Crocodiles are endowed with a multitude of highly sensitive skin receptors in their robust armored skin. Specialized cutaneous mechanoreceptors in “integumentary sensory organs” (ISOs) are scattered on the head in Alligatoridae and along the body costume in Crocodylidae and Gavialidae. Physiological measurements have shown their exceptional sensitivity, which may even exceed that of primate fingertips (Leitch and Catania, 2012). Their morphology has been thoroughly studied over the years using both light and transmission electron microscopy (TEM) (von Düring, 1973; Monica von Düring and Miller, 1979; Jackson Butler, 1996; Di-Poï and Milinkovitch, 2013). Free nerve terminals or discoid receptors (DRs) are closely associated with a tonofibril network in the upper layer of the epidermis derived from keratinocyte and transitional desmosomes (TDs). More recent studies have shown that junctional complexes, such as desmosomes, are dynamic and not static (Han Yoon et al., 2001; Windoffer et al., 2011) and may even be integrated parts of a cell signaling pathway where cadherin-based mechanotransduction and keratin intermediate filament dynamics play essential roles (Green and Jones, 1996; Han Yoon et al., 2001; Leckband et al., 2011; Todorović et al., 2013; Hatzfeld et al., 2017; Green et al., 2019). This motivated us to further analyze the fine structure to enhance our understanding of how mechanical force is relayed across skin corneocytes and keratinocytes to the sensory receptors in the outermost layer of the epidermis in the Cuban crocodile (Crocodylus rhombifer).

Two male juvenile specimens of the Cuban crocodile (Crocodylus rhombifer) approximately 1 year of age were anesthetized using Ketamin 5 mg och medetomidin 0.05 mg and euthanized using an intracardial injection of T-61 0.4 mL. Macroscopic images were taken of cranial and abdominal regions using a Samsung Galaxy A56 5G Camera to document ISO receptors. The skin over the maxiallae, mandibulae and abdominal skin were removed and immersed in 2.5% glutaraldehyde and 1% paraformaldehyde (PFA) in 2.5% phosphate buffer. The tissue was placed in 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon. The embedded specimens were divided into different pieces and mounted for semi-sectioning. Sections were stained in toluidine blue and photographed using an Olympus BX63 microscope. Areas of interest were thin-sectioned, and the sections were stained in lead citrate and uranyl acetate and examined at 80 kV in a Tecnai™ G2 Spirit transmission electron microscope (Thermo Fisher/FEI Company, Eindhoven, NL). Images were acquired using an ORIUS™ SC200 CCD camera (Gatan Inc., Pleasanton, CA, United states) using Gatan Digital Micrograph software.

During their evolution, crocodylians have developed robust keratinized skin for their protection and homeostasis at land dwelling and under aquatic conditions. Nonetheless, molecular and electrophysiological analyses show that their skin is highly sensitive to mechanical, thermal, and pH stimuli but not to osmotic stimuli. This is accomplished through a multisensorial ISO containing neural complexes expressing a set of specific transduction channels (Leitch and Catania, 2012; Di-Poï and Milinkovitch, 2013).

We focused on the free nerve endings located in the granular keratinocyte layer, which are alleged to serve as mechanoreceptors. The present data validate earlier notions that the crocodile possesses a complex epidermal desmosome/tonofibril force-transmitting system in the cutaneous ISO receptors that may stimulate nerve endings as integrated mechanosensing units (Monica von Düring and Miller, 1979). Novel investigations have revealed desmosomes’ roles not only as cell‒cell adhesives to preserve tissue integrity but also as signaling pathways controlling epithelial shape, polarity, function, and modulation of cell behavior (Hatzfeld et al., 2017; Müller et al., 2021). We speculate therefore that the crocodiles desmosomes’ role is to relay external force reception (e.g., pressure and vibration) that deforms the surface of the stratum corneum followed by conduction via a system of CDs and TDs into a keratinocyte TF network surrounding the free nerve endings in the stratum granulosum. This meshwork may act as a mechanical lever system, collecting and focusing force directly onto the intraepidermal nerve ending along desmosome cadherin complexes in response to tension. Different types of desmosomes may anchor the keratinocyte cytoskeleton via the TF system. The keratin intermediate filaments organize into bundles of TFs that exhibit remarkable motile and dynamic properties in vivo (Han Yoon et al., 2001). In crocodiles, the desmosome TFs seem to create a continuous, mechanically-linked web that runs through the surrounding keratinocyte. Cadherin proteins within the desmosomes could be critical molecular links in this chain (Kowalczyk and Green, 2013). When desmosomes are displaced, they may pull on the TF network, creating tension. The variety of desmosomes and CDs may focus and amplify gradient force via the TF “hugging” system of the receptor, thereby gathering mechanical strain from a relatively large area of the epidermis and concentrating it onto the small surface of the DR. This may ensure that any deformation of the surrounding keratinocytes is efficiently and reliably transferred to the nerve membrane, allowing the crocodile to detect very subtle stimuli (Figure 9).

