INHAND: Non-proliferative and proliferative lesions of fish – Chapter 7. Fish integumentary system

Introduction

The skin is the largest mucosal surface of the fish and is continuous with the respiratory, olfactory, digestive, and urogenital systems. It serves not only in protection (physical barrier, mucosal immunity), but in communication, sensory perception, respiration, ion regulation, locomotion, and thermal regulation^1,2,3. The skin is a semipermeable membrane that functions in biotransformation and excretion⁴. Variation between species is wide. In general, the skin is comparatively similar to mammals, with some important differences which will be described.

The thickness and character (pigmentation, breeding tubercles) of the skin varies according to the area of the body examined as well as the age, environmental conditions, gender, and season (reproduction)^{2, 5}. Adnexal structures are lacking. This chapter deals with the skin, including epidermis, dermis, chromatophores (pigment cells), and scales. The lateral line system and other mechanosensory and chemosensory tissues associated with the skin are further described in the ‘Special senses chapter’. Fins are described in the ‘musculoskeletal’ chapter.

Response to Injury

The healing response of fish skin is rapid; the initial response is the production of excess mucus with rapid migration of epithelial cells to cover any defects, which can be accomplished within hours^{6, 7}. Teleosts are also capable of regenerating fins entirely. Grossly the skin may develop distinct color changes as a result of alterations in the cuticle (increased mucus-blue/grey sheen), epidermal and dermal edema (white/milky), necrosis (white/milky), inflammation/neoplasia, pigment accumulation/loss/dispersion, bacterial biofilms (yellow, i.e. Flavobacterium columnare), or epithelial hyperplasia (white, yellow)^8,9,10.

Compared to mammals the histopathological changes exhibited by fish skin are limited and the effects induced by different stressors are often similar^{11, 12}. However, this does not mean skin disease in fish is a simpler matter. The physiology and nature of the mucus/cuticle layer, which is invisible/lacking on H&E slides, is of vital importance to tissue health and disease pathogenesis yet it is poorly understood and difficult to investigate. Compared to terrestrial animals the effects of water in terms of removing/flushing away substances, cells and pathogens from the surface, as well as the osmotic forces (waterlogging in freshwater fish) imposed on the skin, adds considerable complexity to disease mechanisms and interpretation in aquatic species.

Often the first indicator of disease is the production of abundant mucus, which is a general sign of skin irritation and is often accompanied by secretory cell hypertrophy or hyperplasia, and generalized epidermal hyperplasia. Such changes often manifest in parasitic, bacterial, viral, hormonal, and toxin related injury^{4, 13, 14}. The initial secretion of abundant mucus provides an enhanced barrier to bind and remove irritants, and the character of the mucus may change over time or in the presence of toxins^{13, 15,16,17,18,19,20,21,22,23,24}. Mucous cell and other secretory cell differentiation is continuous and adaptive⁴. Hyperplasia in fish skin occurs at all cell levels, not just the basal layer.

Inflammation in fish skin is evidenced in the early stages by intercellular edema/spongiosis and intracellular edema, which may progress to keratinocyte discohesion, vesicle formation, necrosis, and erosion or ulceration¹⁰. Neutrophils are briefly present in very early stages of inflammation; lymphocytes tend to predominate thereafter⁹.

Certain areas of a fish’s body may be more susceptible to injury than others, whether due to perfusion, variation in epithelial cell types (natural defenses), or varied water turbulence^{16, 25, 26}. Ulceration is a very common outcome of skin inflammation and can, with secondary infection by environmental microbes, progress rapidly. A 10% loss of skin surface integrity (scale loss/ulceration) can cause mortality, likely through osmotic stress²⁷. Inadequate mucus production as a consequence of ulceration may also increase the energy cost of locomotion²⁸. Once established, an ulcerative process will involve the deep dermis and hypodermis, sometimes extending into muscle. The shape of an ulcer, often triangulate when viewed in cross section from epidermis to hypodermis, varies depending on an external or internal origin; the narrowest portion tends to be the origin²⁵. For example, a small focal lesion originating in the epidermis may develop into a much larger broad lesion in the hypodermis due to spread of infection²⁵.

The classical stages of wound healing (re-epithelialization, inflammation, proliferation, granulation tissue, remodeling) are conserved in fishes^{29, 30}. Re-epithelialization is very rapid and is not dependent on blood clotting or inflammation.

Epithelial cells at wound margins maintain their desmosome attachments to one another, moving as a group rather than as individualized cells^{6, 29,30,31}. The epithelium along the edges of a wound may separate from the basement membrane in early stages, possibly as a result of reduced hemidesmosomal attachments^{32, 33}. Filament cells in the intermediate portion of the epidermis migrate over, or intercalate with, the basal cell layer; mitoses are not evident until wound closure is complete^{34, 35}.

