Micro- and nano-structural details of a spider's filter for substrate vibrations: relevance for low-frequency signal transmission

Abstract

The metatarsal lyriform organ of the Central American wandering spider Cupiennius salei is its most sensitive vibration detector. It is able to sense a wide range of vibration stimuli over four orders of magnitude in frequency between at least as low as 0.1 Hz and several kilohertz. Transmission of the vibrations to the slit organ is controlled by a cuticular pad in front of it. While the mechanism of high-frequency stimulus transfer (above ca 40 Hz) is well understood and related to the viscoelastic properties of the pad's epicuticle, it is not yet clear how low-frequency stimuli (less than 40 Hz) are transmitted. Here, we study how the pad material affects the pad's mechanical properties and thus its role in the transfer of the stimulus, using a variety of experimental techniques, such as X-ray micro-computed tomography for three-dimensional imaging, X-ray scattering for structural analysis, and atomic force microscopy and scanning electron microscopy for surface imaging. The mechanical properties were investigated using scanning acoustic microscopy and nanoindentation. We show that large tarsal deflections cause large deformation in the distal highly hydrated part of the pad. Beyond this region, a sclerotized region serves as a supporting frame which resists the deformation and is displaced to push against the slits, with displacement values considerably scaled down to only a few micrometres. Unravelling the structural arrangement in such specialized structures may provide conceptual ideas for the design of new materials capable of controlling a technical sensor's specificity and selectivity, which is so typical of biological sensors.

Keywords: chitin; mechanosensing; spiders.

Figure 1.

(a) Adult female spider Cupiennius salei. (b) Magnification of the two last (distal) leg segments; the metatarsus and the tarsus. The cuticular pad is situated at joint between the two segments (arrow). (c) Optical light microscope image of the cuticular pad and the vibration receptor of the spider. Top view on the dorsal side of the pad. The distal direction is marked by a grey arrow. White arrowhead indicates vibration-sensitive metatarsal lyriform organ. (d) CLSM view of the pad in (c). The image is constructed by a superposition of the auto-fluorescence signals (as maximum intensity projection) of excitation/detected emission wavelengths: 488/499–555 nm (green channel); 561/578–678 nm (red channel).

Figure 2.

(a) Surface rendering of the reconstructed µCT data of the pad. Virtual sections representing sample sections used in this study are indicated by three differently coloured slices: pink, blue and red (b) Schematic of the shape for the slices shown in (a). (c–h) µCT virtual slices along the long axis of the leg (sagittal plane) laterally extending from the pad centre (c) to the pad lateral edge (h). The dashed lines show the outline of the pad traced along the organ. The line was determined using a number of successive images.

Figure 3.

(a) Surface rendering of reconstructed µCT data showing three selected components of the metatarsal vibration receptor including the tarsus (blue), the pad (green), and the slit-sensilla lyriform organ (pink) measured during contact. The deflection angle between the tarsus and metatarsus was 9°. (b) Three-dimensional shape of the cuticular pad extracted from (a). Grey regions at the distal side of the pad indicate the contact area with the tarsus. (c) Three-dimensional shape of the cuticular pad under load with a slight lateral component. The tarsus–metatarsus angle was 8°. Grey regions at the distal side of the pad indicate the contact area with the tarsus. (d–f) µCT virtual slices of the sample in a–b sectioned in the sagittal plane in the centre of the pad in relaxed (d) state (less than 0°), and deflected by 9° (e). The dashed lines indicate the outline of the cuticular material of the pad. The white arrows indicate one slit of the metatarsal lyriform organ. Darker region below the ventral side of the pad is caused due to reduction in vapour pressure; the pad itself however is still moist. (f) An overlay of the pad shape from d and e.

Figure 4.

