CETACEAN EPIDERMAL SPECIALIZATION: A REVIEW

Summary:

Cetacean skin continues to be the investigative focus of researchers from several different scientific disciplines. Yet, most research on the basic functions of lipo-keratinocytes, which constitute most of cetacean epidermis, providing the first layer of protection against various environmental aggressors (including an ever-increasing level of pollutants), is restricted to specialized literature on the permeability barrier only. In this review, we have attempted to bring together much of the recent research on the functional biology of cetacean skin, including special adaptations at the cellular, genetic and molecular level. We have correlated these data with the cetacean permeability barrier’s unique structural and metabolic adaptations to a fully aquatic life, including the development of secondary barriers to ward off challenges such as biofouling as well as exposure to extreme cold for the epidermis, which is outside of the insulation provided by blubber. An apparent contradiction exists between some of the reported gene loss for lipogenic enzymes in cetacean skin and the high degree of cetacean epidermal lipogenesis, as well as loss of desmocollin 1 and desmoplakin genes [while immunolocalization of these proteins are reported (Rehorek et al., 2019)], warrants a re-evaluation of the gene loss data..

Introduction:

Cetaceans are a unique group of marine mammals whose adaptations have attracted much attention from researchers in diverse fields of study, evidenced by a quick Google Scholar search (22,800 references) of ‘cetaceans.’ When the term ‘marine pollution’ is added, the search reveals about 7,980 papers; ‘cetacean skin’ scores about 5,470; and ‘cetacean epidermis and its adaptations’ brings up about 1,960 citations. The term ‘adaptation’ is often interpreted in vastly different ways, but it refers to the biological mechanism by which organisms adjust to new environments or to changes in their current environment. Despite being less studied than these other topics, cetacean epidermis has a substantial body of published research regarding its specialization, histology and fine structural features. In cetaceans, epidermis, the interface between the organism and its environment, lies outside of the insulation provided by the blubber layer (located in the hypodermis), and hence it is maximally exposed to frigid temperatures, as well as other physical, chemical and biological stressors.

As the first line of defense to cope with and survive environmental challenges, mammalian epidermis has developed a diverse set of barriers that are interdependent (Elias & Choi, 2005). In terrestrial mammals (from whom cetaceans evolved), the major challenge is to restrict evaporative water loss, and they have developed and evolved an efficient permeability barrier that has been well-characterized (Elias & Feingold, 2006). Permanently aquatic mammals, cetaceans have glabrous skin, and their permeability barrier needs in frigid and hyperosmotic environments (Birukawa et al., 2005) would differ from their hairy, terrestrial ancestors.

There is a renewed and expanding interest in cetacean skin from a variety of viewpoints: i) gene loss related to aquatic adaptations and evolution (Cabrera et al., 2021; Hecker, Sharma, & Hiller, 2017; Lopes-Marques et al., 2019; Sharma et al., 2018; Springer et al., 2021); ii) skin molting as a driving force for global migration (Pitman et al., 2020); iii) as a model to evaluate age estimation techniques (Beal, Kiszka, Wells, & Eirin-Lopez, 2019; Hartman, Wittich, Cai, Van Der Meulen, & Azevedo, 2016); iv) in assessing marine pollution (Lunardi et al., 2016); v) to measure stress in the marine ecosystem (Fossi et al., 2014), including UV damage (Martinez-Levasseur et al., 2013; Martinez-Levasseur et al., 2011); and vi) in wound healing (Kishibe et al., 2012). The morpho-physiological aspects of the cetacean epidermal barrier related to these six areas have received little attention. This review presents a synthesis of available information, which also advances some theories on the significance of epidermal barrier specialization that may aid cetacean survival in an increasingly polluted marine environment.

Cetacean Epidermis: General Features:

The dermal-epidermal junction (DEJ) of cetacean skin is complex, as the dermis extends as ‘dermal papillary ridges’ far into the overlying epidermis, interdigitating with overlaying epidermal pegs. This deep, extensive dermo-epidermal interface characterized by deep interdigitations of dermal and epidermal compartments robustly anchors the epidermis to the dermis, as seen in histology sections (Fig. 1). Excellent descriptions of these features and a further discussion about their functional significance have been reported (Chernova, Shpak, Kiladze, & Rozhnov, 2017; Garten & Fish, 2020; Stromberg, 1985, 1989).

Figure 1: Low magnification histology of Harbor Porpoise epidermis showing fat red 7 stained neutral lipids in the epidermis.

The dermal epidermal junction (DEJ) is characterized by extensive interdigitations of dermal and epidermal rete pegs. D = dermis, E= epidermis, R= rete peg. Mag bar = 100 μm.

