The secretion process of liquid silk with nanopillar structures from Stenopsyche marmorata (Trichoptera: Stenopsychidae)

Abstract

Stenopsyche marmorata larvae spin underwater adhesive silk for constructing nests and capture nets. The silk can be divided into fiber and adhesive regions, according to their function. The silk fiber region has a two-layer structure: a core layer situated at the center of the fiber and S. marmorata fibroin, the major component of the silk. In the anterior part of the anterior silk gland, the morphological characteristics suggest that the silk insolubilization leading to fibrillation occurs by luminal pH neutralization. The adhesive region is composed of three layers: the outermost (OM), B, and C layers. On the B layer, coated with the OM layer, numerous nano-order pillar structures (nanopillar structures) are located at regular intervals. A nanopillar structure is approximately 40 nm in diameter and 125 nm in length. The precursor materials of the nanopillar structure are electron-dense globules of approximately 25 nm in diameter that are located in the A layer of the lumen of the middle silk gland. The precursor globules autonomously connect to one another on the B layer when the liquid silk is transported to the lumen of the bulbous region. The nanopillar structures probably contribute to the strong underwater adhesion of S. marmorata silk.

Figure 1. Schematic representation of Stenopsyche marmorata silk gland.

The silk gland may be divided into four sections according to differences in silk secretion and anatomical features. Scale bar, 0.5 cm. The inset shows the fifth instar larva. Scale bar, 1 cm.

Figure 2. Posterior silk gland of S. marmorata.

Above is a light micrograph of the longitudinal section. Scale bar, 20 μm. Below are the electron micrographs of the boxed area in the upper figure. (a) Electron-dense secretory globules (SG) of core layer components are seen in the lumen. Scale bar, 1 μm. (b) The lumen is completely filled with core-layer components by accumulation of the secretory globules. Core-layer components are seen in the apical cell (arrow). RER: rough endoplasmic reticulum; L: lumen; Mv: microvilli; Nu: nucleus. Scale bar, 1 μm.

Figure 3. Light micrograph of the middle silk gland.

(a) Transverse section of the silk gland. Core layer (C) stained strongly with azure B is located in the center of the S. marmorata fibroin layer (F). The outer layer (O), stained faintly, is situated adjacent to the apical cell. Scale bar, 50 μm. (b) Longitudinal section of the silk gland. The core layer (C) is observed as a column shape in the center of the lumen. Scale bar, 50 μm. (c) Enlargement of the boxed area in left micrograph. A large number of S. marmorata fibroin secretory globules (arrow) pass through the outer layer and accumulate in the middle silk gland lumen. Scale bar, 10 μm.

Figure 4. Electron micrograph of the middle silk gland lumen.

(a) The outer layer of Figures 3 is separable into three layers (A–C) by TEM observations. A layer: composed of a large number of electron-dense globules. B layer: electron-lucent elliptical materials are present in an electron-dense matrix. C layer: located adjacent to the S. marmorata fibroin layer (F) and has no internal structure. Numerous fine granules (S) and electron-dense globules (arrow) are seen around the apical cell, but it is not clear to which layer of the liquid silk these secretions correspond to. Scale bar, 1 μm. RER: rough endoplasmic reticulum. (b) High magnification of the A layer of the middle silk gland lumen. The electron-dense globules are approximately 25 nm in diameter and have almost uniform size and shape (arrow). Scale bar, 500 nm. B: B layer; FSG: S. marmorata fibroin secretory globules.

Figure 5. The bulbous region and the anterior part of the anterior silk gland.

(a) Light micrograph of the bulbous region and the anterior silk gland in longitudinal section. Liquid silk transported from the middle silk gland is coated with outermost (OM) layer components. Scale bar, 20 μm. F: S. marmorata fibroin layer; Nu: nucleus; I: intima. (b–d) Electron micrograph of the bulbous region in longitudinal section (boxed area 1 in Figure 5a). (b) Numerous nanopillar structures (arrow) are seen on the B layer (B) covered with the outermost (OM) layer. The electron-dense globules of the A layer cannot be observed. Scale bar, 2 μm. C: C layer; F: S. marmorata fibroin layer; RER: rough endoplasmic reticulum; Mv: microvilli. (c) The nanopillar structures are located on the separated liquid silk (probably the B and C layers), which has accumulated the outermost (OM)-layer components as well. Note that the OM-layer components are observed as a fine fibril (arrow). Scale bar, 1 μm. (d) The nanopillar structure has several round segments that look like a string of beads, which appears to be composed of the globules contained in the A layer of the middle silk gland lumen. Scale bar, 500 nm. B: B layer; C: C layer; OM: outermost layer. (e) Electron micrograph of the anterior part of the anterior silk gland (boxed area 2 in Figure 5a). The intima (I) is composed of bundled microvilli-like structures and has many vesicles that include electron-dense granules in the apical surface (arrow). Scale bar, 5 μm. F: S. marmorata fibroin layer; OM: outermost layer.

Figure 6. Electron micrograph of the middle part of the anterior silk gland.

(a) Longitudinal section of the silk gland. The liquid silk just before spinning underwater is seen in the lumen. In the anterior silk gland, which functions as a duct, the inner surface is covered with a thick cuticular intima (I). Scale bar, 1 μm. C: core layer; F: S. marmorata fibroin layer; OM: outermost layer. (b) Transverse section of the liquid silk. The nanopillar structures located at regular intervals (arrow) were observed on the B layer (B), which is coated by the outermost (OM) layer. The average diameter and height of the nanopillar structures are approximately 40 nm and 125 nm, respectively. Scale bar, 500 nm. C: C layer; F: S. marmorata fibroin layer.

Figure 7. Electron micrograph of hardened silk.

(a) Oblique section of the hardened silk. The silk consists of a pair of silk fibers 6–8 μm in width with an oval cross section, which can be divided into four layers: core layer (C) with a shape of column in the center of the fiber, S. marmorata fibroin layer (F) composed of numerous fibrils, outer layer (O) containing minute spherical materials, and the outermost (OM) layer gluing the pair of fibers. Scale bar, 2 μm. (b) Longitudinal section of the hardened silk. Numerous fibrils are seen in S. marmorata fibroin layer (F). Scale bar, 2 μm. C: core layer. (c) Transverse section of the outermost layer. The outermost layer components adhere to nanopillar structures (arrow). Scale bar, 500 nm. F: S. marmorata fibroin layer; O: outer layer. (d) Transverse section of the silk used as capture net. In the surface of capture net silk, usually the outermost layer is peeled off by a water stream, and the nanopillar structures are exposed on the surface. Microorganisms as feed for the larvae are captured on the nanopillar structures (arrow). Scale bar, 1 μm. F: S. marmorata fibroin layer; O: outer layer. (e) Cross section of the adhesion layer between the silk and a substrate (leaf). The silk adhesive region is observed as a nonstructural monolayer. The nanopillar structures appear to enter into fine surface roughness of the substrate (arrow). Scale bar, 1 μm. F: S. marmorata fibroin layer; L: a leaf.

Figure 8. Schematic representation of the nanopillar formation process.

(a) The nanopillar precursor materials are present as a large number of electron-dense globules in the A layer of the middle silk gland lumen. (b) Several globules connect to each other to form nanopillar structures on the B layer when the liquid silk is transported to the lumen of the bulbous region. (c) In the anterior silk gland, nanopillars of almost uniform size and shape are situated at regular intervals on the B layer, which is coated with the outermost layer. A: A layer; B: B layer; C: C layer; OM: outermost layer.