The innermost layer of the electron-dense corneocytes was connected to the outermost keratinocytes via a pearl-string-like layer of TDs, from which dense bundles of intermediate filaments coalesce into the keratinocytes´ cytoplasm. TDs may serve as an important bridge to transfer forces from the stratum corneum to the stratum granulosum. TDs lack a cytoplasmic plaque on the corneocyte side and are restricted to the interface between the stratum granulosum and the stratum corneum (Al-Amoudi et al., 2005). Their intercellular midline changes with thickening of the desmosome plaque into electron-dense structures in the stratum corneum. TDs transform into CDs in the stratum corneum, providing strong intercellular adhesion. The extracellular core of CDs has been shown to contain a basic glycoprotein named corneodesmosin, which is specific to cornified epithelia (Matsumoto et al., 2008). CDs seem not only to represent desmosome remnants but also play specific roles in the upper layers of the epidermis (Jonca et al., 2010). The TDs reinforced with proteins such as corneodesmosin are believed to be broken down by proteases to allow for skin shedding or desquamation (Lin et al., 2012). During transition, desmosome plaques fuse, ultimately leading to their degradation and shedding at the surface of the skin.

Mechanical tension generated in the TF system may physically deform the nerve endings’ plasma membrane; a key event that opens mechanosensitive ion channels leading to an influx of ions, depolarization, and ultimately an action potential (Kung et al., 2010; Haswell et al., 2011). Even though large clear vesicles occur in the DR, evidence strongly points to a primarily mechanical activation of the DRs. The direct and extensive physical connection between the TF network and the nerve terminal is the most compelling evidence (Leitch and Catania, 2012). Furthermore, there were no gap junctions, which argues against direct electrical signaling between granular keratinocytes and the nerve. The dense arrangement of TFs “hugging” the receptor is likely crucial for the receptor’s function and sensitivity. There may be two primary functional reasons, such as force focusing and amplification by the TF network acting to gather mechanical strain from a relatively large area of the epidermis and concentrate it onto the small surface of the discoid receptor. This would significantly amplify the signal, allowing the crocodile to detect subtle stimuli. The tight coupling ensures that any deformation of the surrounding keratinocytes is efficiently and reliably transferred to the nerve membrane, suggesting a high degree of sensitivity and fidelity where the keratinocytes and the nerve ending function as a single integrated system.

The presence of larger vesicles in the nerve terminal does not necessarily imply classical chemical neurotransmission. These findings contrast to the Merkel cell complexes in the dermis, which have clear synapse-like structures and dense-core vesicles. This suggests that the crocodile has at least two distinct mechanosensory systems: one based on direct mechanical linkage (DR, Pacini corpuscles) and another (MCs) involving chemical synapses likely for detecting different qualities of touch. Notably, the MCs were located in the dermis layer, unlike in mammals, where they are mostly located at the stratum basale.

Intriguingly, a thin mucous coat was observed along the outer border of the skin, which has also been documented in amphibian skin by Farquhar and Palade (1965). Its role in transmitting mechanical force under different aquatic and terrestrial conditions remains speculative.

Crocodiles seem to have reached the most sophisticated and elegant solutions for achieving high mechanical sensitivity in a tough armored skin. Fine structure organization suggests that a plethora of transitional keratinocyte desmosomes and CD variants gather mechanical strain from a relatively large area of the epidermis onto the small surface of the DR via TFs. Desmosomes are capable of mechanotransduction and play a significant role in sensing and responding to mechanical forces, particularly in epithelial tissues. The desmosome-intermediate filament system may function as a mechanosensing unit, where mechanical stress is focused and amplified via the TF system “hugging” the receptor. Any deformation of the surrounding keratinocytes may be efficiently transferred to the nerve membrane, allowing the crocodile to detect very subtle stimuli. This arrangement seems to work in concert with the more deeply located dermal encapsulated and free nerve endings. The crocodile system appears to be a far more structured and specialized adaptation for high-fidelity mechanosensation than that of humans. The key difference is that in humans, these endings typically lack the highly organized, specialized superstructure of keratinocytes and TFs documented in crocodile ISOs. The advance of these refined and highly specialized sensory skin receptors have allowed crocodilians to discriminate and detect minute motions even under conditions where vision and sound localization are restricted. These unique multi-sensory arrangements have contributed to the crocodilians evolutionary success over millions of years.