Cetacean epidermis is 15 to 20 times thicker than the epidermis of most terrestrial mammals (Giacometti, 1967). Additional callosities are often seen in whale skin, likely offering further protection against mechanical injury, such as when contacting hard surfaces (ice and rocks) (Reeb, Best, & Kidson, 2007). Significant cetacean epidermal variations from that of terrestrial mammals lie in differences in gross morphology (glabrous; lacking both hair follicles and skin glands); epidermal proliferation (Brown, Geraci, Hicks, St Aubin, & Schroeder, 1983), and ultrastructural features that reflect altered differentiation patterns during keratinization (C.J. Pfeiffer & Menon, 2002), as well as evidence of very high levels of cellular lipogenesis (Menon, Grayson, Brown, & Elias, 1986; C. J. Pfeiffer & Rowntree, 1996). The last two features prompted the designation of cetacean keratinocytes as ‘lipo-keratinocytes’ (Elias, Menon, Grayson, Brown, & Rehfeld, 1987; Menon et al., 1986) and are discussed in further detail below. Cetacean lipo-keratinocytes are three to five times more voluminous than human keratinocytes, with some variability between and within cetacean (mysticete and odontocete) species (Garten & Fish, 2020). However, there appears to be no correlation between lipo-keratinocyte size and body mass – the largest keratinocytes occur in the Bryde’s whale, which was not the largest species studied in a comparative investigation (Morales-Guerrero et al., 2017).

Epidermal proliferation:

The estimated cell proliferation rate in bottlenose dolphins, Tursiops truncates, evaluated via radio-labeled thymidine incorporation is a proliferation index (PI) of 7.4 ± 0.6 %, which is 1.3 to 1.9 times that reported for terrestrial mammals (Brown et al., 1983). The size of the proliferative pool, which is a function of the ratio of surface area of basal layer to that of the epidermal surface, is over 13:1. Though this likely explains the unusual thickness of the bottlenose dolphin epidermis, the thickness of cetacean epidermis varies considerably among and within species. As an example, the epidermis of most body regions of the bowhead whale is substantially thicker than that reported for other cetaceans, with both regional and individual variations in thickness (Haldiman et al., 1985). The epidermis is also particularly thick in the harbor porpoise, belugas and humpback whales (Jones & Pfeiffer, 1994). In dolphins, the desquamation rate of the outermost epidermal cell layers is 8.5 times faster than in humans, which likely serves to maintain a smooth surface and to limit microbe colonization in an aquatic environment (Hicks, St Aubin, Geraci, & Brown, 1985). On the other hand, it has been estimated that the cetacean epidermis takes about 72–75 days in its transit from the basal layer to the outermost layer of the stratum externum (the ‘stratum corneum’ in terrestrial mammals) (St. Aubin, Smith, & Geraci, 1990). In conventional terms, this indicates slow rates of epidermal cell turnover. However, Bechshoft et.al (2020) considered the turnover time to be around 45–60 days based on their observations (Bechshoft, Wright, Styrishave, & Houser, 2020). Because the stratum externum loses many cells in large flakes (due to friction at the boundary layer), particularly as some polar cetaceans molt during migration through shallower and warm waters (Chernova et al., 2017) and have remarkable wound healing abilities (Zasloff, 2011), the reported rates of cell turnover warrant re-evaluation. Much still needs to be learned about species and anatomical location, since both could influence cetacean epidermal proliferation rates and transit time.

Histological and histochemical features at the light microscopic level:

Histological skin features of several different species of odontocete and mysticete cetaceans have been reported. These species include: bottlenose dolphins, Tursiops truncates (Harrison & Thurley, 1974; Williams, 1968); spotted dolphins, Stenella attenuata (Morales-Guerrero et al., 2017); harbor porpoise, Phocaena phocaena (Harrison & Thurley, 1974; Menon et al., 1986; Sokolov & Kalashnikova, 1971), spinner dolphins, Stenella longirostris (Morales-Guerrero et al., 2017), beluga whales, Delphinapterus leucas (Jones & Pfeiffer, 1994); Bryde’s whales, Balaenoptera edeni (Morales-Guerrero et al., 2017); fin whales, Balaenoptera physalus (Giacometti, 1967); humpback whales, Megaptera novaeangliae (Jones & Pfeiffer, 1994); southern right whales, Fubalaena australis (C. J. Pfeiffer & Rowntree, 1996; Reeb et al., 2007), and bowhead whales, Balaena mysticetes (Haldiman et al., 1985).

Statistically significant differences were observed in both epidermal thickness and height of dermal rete pegs in the dorsal skin versus fluke skin of cetaceans by Garten and Fish (2020). They provide an excellent discussion on modifications (in five species of cetaceans), such as taller rete pegs and a relatively thicker epidermis along the leading edge of the fluke (which is exposed to greater water pressure), that reflect an adaptive feature such as improved resistance to shear forces. In addition, the deep interdigitations of the dermal pegs and epidermis increase the area of the basement membrane, and hence the pool of proliferative cells in the basal layer, as well as providing adequate blood vessels and nerve supply to the very thick epidermal layer. Histological studies examining horizontal sections of this area revealed a highly complex and diverse pattern of the arrangement of dermal rete pegs, which notable variation among different species (Chernova et al., 2017).

A concise description of the epidermis from the dorsal flank of the harbor porpoise (Phocaena phocaena) can be found in Menon et.al. (1986) and in Pfeiffer and Menon (2002). Figure 1 shows a frozen section, stained with fat Red 7 to detect the histochemical distribution of lipids. A progressive increase in the lipid content of cells occurs as lipo-keratinocytes differentiate and stratify (Fig. 2). The various perinuclear, cytoplasmic ‘halos’ seen on light microscopy are identified as coalesced lipid inclusions by histochemical staining (Menon et al., 1986), histology (Fig. 3) and electron microscopy (Figs. 3&4) (Menon et al., 1986; C. J. Pfeiffer & Jones, 1993; C.J. Pfeiffer & Menon, 2002; Stromberg, 1985).

