A study of ladder-like silk foothold for the locomotion of bagworms

Bagworm walking method using a ladder-like silk foothold

When bagworms are reared in a plastic or glass cage, they walk not only on the floor but also on the walls or ceiling using only their three pairs of thoracic legs. The method by which they achieve this was clarified by placing a bagworm on black paper. Where the bagworm had walked, a ladder-like silk trace was observed on the black paper (Fig. 2a). Scanning electron microscopy (SEM) observation of one of the steps (or rungs) of the ladder-like trace revealed that each step was made up of a zigzag pattern of silk threads (Fig. 2b). Further magnified SEM observations revealed that the folded parts of the zigzag-spun thread were glued selectively to the substrate with adhesive whereas the remaining straight parts (hereafter, termed ‘bridges’ or ‘bridge threads’) were unglued (Fig. 2c–e).

Notably, the steps of the foothold were not independent but rather always connected with neighbouring steps via a single thread (Fig. 2f). The overall basic construction of the foothold is schematically depicted in Fig. 2g. We found that the foothold was constructed in one continuous movement and always made of a single thread regardless of walking distance or time; therefore, a continuous thread exceeding a length of 100 m could be collected from one foothold¹⁴. We also observed bagworm climbing behaviour on a transparent plastic board, which clarified the important role of the silk trace as a foothold (Fig. 2h). During this behaviour, the bagworm used its sickle claws (Fig. 1e) to hook its second and third pairs of thoracic legs onto the first and second newest steps, respectively, and constructed the next step by spinning silk with a zigzag motion of the head and the skilful use of the first pair of thoracic legs. When the bagworm advanced one step, it always first shifted its third pair of thoracic legs to the next step before then shifting its second pair of thoracic legs to the newest step to avoid overloading this step, which may not yet be fully adhered to the surface (see Supplementary Movie S1). Because of this construction method, the interval distance between neighbouring steps is automatically determined by the interval between the thoracic legs. By repeating this process, the bagworm can advance forward slowly but steadily. This walking method was commonly observed on a horizontal floor surface, vertical wall, or horizontal ceiling. Although we have mainly described and shown observations from E. variegate here, with the exception of Supplementary Fig. S4 and Movie S1, we also observed instances of walking behaviour in other species, namely Eumeta minuscula, Mahasena aurea, Nipponopsyche fuscescens and Bambalina sp. (for a movie on E. minuscula walking behaviour, wherein it climbs a vertical wall, see Supplementary Movie S2). For at least 100 individuals of these bagworm species, we observed essentially identical walking behaviour to that described in the present study without exceptions for locomotion on substrates with slippery surfaces.

Based on our observations, we asked the following question: how do bagworms selectively glue the folded parts of the foothold onto the substrate? Real-time observation of the tip of the spinneret (i.e. the spigot) through a transparent plastic board during the construction of the foothold revealed that adhesive was selectively discharged to attach the folded parts to the substrate; this process could be distinguished from the continuous spinning of the silk thread (for a movie showing construction behaviour, see Supplementary Movie S3). Figure 3a–g shows a time-sequence of foothold construction with enlarged images in the vicinity of the spinneret provided, whereas Fig. 3h depicts a schematic trace of the construction process. It was clearly noted that the bagworm discharged the adhesive only at the folded parts (shown in Fig. 3a–c,e,f; termed the ‘glued turn’) and not at the straight bridge parts (shown in Fig. 3d,g; termed the ‘unglued bridge thread’). From these time-sequence observations, we concluded that the bagworm controls the discharge of adhesive in an ‘on and off’ manner as necessary (essentially the same construction behaviours were confirmed for at least 20 individuals).