Nodules in the skin are common manifestations of microbial disease and parasitic infestations and are dealt with in both this chapter and in the ‘soft tissues’ chapter; however detailed treatment of specific conditions may be found in the literature^{9, 14, 36}.

Necropsy and Trimming

Fish skin is extremely delicate, and artifacts are inevitable, but distortions must be carefully accounted for and minimized whenever possible. The mucus cuticle is generally lost during standard processing and is not available for assessment. The slightest contact will denude the epidermis both before and after fixation. Fish must be handled gently prior to and duration euthanasia, with wet gloved hands to avoid skin damage. Also, be aware that the method of euthanasia can lead to artifactual distortion of tissue; for instance, unbuffered tricaine methanesulfonate solutions are irritating, injuring gills and potentially the skin as well (see Respiratory chapter for further comment).

Davidson’s solution (or the modified formulation) is recommended for skin studies due to its superior preservation of epidermis; however neutral buffered formalin is a common choice. Additionally, Davidson’s solution has decalcifying properties may alleviate artifacts encountered when cutting scales. Saturated EDTA can also be used to good effect and may be a preferable method for decalcification.

Compared to mammals, small fish can be completely decalcified in a short time span.

I. Morphology, Physiology, Nomenclature, Diagnostic Criteria, and Differential Diagnosis

1. Epidermis

The skin of fish is broadly composed of three layers: epidermis, dermis, and hypodermis. The outermost layer of integument is that of mucus, or the cuticle, which is composed of the secretions of surface epithelial cells, goblet cells, sacciform cells, club cells, and sloughed epithelial cells; it is approximately 1–2 mm thick, but is usually lost during histologic processing^{3, 37,38,39,40}. Specialized histologic techniques to retain the cuticle have been published^{41, 42}.

Keratinocytes: The epidermis is delicate, stratified and squamous (from 2, up to 30 or more cells thick); it does not keratinize except in certain species (rarely used in toxicologic studies). The basic unit of the epidermis, the keratinocyte (variably termed filament, polygonal, or Malpighian cells), is metabolically and mitotically active in all layers, however mitoses are most common along the basal layer⁴³. Epithelial cells are generally similar throughout the layers; however, a basal layer (stratum basale), an intermediate/mucous layer, and a superficial (pavement) layer are evident; toward the surface cells flatten. They attach to one another at all levels by desmosomes, which in the outer layer (pavement cells) form part of the water-tight terminal web^{37, 44}. The pavement cell layer contains abundant microfilaments that may help protect the skin from osmotic shocks⁴⁵. The subjacent cells are separated by intercellular spaces that contain lymph fluid but do not communicate with the surface/water². Basal cells are also involved in the development of dermal stroma and scales^45,46. Epithelial cells have the capacity for antigen sequestration or phagocytosis and will, when filled with ingested content, slough from the surface^{47, 48}. Epithelial cell translocation/maturation, from basal layer to the surface, can occur in as little as 4 days⁴⁹.

The pavement cells always have surface modifications in the form of species-specific microridges that impart a fingerprint pattern to the surface for cuticle adherence; they are plastic features that are often modified or altered due to physiology or injury^{2, 50,51,52}. In some species the surfaces are papillate or pitted rather than ridged. Such cells contribute to surface secretions³. Prior to sloughing the pavement cells lose intercellular attachments and round up. Keratinization is exceptional, generally seen in surface specializations (nuptial tubercles, attachment organs, etc.), which are present in medaka⁵³ and fathead minnow. The epidermis will undergo changes in association with reproductive cycle and life stages³⁷.

Modified secretory cells: Various types of unicellular glands (mucous cells and other secretory cells) are abundantly present in the epidermis and derive from keratinocytes^{2, 49, 54, 55}. Mucous cells are the most common and inhabit the middle to outer layers of the epidermis, but they often are attached to the basement membrane. They terminally differentiate from basal epithelial cells and enlarge as they move to the surface; cell shape, nuclear location, and secretory content change through the maturation cycle. Once at the surface, the cell ruptures and dies, releasing its content into the cuticle layer³⁷. Goblet cells may have mucous or serous content and may vary in number according to area of the body, sex, season, stage of maturity, water conditions, and exposure to toxicants^{3, 19, 56}. In some species, they have phagocytic abilities and may retain immunoglobulin. Sacciform cells (granular cells, clear mucous cells), a third type of secretory cell, are present in some species and are generally similar to goblet cells. Club cells (a fourth type of secretory cell) are large, round cells in the middle layers of the epidermis that have abundant homogeneous cytoplasm and one or two large nuclei. They are common in many species, and the cytoplasmic content contains Schreckstoff, or alarm substance, which is a pheromone released when the cells are damaged⁵⁷. In some species, club cell numbers are seasonally dependent. In experimental skin injury and inflammation, club cells may contain leukocytes within their phagosomes^{146, 162}. Venom cells, forming multicellular ductless holocrine glands, may be present at the base of fins spines.