Figure 2: Semi-thick section of Harbor Porpoise stratum externum showing large lipid inclusions in the corneocytes.

LD = lipid droplet. Plastic embedded sample, stained with toluidine blue (modified from Menon et.al. 1986). Mag bar = 50 μm.

Figure 3: Electron microscopy of a basal layer of epidermis from a Pilot Whale shows cuboidal basal cells with large nuclei, cytosolic keratin filaments and perinuclear lipid droplets.

LD = lipid droplets, K = keratin filaments, N = nucleus (unpublished microscopy from Pfeiffer & Menon, 2002). Mag bar = 2 μm.

Figure 4: Electron microscopy of RuO4 stained epidermal lamellar bodies of Pilot Whales.

LB = lamellar body (unpublished micrograph. Pfeiffer & Menon, 2002). Mag bar = 0.2 μm.

Ultrastructural features:

The stratum basale of epidermis is comprised of columnar cells with a large nucleo-cytoplasmic ratio, electron-lucent, cytosolic lipid droplets, abundant mitochondria, and bundles of tonofilaments that link to desmosomes. The cell membranes of adjacent cells interdigitate extensively, with numerous desmosomes present. In pigmented areas, Reeb et.al (2007) reported that melanocytes are interspersed among basal cells and occasionally in the first few layers of the stratum spinosum. These melanocytes are large and well developed with typical dendritic processes and abundant melanosomes.

Suprabasal cells (stratum spinosum) show an increase in cell size, cytoplasmic volume and cytosolic lipid droplet contents, compared to basal cells. With cell stratification, stratum spinosum cells progressively flatten in the upper spinosum, with a concomitant increase in desmosomes, cellular lipid droplet content, and epidermal lamellar bodies (LB) (Figs. 4&5). Lipid droplets tend to become larger due to coalescence of smaller droplets. In view of recent research on cellular lipid droplets (Olzmann & Carvalho, 2019; Renne, Klug, & Carvalho, 2020), it would be appropriate to consider such lipid droplets not merely as a storage depot for fat, but as distinct and metabolically active organelles (Cohen, 2018). These lipid droplets typically possess an organic core of neutral lipids (triacylglycerols and sterol esters) bounded by a monolayer of phospholipids, a structure that segregates the aqueous and organic phases of the cytosol. In addition, several proteins decorate lipid droplets, including the perilipin family of structural proteins (Brasaemle, 2007), lipid synthesizing enzymes (Stone et al., 2009) and lipases, as well as membrane trafficking proteins, including Rab 5 and Rab 18. Different lipid droplets within a cell can contain different suites of proteins (Ducharme & Bickel, 2008), and can display different rates of acquiring triacylglycerol – pointing to morphological and functional heterogeneity of cellular lipid droplets; which can be seen in cetacean epidermis as well (Figs 4&5). Other researchers have shed light on the functions of cellular lipid droplets that are unrelated to energy production (Welte & Gould, 2017), including storage of retinyl esters, hydrophobic proteins, and fatty acid ligands for peroxisome-proliferator activated receptors (PPARs). This last group of receptors has profound effects on epidermal differentiation (Schmuth et al., 2004).

Figure 5: Electron microscopy showing morphological diversity of lipid droplets in Harbor Porpoise epidermis.

(A) Upper SS layer (modified from Menon et.al., 1986). (B) Lower SS layer. Note the much larger sized lipid droplets in A vs. B.(Unpublished: Menon et.al. 1986). LD, lipid droplet, K = keratin filament. Mag bars = 0.2 μm.

Cells in the upper stratum spinosum of the harbor porpoise also show remarkably enriched LB contents, seen with both electron microscopy (EM) and freeze fracture techniques (Menon et.al., 1986). These LBs are small (usually 0.2 to 0.5 micrometers in diameter), membrane-bound organelles, containing stacks of disk-shaped membranes Thus, while cytosolic lipid droplets are unique to cetacean epidermis (among mammals), lipo-keratinocytes share a common feature with terrestrial mammal keratinocytes -- elaboration and secretion of epidermal lamellar bodies (a lysosome related organelle). The lamellar body contents are enriched in glucosylceramides, cholesterol and phospholipids; a battery of lipolytic and proteolytic enzymes; and possibly antimicrobial peptides as occur in terrestrial mammals (Braff et.al. 2005). While epidermal keratinocytes deposit their LB contents in the extracellular domains at the outermost epidermal layer; (i.e., at the SG-SC interface), lipokeratinocytes secrete LBs within the upper stratum spinosum much before they transition into cells of the stratum externum.

Stratum externum:

In the absence of a stratum granulosum, the upper stratum spinosum seamlessly transform into the stratum externum in cetaceans. The lack of a distinct stratum granulosum, due to absence of keratohyalin granules, is a major feature of cetacean epidermis, and a clear departure from the differentiation pattern of terrestrial mammals.

