Tetranychus urticae is a phytophagous mite living in group. Every individual produces silk strands and constructs a common web for the colony. In spite of the silk value for T. urticae survival, silk remains poorly studied. We developed a technique to dye the silk on both inert and living substrates. Fluorescent brightener 28 was used to visualize the silk. This technique will help to carry out future studies about the web architecture of T. urticae and other silk-spinning arthropods.

Introduction

Silk displays a diversity of material properties and chemical constituents. Silks can be derived from several glands with different evolutionary origins [1]. Functions of silk strands in arthropods are numerous: protective shelter, structural support, reproduction, foraging, dispersal [1], life lines or as a support for chemical communication among individuals [2].
Tetranychus urticae is a major pest of many cultures. One particularity of T. urticae is its continuous silk deposition while walking [3]. In the case of T. urticae, silk was reported to play four main purposes: protection against biotic agents [4], protection against abiotic agents [4], sex pheromone substrate [5], lomotion and dispersion [6].
Previous works allowed a better understanding of T. urticae silk roles but the silk itself remains poorly studied. In spite of the silk value for T. urticae survival, web architecture is still unexplored. Visualizing this product could be useful for exploring different domains like the link between mite movement and silk deposition, variation in architecture, role in social organization and influence of the substrate. Johnson et al. [7] have already developed a method to reveal the silk of Helicoverpa armigera neonates on a plant surface using the Fluorescent Brightener 28 (FB). They [7] specified that their protocol could stain all types of arthropod silk including tetranychid mites but they did not get optimal results for silk networks from infested plants and they did not try for silk on glass lenses. Therefore, we developed here new protocols by using the same fluorescent dye in order to optimize silk visualization on inert and living substrates, including web under natural T.

urticae environment.

Materials and Methods

Rearing of mites. Spider mites were reared on bean (Phaseolus vulgaris) in climate room under 26 °C, with a relative humidity of 50-70 % and a photoperiod of L16:D8.
Silk collection. On glass lenses: one individual was deposited onto the lens (L1) during a period of 30 minutes. The mite was then removed from the lens. On bean leaves: one individual was deposited during 48h on a bean leaf circle (B1). After two days, the mite was removed of the leaf. For the web located between two leaves : samples were collected directly on 12-days infested beans.
Staining of silk : the dye used was the Calcofluor White M2R (Sigma-Aldrich NV/SA, Belgium) also known as Fluorescent Brightener 28. Two solvents were tested: water [7] and dimethyl sulfoxide (DMSO) [8]. Two elutions were tested by solvent: 0.1 % [7] and 0.2 % in alkaline-distilled water; 0.5 % and 1 % in DMSO. To facilitate the solubilization process in water, 39 µl of 10 M KOH was added to 100 ml stock solution. Solutions were mixed until the dissolution of the FB powder and filtered twice with 11 µm cellulose filters.
The two substrates, glass lenses and bean leaves, required two different staining methodologies. For the glass lenses : T. urticae silk was sandwiched between two glass lenses. Five µl of staining solution was deposited on a clean glass slide (L2). The lens with the face where the mite laid its silk (L1) was deposited downwards on the solution (5 µl). The glass lens (L1) remained on the glass slide (L2) for 5 min. To speed up the drying processus, the set-up (L1 + L2) was then placed in an oven (100 °C) during 5 minutes for the water solution and 90 minutes for the DMSO solution. For the leaves : the leaf was dipped into water (0.1, 0.2 %) and DMSO (0.5, 1 %) solutions for 20 min [7]. The leaf was then dried out on a glass lens at 26 °C. For the web located between two leaves : the web was carefully sandwiched between a glass lens and a glass slide with 5 µl solution on it. Then, the lenses were placed in an oven (5 min for W and 90 min for DMSO). Table 1 summarizes the methods used to visualize T. urticae silk.
Observation of the silk : silk images were captured using a Konica FT-1 camera placed on a Polyvar Reichert- Jung microscope. Photos were taken using a 10X ocular and a 10X magnification (Fig. 1), under a mercury vapor lamp OSRAM (330-380 nm wavelengths).

Visualization: Results, Discussion and Perspectives