Ionocytes or chloride cells are modified epithelial cells which are found most commonly in the gills but are present in the skin, particularly in larval stages^{58, 59}.

Nerves/Sensory cells: The nerve fibers that penetrate the epidermis may ramify between epithelial cells, or they may be associated with individual or groups of specialized sensory cells⁶⁰. Nerves that penetrate the basement membrane lack Schwann cell investments and are surrounded by epithelial cells. Mechanoreceptors and electroreceptors, termed neuromasts, will be further described in the lateral line system (Special senses chapter). Other multicellular receptors termed taste buds or chemoreceptors are present predominantly in the mouth, but also on the head.

Leukocytes: The skin of fish is an important site of mucosal immunity⁶¹.

Lymphocytes, macrophages, and other leukocytes are commonly present in the epidermis, particularly in the basal layers; B cells and plasma cells are the most numerous^61,62,63. Dendritic cells are present in multiple fish species, and in zebrafish, heterogeneity in tissue-resident macrophage cell types has been described⁶⁴. Eosinophilic granular cells/mast cells may be increased in disease conditions. Rodlet cells may also be present in the skin and in association with neuromasts⁶⁵.

Melanocytes and melanin containing macrophages can occur in the epidermis-see further discussion in the chromatophore section of this chapter⁶⁶.

2. Dermis

The dermis is composed of an outer stratum spongiosum (SS) and a deep stratum compactum (SC) that forms a collagenous sheath around the body, functioning in part as a tendon to aid locomotion and prevent splitting under tensile forces⁴⁵. Dermal thickness varies with species, age, and area of the body². It is separated from the epidermis by the basal lamina/basement membrane (BM), and the deep dermis is separated from the hypodermis by a thin sheet of distinctive fibroblasts attached by desmosomes, termed the ‘dermal endothelium’⁶⁷. The BM is similar to that of terrestrial animals and is positively stained with PAS methods. BM thickness varies across the body, and even between scaled and non-scaled areas. In unscaled skin, the BM may adopt extensive infoldings into the basal layer; a possible adaptation to shearing stress³⁷. In some cases, capillaries penetrate the epidermis, however they retain their investment by the BM⁶⁸.

During development the basal epithelium is involved in the production of early dermal collagen and interacts with dermal mesenchymal cells in the formation of scales and fins^{45, 46}. The fibroblasts that form the dermal endothelium in zebrafish appear at 3 days post-fertilization and begin to contribute to dermal stroma production along the deep aspect. In the next several days, the primary dermal stroma becomes re-organized and more compact but is still acellular. By 26 days post-fertilization, the dermis has changed into the adult secondary dermal stroma (stratum compactum) with numerous fibroblasts scattered within⁴⁵. At this time the dermal endothelial cells are now present in the SS that will develop into scales.

The SS is a loose network of collagen bundles containing fibroblasts, chromatophores (pigment cells), mast cells, and the scale beds. The SC is poorly cellular, with highly organized collagen sheets and little if any elastic fibers. The matrix is penetrated by vessels and nerves periodically, and in such areas melanocytes and mast cells are often present. The laminated collagenous structure of the SC resembles the cornea, allowing it to effectively transmit light to the underlying chromatophores while maintaining rigidity. This feature is important in the interpretation of gross changes in the skin, as edema will cause entering and reflected light to scatter, leading to opacity and the loss of chromatophore coloration macroscopically. The components of the dermal matrix are similar overall to those in mammals, with type I and type V collagens as major and minor collagens respectively. As the fish grows, collagen fibril diameter will increase markedly⁴⁵.

In fish, the term “scale” refers to hard (generally mineralized), flattened skeletal elements in the dermis that arise from mesenchyme^{69, 70}. They have various forms (placoid, ganoid, scutes) according to taxa; however, the most common is the elasmoid scale, which is seen in zebrafish, fathead minnows, and medaka and will be commented on here. Elasmoid scales are generally round and broadly overlap one another with posterior non-overlapping segments that are either smooth (cycloid) or finely toothed (ctenoid). They reside near the surface (in shallow pockets) and may be easily rubbed off or removed/resorbed as a result of natural habits (nest building, spawning). Most teleosts lose their scales at least once in a lifetime⁷¹. They represent a primary calcium reservoir, and minerals are resorbed preferentially from scales compared to the endoskeleton⁷². Osteoclasts of regenerating scales are found on the episcleral surfaces; they exhibit MMP-9, TRAcP and cathepsin K, similar to mammals^{73, 74}.