In his description of whale skin stratum corneum (the term ‘stratum externum’ was not in use then), Spearman (1972) mentions the phospholipid-rich nature of the parakeratotic SC as contributing to the water barrier. Histology from his paper clearly shows prominent intracellular vesicles – subsequently identified as lipid inclusions by us and others - although he did not discuss this feature (Spearman, 1972). In cetacean stratum externum, distinct cornified envelopes appear to be absent, likely due to loss of genes for transglutaminases (Crumrine et al., 2019), AloxE3 and Alox 15, all crucial for corneocyte lipid envelope and corneocyte envelope formation. Within the outer cells, keratin filaments do not pack or aggregate tightly, while often appearing sparse. Due to their intracellular localization, lipid droplets of cetacean epidermis do not contribute to the extracellular permeability barrier, unlike secreted LB lipids. The fate of secreted LB contents is also different in cetaceans. In terrestrial mammals, enzyme-mediated processing of secreted lipids leads to formation of tight lamellar structures that occlude the paracellular domains of SC and serve as the basis of their epidermal permeability barriers (Holleran et al., 1991). Also, concerted activity of various proteases enables the orderly desquamation of corneocytes (Elias & Feingold, 2006) via dissolution of corneodesmosomes (Ishida-Yamamoto, Igawa, & Kishibe, 2018). Such lipid processing is incomplete in cetacean epidermis, and we speculate that the process of desquamation that occurs in cetaceans would also vary in its details as the genes for two desmosomal proteins are reported to have been lost (discussed in further detail in the section on molting). However, free lipid droplets are retained within lipo-keratinocytes of the stratum externum (Fig. 7), and we have not gathered any evidence suggesting that lipid droplets translocate into the extracellular domains of the stratum externum.

Figure 7: Electron microscopy of Pilot Whale stratum externum.

Note the ‘empty spaces” in the extracellular domain, showing non-lipid areas as revealed by RuO4 post fixation. C = corneocyte, LD = lipid droplet, M = melanosomes. Mag bar = 1 μm.

This difference in barrier lipid organization could be due to: a) exposure to an aqueous milieu (hydration disrupts the barrier lipid organization of SC in terrestrial mammals) or b) retention of glycosphingolipids likely makes the stratum externum more compatible with a highly hydrated interface.

Unlike the keratinocytes of terrestrial mammals, which have been extensively studied in vitro, cetacean lipo-keratinocytes have only rarely been cultured. In one such report (Yu et al., 2005), cells progressively lose their melanin content in culture but continue to retain their lipogenic potential. These cells also reportedly stained positively with monoclonal antibodies AE1/AE3 against human cytokeratins.

Keratinization of cetacean epidermis:

Until recently, cetacean epidermal keratins have received little attention. Keratin filaments in the basal layers resemble those in terrestrial mammals, appearing as electron-dense bundles that form the cytoskeleton (C.J. Pfeiffer & Menon, 2002). However, many of the keratins in the suprabasal cells of cetacean lipo-keratinocytes are K17 (a stress-induced keratin in terrestrial mammals (Fig. 8) (Eckhart, Ehrlich, & Tschachler, 2019), which localizes over sparse filaments or small bundles of short filaments. Keratin 17 (K17) is rapidly induced in wounded stratified epithelia and regulates cell growth through binding to the adaptor protein 14-3-3σ (Kim, Wong, & Coulombe, 2006). K17 is also expressed aberrantly in the suprabasal keratinocytes of psoriatic lesions (Jin & Wang, 2014), which are hyperproliferative and exhibit extensive rete pegs at the dermal-epidermal junction, albeit much less developed than in cetacean skin. Again, in the absence of filaggrin, the keratins within the lipo-keratinocytes/stratum externum remain loosely packed and homogeneously distributed (Fig. 6), as part of a suite of physical and chemical modifications that accompany epidermal differentiation in cetaceans. It should be noted that Pfeiffer and Menon (2002) erroneously labeled the clumps of keratin as keratohyalin in electron micrographs, though the text in this chapter referred to keratohyalin as being absent.

Figure 8: The epidermis of the dolphin evolved from a stress-inducible epidermal differentiation program of a terrestrial ancestor.

The cellular organization of the epidermis in terrestrial and fully aquatic mammals is depicted above. Keratins K5 and K14 are expressed in the epidermal basal layer where keratinocytes proliferate before undergoing terminal differentiation in the suprabasal layers. Comparative genomics and transcriptomics of terrestrial and aquatic mammals reveal that keratin markers K6 and K17 of a stressed terrestrial epidermis are constitutively expressed in the skin of dolphins, whereas the genes encoding the keratin markers K1 and K10 of the terrestrial skin barrier are lost in cetaceans. (Modified from Eckhart L et al, 2019, A Stress Response Program at the Origin of Evolutionary Innovation in the Skin, Evol Bioinform -- with approval from Creative Commons Attribution 4.0 License (http://www.creativecommons.org/licenses/by/4.0/)

Figure 6: Electron microscopy of Pilot Whale suprabasal cells showing cytosolic keratin organization.

D = desmosomes, G = golgi complex, K = keratin filaments, N = nucleus (unpublished figures from Pfeiffer & Menon, 2002). Mag bars = 0.5 μm and 0.2 μm.

Studies in gene loss that correlate with aquatic adaptations of cetaceans show that these genes belong to the epidermal differentiation complex (EDC), including S100–fused type protein genes (CRNN, FLG, HRNR, RTN; TCHH, TCHHL1 and TCHHL2) and suprabasal epidermal keratin genes (KRT 1, KRT 2, KRT 10, KRT 77, KRT 23, KRT 24) as well as AWAT 1 and MOGAT3 that are involved in lipogenesis. (Springer et al., 2021).