Passages of fibroin brins and adhesive

We next investigated the spinning mechanism that enables continuous spinning of silk thread together with the selective discharge of adhesive via a single spigot. To this end, we observed the morphology of the bagworm from the silk gland to the spigot. Figure 4a shows the area in the vicinity of the spinneret, dissected and isolated from an E. variegata bagworm, which included a pair of silk glands and plural adhesive glands. As we previously reported²¹, the exterior shape of the silk gland in E. variegata (see Supplementary Fig. S1) is almost the same shape as that in the silkworm Bombyx mori and it is subdivided into three parts: the anterior (ASG), middle (MSG) and posterior (PSG) silk glands. We also previously confirmed that fibroin heavy chain (h-fib), fibroin light chain (l-fib) and fiboinhexamerin genes are expressed dominantly in the PSG, while sericin is expressed in the MSG, which strongly suggests that division-selective production of each protein exists in E. variegata (as has been shown in B. mori²²). Figure 4b shows a magnified image of the spinneret including the end of the ASG. Beyond the pair of ASGs, which are merged into a common tube, a silk press and spinning tube appear before the spigot. This basic passage of silk fibroin from the ASG to the spigot is essentially the same as the passage observed in B. mori²³. However, more detailed morphological observations of the inner structure of the passage revealed several obvious differences between E. variegata and B. mori.

Cross-sectional images along the spinneret are shown in Fig. 4c–j; these focus on the silk brins and their passage (unmagnified versions of the images in Fig. 4f–j are shown in Supplementary Fig. S2). The fibroin brins have an approximately round cross-sectional shape at the end of the ASG (Fig. 4c) and are merged at a common tube, which deforms their round shape slightly (Fig. 4d). The fibroin brins seem to be coated with a thin layer of sericin after the MSG, similar to B. mori; however, we omit the presence of the sericin layer here for convenience. The paired brins are gradually pressed between the ventral and dorsal hard cuticle plates at the silk press, and a gradual diameter decrease and shape deformation follows (Fig. 4e,f). At the exit of the silk press, each brin becomes elliptic and the diameter in the major axis decreases. Interestingly, the elliptical shape and 1.7-axial ratio for the major and minor axes of the fibroin brin cross-section in bagworm silk, which we previously reported¹⁴, are already determined at this stage in the silk press; afterwards, the diameter decreases without any change in the axial ratio of the elliptical cross-section. Notably, the two elliptical fibroin brins are aligned side-by-side so that their major axes are in line horizontally (to resemble a figure of ‘∞’) at the spinning press, and these are followed by the spinning tube (Fig. 4e–h). However, the alignment is twisted by 90° in one direction (to resemble a figure of ‘8’) before the brins are spun from the spigot (Fig. 4i,j).

We found that the spinning tube was surrounded by a hard exoskeleton. Using 3D-X-ray CT observations, we produced clear images of the exterior and interior morphologies of the spinning tube enveloped by exoskeleton (Fig. 4k–m; the exterior shape observed from the dorsal-, ventral- and lateral-sides by optical microscopy is provided in Supplementary Fig. S3). The spigot was not cut perpendicularly to the spinning tube but rather with a slope of around 20°; consequently, it was elliptic. X-ray CT clearly showed the core-sheath structure of the spinneret and a wide expanse of sheath parts (Fig. 4m) between the exterior shell and interior spinning tube (Fig. 4l,m). Using optical microscope observations of the cross-sections, we found that at least three pairs of adhesive ducts were running in the sheath space (Supplementary Fig. S2E). Therefore, while the silk brins pass through the central narrow spinning tube, the plural adhesive ducts pass through the outer space independently of the silk thread. Finally, the adhesive enters a ladle-like reservoir located at the spigot and is released together with the silk thread (Fig. 4o). The presence of definitive routes connecting the adhesive passage and the spigot were not clearly observed in our X-ray CT images, probably due to the small structural scale relative to the space resolution used in our analysis (i.e. 0.31 μm). We speculate that the adhesive merges into the spigot via a fine, porous sponge-like structure, and we indicate assumed routes in Fig. 4l,m. X-ray CT observations also revealed a sophisticated structural design involving gradual twists in the silk brins by 90° from ‘∞’ to ‘8’ (Fig. 4n and Supplementary Movie S4). Essentially identical spinneret structures were observed by X-ray CT images for all of eight observed individuals from the third to final instars of E. variegata.

Source: Ecology - nature.com