The matrix of the scale may include various mineralized tissue types, including dentine and enamel/enameloid in some taxa. The term “scleroblast” has been coined to generally describe the cells that produce any of the mineralized tissues in a scale. Elasmoid scales are thin, flexible and transparent with a basal plate (also internal layer or fibrillary plate) of highly-ordered, variably mineralized collagenous tissue (composed of elasmodine), an external layer (superficial mineralized layer, osseous layer), and an outer limiting layer; the upper surface is marked by concentric elevated mineralized circuli (striae) and intervening non-mineralized sulci⁶⁸ (Figs. 7.11 and 7.12). They usually overlap one another within the dermis; the non-overlapping posterior segment is covered by epidermis. In most cases, scales exhibit continuous growth, with addition of annular striae (annuli); alterations in annular rings/overall growth are indicative of various stressors². Scales are composed of three components ultrastructurally; collagen, a cement-like non-fibrillar collagen material “ichthylepidin”, and hydroxyapatite-like crystals^{75, 76}.

Fig. 7.11.

Fathead minnow, male. Nuptial tubercle, pre-estrogen. H&E, 100×. Image courtesy of Jeffrey Wolf.

Fig. 7.12.

Fathead minnow, male. Nuptial tubercle, decreased. Reduced size was caused by experimental exposure to exogenous estrogens. H&E, 100×. Image courtesy of Jeffrey Wolf.

Each scale resides within a scale pocket in the SS, and the pocket is lined by layers of mesenchymal cells (dermal endothelium) which differentiate to form scales within dermal papillae. The upper layers develop into scale-forming scleroblasts (marginal zone, episquamal, and hyposquamal populations), while a deeper population constitutes the scale-pocket lining (SPL) cells, which serve to produce scale plate collagen and to regenerate scales^{68, 70, 77} (Figs. 7.1-a–e and 7.2). The SPL cells (dermal endothelial cells) appear similar to fibroblasts but are joined by desmosomal attachments as are the scleroblasts, sometimes termed osteoblasts or elasmoblasts^{78, 79}. Scleroblasts/osteoblasts produce alkaline phosphatase, and scale matrix contains osteocalcin and osteonectin⁷⁴. Histologically, mature scales in fish are typically non-descript, being slender with tightly lamellated collagen layers and few associated mesenchymal cells.

Fig. 7.1.

Microscopic structure of the ontogenic and regenerating scales of a goldfish. a) Scales (ontogenic) in pockets, with anchoring bundles of collagen, scale forming cells (scleroblasts) and scale pocket lining cells. b) Enlargement of the posterior (non-overlapping) region of a scale, showing elements of growth and mineralization. c) The skin immediately after scale removal, with empty scale pockets. The scleroblasts along with epidermis and some dermal tissue have been removed, but scale pocket lining cells remain. d) Enlargement of a scale showing the lamellar, plywood-like structure where the basal plate elasmodine is crossed by sheets of vertical thin collagen fibers, characteristic of this species. e) Enlargement of a regenerating scale showing the orthogonal plywood-like structure of basal plate elasmodine typical of regenerating scales in teleosts and ontogenic scales in some species. Hyposquamal scleroblasts that produce the collagen of the basal plate are cuboidal in regenerating scales. The outer limiting layer is not present in newly regenerated scales. Abbreviations: bp: basal plate; cf: calcification front; ch: chromatophore/melanocyte; co: circulus; dct: dense connective tissue (stratum compactum); ep: epidermis; es: episquamal scleroblast; ex: external layer; hs: hyposquamal scleroblast; lct: loose connective tissue (stratum spongiosum); mc: Mandl’s corpuscle; ms: marginal scleroblast; ol: outer limiting layer; r: radius; sca: scale; sp: scale pocket; spl: scale pocket lining cells; tc: vertical thin collagen fibers. Sources: From Elliott, 2000³⁸, after Bereiter-Hahn and Zylberberg, 1993 (A,C,D,E)⁷¹, Elliott 2000 (B–adapted from others, cited)³⁸, with permission from Elsevier.

Fig. 7.2.