Comparative genomic studies of epidermal keratin genes in terrestrial and fully aquatic mammals show that keratins K5 and K14 of the basal (proliferation-competent) layer of the epidermis are conserved in all mammals investigated to date (Ehrlich et al., 2019). However, K1 and K10, which are the main component of the cytoskeleton of the suprabasal cells of terrestrial mammals, are lost in cetaceans (as well as in an unrelated aquatic species, the manatee), where they were replaced instead by K6 and K17, which typically are expressed under stress-induced conditions (wounding) in the epidermis of terrestrial mammals. The constitutive expression of high levels of K6 and K17, and the evolution of alternate splicing of K10 in cetaceans suggest a complete replacement of the quantitatively predominant, epidermal proteins of terrestrial mammals by stress-inducible keratins in cetaceans (Eckhart & Ehrlich, 2018; Ehrlich et al., 2019). This differential expression of hyperplastic keratins is likely an aquatic adaptation and is consistent with the thickened epidermis of cetaceans.

As noted above, the lack of keratohyalin granules (profilaggrin) as markers of a distinct stratum granulosum correlates with a loss of genes for filaggrin in most cetaceans (Strasser, Mlitz, Fischer, Tschachler, & Eckhart, 2015). Although filaggrin genes are detected in dolphin epidermis, they are completely absent in whales, likely because ‘natural moisturizing factors’ (NMF), a filaggrin breakdown product irrelevant for fully aquatic species. Hence, both the paucity of the filaggrin gene, as well as caspase 14 (which initiates filaggrin catalysis into NMF) can be considered aquatic adaptations of cetaceans. LCEs (genes for corneocyte envelope proteins) form a second large gene family in the EDC of cattle and human; but only a single LCE gene was identified in dolphin and porpoise and no LCE is present in the EDC of the minke whale. The evolutionary changes in the EDC, as well as those genes involved in skin barrier function (CASP 14, TGM 5, DSG4 and DSC 1), and skin immune responses (CCL 27, IL20, IL36A, IL36B, IL37, IL38, NLRP10, PYDC1 and PSORS1C2) (Holthaus, Lachner, Ebner, Tschachler, & Eckhart, 2021) amply demonstrate that cetacean skin barrier functions are unique and very different from that of terrestrial mammals. Still there is much to be learned about the multifaceted cetacean skin adaptations to marine environment.

Uniqueness of cetacean epidermal permeability barrier:

The epidermal permeability barrier; crucial for survival on land, should not be expected to be comparable in truly aquatic mammals, as it has been shown that hydration disrupts the lamellar organization of mammalian stratum corneum (SC) lipids (Warner, Stone, & Boissy, 2003). Though absorption of water through the skin was thought unlikely to occur in cetaceans (Telfer, Cornell, & Prescott, 1970), later studies reported that the skin is a major avenue of water flux that may account for as much as 70% of total flux in fasting dolphins (Hui, 1981). Water flux across the skin was also reported to be dependent on the osmotic gradient of the environment, suggesting that delphinids with access to a hypo-osmotic habitat could experience a net gain in fresh water (Andersen & Nielsen, 1983). This also raises the question of loss of water through the skin in hyperosmotic marine environment, which seemingly is not adequately studied. Thus it appears that the cetacean skin barrier is less ‘water-tight’ than in the case of terrestrial mammals, as supported by ultrastructural findings of a lack of organized extracellular lamellar bilayers in the cetacean stratum externum (Menon et al., 1986). We propose that while LB secretion takes place in cetaceans as in other mammals, the crucial step of deglucosylation of glycosylceramides (to generate ceramides) is lacking in cetaceans, leaving the water barrier ‘imperfect’ in comparison to terrestrial mammals. We have speculated previously that retention of glucosylceramides provides an advantage in making the SC more compatible to an aquatic habitat. Lipid extraction and chromatographic analysis (Menon et al., 1986) have shown that cholesterol and triglycerides predominate in neutral lipids (with lesser quantities of free fatty acids and sterol esters). While glycosphingolipid fractions co-migrating with acylglucosylceramides (as in terrestrial mammalian SC), are prominent, there is a clear paucity of bulk ceramides, in sharp contrast to terrestrial mammals. However. whether the retention of the more polar glucosylceramides keeps the stratum well- hydrated in a hyperosmotic environment has not been tested. Yet another possible waterproofing strategy, employed by some terrestrial vertebrates such as tree frogs and birds, is the use of triglycerides to coat the external surface (Lillywhite, 2006; Menon & Menon, 2000). If the intracellular triglycerides could escape the cells of the stratum externum (through physical breaks in membranes as occurs in the stratum corneum of birds) (Menon, Aggarwal, & Lucas, 1981), thus coating the external surface; it could reduce desiccation of the stratum externum. At present, we do not have evidence for such an occurrence.

In addition to their role in permeability barrier, the secreted LB contents in cetacean skin provide another type of protective function- defense against biofouling by the larvae of sessile organisms like barnacles that settle on the skin (Baum, et.al. 2001, 2003).

