Nanopores in the ventral scales of Bitis rubida and Bitis armata cause white venters

Recent studies speculated that some snakes developed white venters to avoid overheating caused by highly radiative soil and rocks. Here, we present the scale-embedded porous nanostructures through which some snake species of the genus Bitis achieve such whiteness. Our analysis reveals nanopores causing scattering underneath the external surface of the white ventral scales of Bitis rubida and Bitis armata. Such nanopores are not present in the scales of Bitis parviocula, Bitis arietans, and Bitis rhinoceros that appear transparent or translucent to the naked eye. White ventral scales with nanopores reflect up to 40% of light in the visible regime. The reflection, however, decreases for longer wavelengths and drastically reduces in the infrared. In contrast, a much lower, almost constant reflection around 8% between 250 nm and 2500 nm is observed for the transparent or translucent ventral scales without nanopores. Our study demonstrates that some snake species of the genus Bitis utilize a light scattering network of nanopores underneath their external surfaces to create white ventral scales.

Methods
Optical Spectroscopy: The total transmittance and reflectance spectra of the ventral scales were determined using a Cary 7000 spectrophotometer with an integrating sphere (DRS attachment, Agilent, USA). Unpolarized light was used to measure spectral properties, shining incoming light beams on the outer surfaces of the moulted snake scales close to normal incidence. The measured spectrum range was set to 250 to 2500 nm with a spectral resolution of ≈ 1 nm and a beam spot diameter about 2 mm. This spectroscopy range covers most of the solar spectrum. The NIST calibrated Spectralon® diffuse reflectance standard (Labsphere, USA) was used to define a reference reflection.

Atomic Force Microscopy: To conduct a topological analysis of the scales’ surface by atomic force microscope (AFM, Dimension Icon, Bruker), the scales were cut into small pieces. Two-component glue (UHU End-fest, UHU GmbH & Co. KG) was used to attach the samples to a glass slide for AFM imaging. The prepared samples were carefully cleaned with pressured air. Afterward, the samples were imaged in tapping mode utilizing rectangular silicon cantilevers (All-in-One-Al, Budget Sensors, Type C) as sensors.

Scanning Electron Microscopy: Scanning electron microscopy (SEM, SUPRA 60 VP, Zeiss, Germany) was applied for imaging the cross-sections of ventral scales. For that, the samples were carefully cut into pieces with a sharp razor blade and sputtered with a thin silver layer. The imaging was conducted with an acceleration voltage of 5 kV and the detector was placed at a working distance of 5 to 7 mm.