Color-specific porosity in double pigmented natural 3d-nanoarchitectures of blue crab shell

Abstract

3D-engineered nano-architectures with various functionalities are still difficult to obtain and translate for real-world applications. However, such nanomaterials are naturally abundant and yet wasted, but could trigger huge interest for blue bioeconomy, provided that our understanding of their ultrastructure-function is achieved. To date, the Bouligand pattern in crustaceans shell structure is believed to be unique. Here we demonstrated that in blue crab Callinectes sapidus, the 3D-nanoarchitecture is color-specific, while the blue and red-orange pigments interplay in different nano-sized channels and pores. Thinnest pores of about 20 nm are found in blue shell. Additionally, the blue pigment co-existence in specific Bouligand structure is proved for the green crab Carcinus aestuarii, although the crab does not appear blue. The pigments interplay, simultaneously detected by Raman spectroscopy in color-specific native cuticles, overturns our understanding in crustaceans coloration and may trigger the selective use of particular colored natural nanoarchitectures for broaden area of applications.

Figure 1

Summary display of the experimental approach comprising resonance and non-resonance Raman micro-spectroscopy, HR-SEM, EDX, XRD of blue, red, white and green exoskeleton of C. sapidus and C. aestuarii crabs in native form. Blue shells turning red when cooked or solvent extracted is molecularly illustrated by Raman spectroscopy.

Figure 2

Multi-peak Lorentzian fit of raw RR spectra (1425–1600 cm⁻¹ range) acquired from blue Callinectes sapidus claw shell (a,c), and green Carcinus aestuarii claw shell (b,d), using two laser lines, as indicated. Profile broadening via concentration was assimilated with pressure broadening, thus, Lorentzian fit was considered for multi-peaks deconvolution. Corresponding coefficients of determination (R²) are displayed in the figure. Note the effect of resonant excitation of pigments: deconvolutions of spectra excited with 532  nm featured stronger carotenoid modes above 1500  cm⁻¹, while ncb-ATX mode at 1492  cm⁻¹ is always dominant as “blue band” in spectra excited with the 632.8  nm line. Additional -CH₂ mode (filled black band) at 1445  cm⁻¹ along with other carotenoid species with minor overall contribution, are highlighted.

Figure 3

Mapping the Raman intensity distribution of the astaxanthin (ATX) v₁(C=C) band at 1514  cm⁻¹ and its non-covalently bound counterpart (ncb-ATX) at 1492  cm⁻¹ from spectra collected from the blue Callinectes sapidus and green Carcinus aestuarii crab cuticle surface. Excitation: 532  nm. A1,2 and B1,2 show the light microscopy images taken via Raman microscope with 5x or 20x objective respectively, while A3 and B3 display the two pigments interplay over the mapped area highlighted in rectangle in A2 and B2. A4 and B4 show the rough Raman signal of the respective cuticle at the cross-hair of the maps. Note the spatial interplay of the two pigments contribution.

Figure 4

Pigments distribution in the cross section of blue cuticle (a) along with the normal-to-surface direction: (b,c) micrographs taken via Raman microscope during measurements; (d) RR spectra of ATX collected from surface points along the shell transect, from margin to the inner layers (shell depths) from 20 to 420  µm as indicated on each spectrum; (e) ATX Raman C=C mode (1514  cm⁻¹) intensity distribution against depth; the data fit showed a Lorentzian profile with two components, peaking at 130 and 260 μm respectively; (f) RR spectra of ncb-ATX taken from cross section surface (shell depth from 20 to 240 μm); and its main band (1492  cm⁻¹) intensity variation along cross section (g), showing the highest intensity in epicuticle. Error bars show the standard deviation.

Figure 5

XRD diffractograms of the powdered crab cuticles as indicated, showing the Mg-calcite pattern. FWHM values of the main peak are inserted. Additional signal from crab tooth (e) is showed to highlight its higher crystallinity compared to the claw cuticles. Q- denotes quartz peak probably originated from benthic diatoms inhabiting the cuticle and/or spurious sand micro-grains (especially in the claw teeth). The weak band centred at 19° 2θ was assigned to α-chitin.

Figure 6

(A) Representative SEM images collected from blue cuticle, highlighting the channels and the regular arrays of nano-pillars separated by pores in the ultrastructured channel walls. Schematic representation of the pores and channels arrangement is displayed in the top right corner. The channel width of about 450  nm is comparable with the blue light wavelength. (B) Comparative SEM images from the four coloured shell, blue, green, white and red, showing nanoscale details of color-characteristic pores. (C) The averaged distances between pores and canals and their diameter for the white, blue, green and red cuticle, as indicated on each graph. Error bars show the standard deviation; statistical difference for values of p ≤ 0.05 were considered significant (and represented by*), p ≤ 0.01 very significant (and represented by**), p ≤ 0.001 extremely significant (and represented by***).