Permeability barrier functionality is measured in terrestrial mammals by rates of transepidermal water loss (TEWL) (Fluhr, Feingold, & Elias, 2006), a methodology that cannot be employed in the cetacean aqueous environment – making direct comparison by TEWL impossible. Alternatively, ultrastructural demonstration of tracer permeation to visualize the barrier to water flux, commonly seen in terrestrial mammals, is possible with cetacean skin, although we have not encountered any published reports to date. Another gap in our knowledge of cetacean epidermis is information relating to the presence of tight junctions or transmembrane water channels, such as aquaporins, which could provide further insights into the cetacean epidermal water barrier. Additionally, comparative studies, using marine vs river dolphin skin could bring out specific modifications in barrier in aquatic vs. marine habitat of cetaceans.

Potential roles of lipid droplets in cetacean epidermis:

Free lipid droplets have been traditionally considered as cellular fat storage depots. However, in view of recent research on cellular lipid droplets (Olzmann & Carvalho, 2019; Renne et al., 2020), it would be appropriate to consider epidermal lipid droplets as distinct, metabolically active organelles (Cohen, 2018). Such droplets possess an organic core of neutral lipids (triacylglycerols and sterol esters), bounded by a monolayer of phospholipid, which segregates the aqueous and organic phases of the cell.

We speculate that the lipid droplets in cetacean epidermis could provide a source for: a) metabolic water, b) thermogenesis, and c) glycerol, a known antifreeze compound. They also could be sequestering lipophilic xenobiotics, while eliminating these from the body by exfoliation of corneocytes (an excretory function) based on the well-known fact that fat in the blubber serves as a sink for organochloride pesticides, a major marine pollutant. An analogous role for epidermal lipids occurs in the sebokeratinocytes of Pitohui, a ‘toxic’ bird that sequesters diet-derived homobatrachotoxin in the epidermis and feathers, serving as a chemical barrier against predation (Dumbacher, Beehler, Spande, Garraffo, & Daly, 1992; Menon & Dumbacher, 2014). In cetaceans, it is also possible that lipophilic pollutants that permeate across the stratum externum (lacking a tight permeability barrier) could be partitioned into free lipid droplets and eliminated by exfoliation, in the rate of which is higher in cetaceans compared to land mammals.

Cetacean skin molting, migration, and sun damage:

Molting:

Early reports on epidermal molting in whales, mainly by whalers and some researchers, initially were considered as unreliable. However, there has been renewed interest in this subject in recent years. Beluga whales (Delphinapterus leucas) undergo a seasonal epidermal molt during summer, facilitated in part by the warm and low salinity environmental conditions found in seasonally occupied estuaries, postulated to influence the growth and turnover of epidermis by increasing metabolic activities (St. Aubin et al., 1990).

Southern right whale (Eubalaena australis) calves are known to shed multiple layers of their epidermis, similar to beluga calves that conduct a multilayered molt to remove fetal epidermis (Reeb, Duffield, & Best, 2005).

Histological analysis has revealed that bowhead whales belonging to the Okhotsk Sea population molt during summer months while occupying a warm, shallow bay. Shedding of epidermal sheets of varying sizes and thickness, enhanced by tail slapping and breaching was classified as proper molting in bowhead whales (Chernova et al., 2017; Fortune et al., 2017; Shpak & Paramonov, 2018). The detailed histological study by Chernova et.al., (2017), comparing molting to non-molting skin, showed differences in features of the typical adult skin molt from the temporal molt in bowhead whales. In the latter, molts begin as a generation of newly keratinized (parakeratotic) cell layers in the lower epidermal layers (halfway between the apices of the rete pegs and the external surface) which moves outwards towards the skin surface as the outer cell layers are desquamated, to replace the upper layers, reminiscent of the reptilian molting process (Alibardi, 2014). A detailed study of Rehorek et.al (2019) on the shedding of the ear plug in Bowhead whales also revealed some intriguing findings (Rehorek et al., 2019). In the external auditory meatus (continuous with the skin) of Bowhead whales, the parakeratinized epithelium is shed entirely, via the formation of annual deep intraepithelial and subepithelial fissures which correlate with the annual molt. Using immunostaining techniques for desmosomal proteins, they found season-dependent decreases in the levels of desmocollin, consistent with skin shedding. This finding raises questions whether there is truly a loss of the desmocollin gene or is there a seasonal gene inactivation.

Sun Damage and Migration:

Martinez-Levasseur, et.al. (2011) documented various degrees of sunburn in different species of whales and correlated these levels with time spent on the surface during migration and social interactions. Hence, it is worth considering whether cetacean molting could be linked to sun damage as well. But Pitman et al. (2020) advanced an alternate view that molting has an important role in eliminating diatoms and other organisms that attach to cetacean skin, and that this function could be a ‘driver’ of long-distance migration of whales. If this argument is tenable, could the sun damage that occurs during migration provide an additional driver for molting? As in humans, cetacean keratinocytes repair DNA damage in response to UV radiation, while also deploying adaptive pigmentary responses (Martinez-Levasseur et al., 2013). Melanosomes capping over the nuclei (micro-parasols) in epidermal cells are assumed to be cetacean adaptations for sun protection (Eroh et al., 2017), as they are in terrestrial mammals. In addition, an abundance of melanophages support the notion that cetaceans evolved specific modifications to deal with excessive sun exposure (Martinez-Levasseur et al., 2013). Melanophage proliferation has been further associated with chronic or transient UV-associated inflammation, where these macrophages in turn can act as antigen-presenting cells to T lymphocytes (Morales-Guerrero et al., 2017).

