel A stylet sheath (ss) region minus fluorescence, indicating the bssf areas as well as the apparent stylet canal traveling parallel to the stylet sheath core. Panel C illustrates bssf continuing within the phloem tissue and additionally illustrates the multi-branching of the sheath portion within the phloem tissue. Micrographs are DIC.