(a,b) Autofluorescence signal (maximum intensity projection) of a longitudinal section (thickness 30 µm) of the pad in (a) wet and (b) dry states. Excitation/detection emission wavelengths: 488/499–555 nm (green channel); 561/578–678 nm (red channel). (c) Maximum intensity projection of a longitudinal section of the metatarsal exoskeleton (thickness 30 µm) in wet state. (d) Polarized light microscopy image of the pad in wet state. The white line indicates the outline of the pad. The orientation of the polarizer-analyser is indicated by the white cross. (e) SAM image of the pad sagittal section. The picture consists of two merged images (white line) obtained from two samples measured at the same experimental conditions. The colour code indicates the reflectivity distribution for acoustic waves. Regions a–d indicate the positions chosen for nanoindentation measurements on the same samples.

Figure 5.

X-ray scattering analysis of the pad slice cut in sagittal plane. (a)(i) Characteristic pattern of radially integrated X-ray scattering measured at the dorsal part of the pad in its wet state. The pattern contains both SAXS and WAXS scattering regions. The SAXS peak around q = 1.3 nm⁻¹ is assigned as the packing peak from chitin fibrils (marked with *). The main chitin diffraction peaks in the WAXS region are indicated. Right: SAXS region radially integrated X-ray scattering pattern plotted in a double log scale, extracted from the distal region of the pad in wet and dry states. The dashed lines represent slopes of −1 (red), and of −2 (black). (b) Intensity map assigning different components contributing to the X-ray scattering patterns as shown by example in (a). Cyan colour intensity: diffuse scattering intensity at lowest measured scattering vectors (q = 0.37–0.45 nm⁻¹) and representing scattering from nanometre-sized cuticle components. Yellow colour intensity: integrated scattering intensity from (110) and (013) chitin diffractions (fitted peak areas), representing the distribution of chitin. Magenta intensity: the packing peak intensity (fitted peak area) in the SAXS region, representing the in-lattice ordering degree of chitin fibrils. Blue solid line indicates the outline of the pad. (c) Representation of three-dimensional orientation of chitin fibrils in the pad. Data analysis was based on the non-symmetric azimuthal distribution of the (110) chitin crystal peak in the WAXS region ([31]). Bars show the mean orientation of the chitin fibrils in different parts of the pad. The colour code indicates the chitin tilting angle out of the sample plane. Left: examples of the two-dimensional scattering pattern: in-plane fibres (upper) and out of plane (lower). (d) Orientation of the nanometre-sized objects extracted from the low-q signal and that of chitin fibrils extracted from the packing peak. Both orientations were determined from the non-isotropic azimuthal distribution of the respective SAXS signal. Note that only measurement points where both components show preferred orientation simultaneously are shown.

Figure 6.

(a) SEM image of the dry fractured pad section (in sagittal plane). The distal and the dorsal directions are indicated by black and white arrowheads, respectively. The white arrows point to micro-channels within the chitin sub-structure of the pad. (b,c) AFM images: topography (b); phase (c) of the distal surface of the pad. (d,e) AFM images: topography (d); phase (e) of the dorsal surface of the pad.

Figure 7.

A scheme of the pad's performance in the low- and high-frequency working ranges of the metatarsal vibration sensor. Blue line: typical threshold (tuning) curve, adapted and schematized from ref. [8], showing the displacement of the substrate (and tarsus) necessary to elicit a response of the sensory cells at different frequencies. At low frequencies below ca 40 Hz large displacements are required. According to our present results this is because much of the tarsal (T) displacement at its contact area with the pad (P) is used to compress the pad's hydrated and soft frontal part (upper inset). At high frequencies, however, minute displacements suffice to elicit a nervous response (lower inset). Then the epicuticle on the pad's front side undergoes glass transition, implying sufficient stiffness to transmit the mechanical stimulus to the slits (S) of the vibration receptor much more effectively than at low frequencies [15]. Arrows show load direction. Yellow lines indicate the contact area between the tarsus and the pad. T, tarsus; P, pad; S, slits of the vibration receptor; MT, metatarsus.