Cetacean epidermis as a ‘biomonitor’ of stress, marine pollution and detoxification:

With irrefutable evidence that the marine environment is becoming a repository of toxic waste (agrochemicals, pesticides, drugs, nanoplastics) (Hader et al., 2020; Nelms et al., 2019), numerous ecotoxicological studies have utilized cetacean skin to monitor the impact of pollution upon cetacean physiology (Aubail et al., 2013; Fossi et al., 2014; Marsili, Di Guardo, Mazzariol, & Casini, 2019; Monaci, Borrel, Leonzio, Marsili, & Calzada, 1998). Increased harvesting of cetacean skin biopsies has led to evaluation of their body content of marine pollutants, metabolic adaptations to pollutants, and resultant genetic changes. Some of these studies have focused on P450 (CYP 1A1) induction. Transcriptomic analyses of skin biopsies treated with topical contaminants show that genes which regulate immunity, endoplasmic reticulum stress responses (ER stress), tissue development and lipid homeostasis can be impacted (Lunardi et al., 2016).

Other researchers have utilized skin biopsies to localize the expression of stress markers (Bechshoft et al., 2020; Bechshoft et al., 2015). As surmised by Van Dolah et.al (2015), immunostaining showed Cyp1A1 protein, the most widely studied biomarker of exposure to organic contaminants in cetaceans, is expressed primarily in endothelial cells of the arterioles and capillaries of the dermis, rather than in the epidermis (Van Dolah et al., 2015). Yet, Cyp1A1 transcripts are also expressed in the epidermis, as measured by qPCR. In killer whales, both skin and blubber express mRNAs for several nuclear receptors, such as the aryl hydrocarbon receptor (AhR), thyroid receptor (TR), retinoid x receptor (RxR), and glucocorticoid receptor (GR), known to activate Cyps as well as additional xenobiotic pathway genes. Similarly, striped dolphin skin (Stenella coeruleoalba) expresses mRNAs encoding AhR, Cyp1A, Cyp2B, and ER stress (Panti et al., 2011).

Wound healing and inflammation in cetaceans:

Due to both aquatic habitat and the inherently greater epidermal proliferation potential of cetaceans in comparison to terrestrial mammals, their wound healing could be considered as a unique model system. Additionally, threats of large skin wounds caused not only by natural predators (sharks) but also by boat propellers are increasing. Hence, understanding wound healing processes in cetaceans is critical for further conservation efforts.

In an early study, Williams (1968) reported that glycogen was found in the regenerating epithelium of the dolphin Tursiops truncatus following injury. Bruce-Allen & Geraci (1985), in a limited experimental study on three bottlenose dolphins (Tursiops truncates), examined histology of incision wounds (Bruce-Allen & Geraci, 1985). They reported a mixed cell infiltrate predominated by neutrophils, and extensive areas of intraepidermal vesicles in early stages (6 through 72 hrs) and that by the second day, migrating epidermal cells bridged the incisional gap. By day 7, elevated mitotic activity of basal cells restored full epidermal thickness. A unique feature was the absence of a solid fibrin clot or typical scab formation (essential for healing wounds in dry environment); its purpose being served by a transformed barrier layer of epidermal cells (Su et al., 2022). A good histological description of the process of wound healing is provided by Su et.al (2022).

The absence of epidermal scabs indicates that the usual inflammatory mechanism that underlay wound healing in terrestrial mammals may be less active in cetaceans and more reminiscent of scar-less wound healing in terrestrial mammalian fetuses. Although a detailed discussion of the role of inflammation in wound healing is outside the scope of this review, cetaceans could indeed offer a good model system for such investigations. Especially important here is the finding that cetaceans are natural genetic knockouts for IL-20 (Lopes-Marques et al., 2018), and that inactivation of IL 20 facilitates specific adaptations of cetacean skin to the aquatic environment. Increased expression of activated IL-20 receptor in terrestrial mammals is known to alter interactions between endothelial cells, immune cells and keratinocytes – leading to dysregulation of keratinocyte proliferation and differentiation (Stromberg, 1985). Whether IL-20 is defunct in cetaceans as a consequence (or cause) of altered epidermal proliferation and differentiation of lipo-keratinocytes is not clear at present. Finally, the chemokine ligand CCL-27 (associated with homing of memory T lymphocytes to the skin, and hence T cell-mediated inflammation) is also lost in cetaceans (Lopes-Marques et al., 2019).

Immunostaining of Langerhans cells (LC) in the skin of Atlantic bottlenose dolphins (Tursiops truncatus) has shown an antibody against human major histocompatibility complex (MHC) class II molecules labeled cells with a dendritic cell-like morphology (Zabka & Romano, 2003). The LC-like cells occurred predominantly in rete pegs, primarily along the epidermal–dermal junction, a pattern that differs from the suprabasal location of LC in terrestrial mammals. Other MHC II(+) cells of varying morphology were observed deeper in the dermis, with a perivascular concentration, which displayed characteristics of both macrophages and dermal dendritic cells. Toll-like receptor 2 positive cells were also found within the stratified epidermis of striped dolphin (Stenella coeruleoalba) skin (Lauriano et al., 2014). Marsili et.al (2019) concludes that the ‘magnitude’ of environmental stress to which cetaceans are subjected is a key determinant of specific immune responses.

Other changes in gene expression aiding aquatic adaptation:

There is much interest in the role of gene loss during evolution, both as a predictable consequence of phenotypic variation, and as a ‘driver’ for a variety of mammalian adaptations - such as the process of transition to a fully aquatic life. A brief account of loss of epidermal genes that shaped the evolution of uniqueness of cetacean epidermis was already discussed in relation to epidermal differentiation. In addition to the gene loss, cetaceans appear to have alternately spliced genes, such as Aquaporin 2 (AQP 2 and alternative AQP2), both of which are upregulated by hypertonicity due to increased NaCL. The alternative AQP2, with a lower water transport, is expressed in every cetacean organ examined, including epidermis, in contrast to normal AQP2, which is expressed only in the kidney (Suzuki et al., 2016). Suzuki et.al (2016) also suggest that the alternately spliced AQP2 is a presumptive unique adaptive trait that evolved in cetaceans. Further characterization of the functional significance of these isoforms would undoubtedly contribute to their importance in the permeability barrier of cetaceans, as they have important roles in barrier properties of terrestrial mammals (Hara, Ma, & Verkman, 2002; Qin et al., 2011).

Desmocollin-1 (Dsc1) and its specific binding partner Desmoglein-4 (Dsg4) are components of desmosomes that promote cell adhesion in the upper epidermis. Both of the genes coding these proteins are reportedly lost in cetaceans (Sharma et al., 2018). Transglutaminase 5 (TGM5), which contributes to cross linking of structural proteins to form the cornified envelope of terrestrial mammals, is also lost early in the evolution of cetaceans (Sharma et al., 2018). Two other lost genes that encode enzymes crucial for the formation of the superjacent, corneocyte lipid envelope of the corneocyte (Krieg & Furstenberger, 2014) are epidermis-type lipoxygenase e3 (Aloxe3) and the arachidonate-15-lipoxygenase (Alox15). Alox 15 regulates inflammation and immunity, and the absence of this gene is also consistent with a near absence of any inflammatory response during wound healing in cetaceans.

Another interesting gene loss relates to kallikrein related peptidase 8 (Klk8) during the transition of mammals to an aquatic habitat (Hecker et al., 2017). In the epidermis of terrestrial mammals, this serine protease is highly expressed in the suprabasal layers. Klk8 activates the convergence of the inactive proenzyme form of both Klk1 and Klk11, which influences corneocyte desquamation likely by cleaving corneodesmosomes in conjunction with additional Klks (Kishibe et al., 2012). In light of the loss of some desmosomal proteins in cetaceans (Sharma et al., 2018), loss of Klk8 may not appear consequential for desquamation. Yet, because Klk8 is also involved in wound healing in terrestrial mammals, as shown by hampered wound healing in Klk8^−/− mice (Kishibe et al., 2012) the admirable wound healing ability of cetaceans in the absence of Klk8 is worthy of further evaluation.

Lopes-Marques et.al (2019) reported that genes central for the unique lipid production in sebaceous glands, such as Mogat3, Dgat2l6, Awat1, Awat2, ElovL3, and Fabp9 present inactivating mutations in cetaceans. This finding is surprising, in light of the crucial role of DGAT enzymes in control of the final, rate-limiting step of triacylglycerol biosynthesis, and the high lipogenic activity of cetacean epidermis. This subject should be an area for future investigations using lipo-keratinocytes in vitro.

Conclusions:

Several structural components of the cetacean epidermal barrier have evolved specializations that are evidently adaptive traits for marine life. The lipids and keratin components of lipo-keratinocytes have modified functions that subserve various specific aquatic challenges. These include loss of components critical for the barrier in their terrestrial ancestors, such as specific keratins, transglutaminases, and filaggrin - now defunct for aquatic life, along with the presence of alternately spliced aquaporin 2 which alters the transcutaneous passage of water and possibly glycerol (an antifreeze molecule) with implications for the higher osmolarity and frigidity of a marine environment. The high rate of cetacean epidermal lipogenesis potentially promotes acquisition of secondary barrier functions against biofouling agents and for xenobiotic elimination. Yet, some of the gene expression studies reporting large scale loss of lipogenic genes (Lopes-Marques et al., 2019) contradict the morpho-physiological evidence for the high lipogenic activity of cetacean lipo-keratinocytes. Reported loss of certain desmosomal genes contradicts immunostaining evidence of seasonal changes in these proteins being involved in the process of skin molting in cetaceans. A reexamination of the gene array results is required to resolve this fundamental issue. The role of cetacean migration in the annual molt and the possible role of sun damage and UV repair during migration are also subjects that need further in-depth analysis.

Abbreviations:

DEJ

dermal-epidermal junction

EDC

epidermal differentiation complex

EM

electron microscopy

LB

lamellar body

NMF

natural moisturizing factor

PI

proliferation index

SC

stratum corneum

TEWL

transepidermal water loss

Data Sharing and Data Availability:

n/a