Histological section of the skin of a juvenile chinook salmon, showing elements of scale growth. The ends of the scales (sca) are visible with episquamal scleroblasts (es), marginal scleroblasts (ms), and hyposquamal scleroblasts (hs). The basal plate (bp), external layer (ex), and circuli (ci) of the scales are evident. Other elements in the loose connective tissue layer (lct) include scale pocket lining cells (spl), melanocytes (me), and the basement membrane (bm). Cells in the epidermis include attenuated surface epithelial cells (sec), mid-level epithelial cells (ec), and cuboidal basal cells (bl) as well as goblet cells (gc), and serous cell (ser) that may be a serous goblet cells or sacciform cell. Scale bar is 10 micrometers. From Elliott, 2000³⁸ with permission from Elsevier.

The three layers of the scale have structural similarities to fish teeth; the outer limiting layer is similar to enamel while the external layer and basal plate are composed of dentine-like material⁶⁹. The external layer is formed first and appears similar to woven bone; it is entirely formed and mineralized at the time of development with no further thickening, only lateral growth. The basal plate is then deposited deep to the external layer by deep scleroblasts (elasmoblasts) and continues to thicken throughout the life of the scale; it is partially mineralized (superficial layers) and is composed of orderly stacks of elasmodine, which imparts transparency⁸⁰.

Elasmodine is a form of lamellar dentine rather than of true bone, with thick collagen bundles and rare Sharpey’s fibers⁸¹. In some species there are vertically oriented fibers that run perpendicular to the layers in the basal plate (TC fibers), which are processes of the hyposquamal scleroblasts^{82, 83}. As a rule, scale matrix is acellular, however occasional scleroblasts may become entrapped in lacunae⁸³. A third layer, the hypermineralized outer limiting layer, is devoid of collagen (lacks Sharpey’s fibers) and is deposited on the non-overlapping scale regions in very close proximity to the epidermis; it also thickens, in periodic fashion, throughout life^{81, 84, 85}. The scale itself is anchored within the pocket by collagen bundles arising from the posterior aspect of the upper surface. When they are shed, the epidermis is lacerated but heals quickly (as early as 3 hours)^{71, 78}. The mineralization of the scale layers occurs by different mechanisms; scleroblasts direct the mineralization of the external layer, while scleroblasts and SPL cells are involved in mineralization of the basal plate^{82, 86,87,88}. The limiting and external layers may stain with Alcian blue, while the external and basal layers are PAS positive⁸⁹.

Scales are also intimately associated with the lateral line mechanosensory system (described special senses chapter), where canals penetrate scales. A lateral line scale (tubular scale, pored scale) is composed of a scale plate and a canal tube (bony vault, canal housing) which is the bony tissue surrounding the canal⁹⁰. Additionally, in fish that lack scales (certain catfish like Ictalurus punctatus) bone (canal tubes) may partly or completely surround lateral line canals, suggesting that the tubes may be of different origin than scales; a point raised for other species as well⁹¹.

The hypodermis is a fatty, well vascularized, areolar tissue that blends into the subjacent musculature. Adipocytes, chromatophores, blood vessels, lymphatics, and nerves are present in this layer⁶⁸.

The lymphatic system/secondary vascular system is an important component of the integument of the fish and is dealt with in greater detail in the cardiovascular system chapter⁹². The lymphatic volume of the skin and musculature is more than 4 times the blood volume in fish; disease, especially ulcers, can severely affect blood parameters and animal health in ways not readily appreciated by investigators more familiar with terrestrial laboratory species⁹.

3. Chromatophores

Color in adult fish is determined by pigmentation patterns that depend upon the presence of pigmented cells (chromatophores) in the skin, and exhibit plasticity over time through two mechanisms: ‘physiological’ color change and ‘morphologic’ color change⁹³. Not only do fish have an embryologic color pattern, but as adults they have a dorso-ventral countershading pattern as well as a striping pattern (particularly zebrafish)⁹³. The striping pattern of zebrafish is formed via the interactions of melanophores, xanthophores, and iridophores through mutual attraction, repulsion, clonal expansion, and cell survival^{94, 95}.

Color change mechanisms are mediated largely by the endocrine system (including melatonin, melatonin concentrating hormone, ACTH, prolactin, estrogen, and testosterone) and the sympathetic nervous system (nerve endings and neurotransmitters-catecholamines)^{96, 97}. “Physiological” change is a rapid process whereby pigment organelles are shifted within the cells, while “morphological” change is accomplished over weeks and is a result of pigment cell loss, altered morphology, and variation in pigment synthesis/amounts⁹⁸. In addition, pigment cells can respond directly to incident light. Morphological change may be a response to diet, stress, temperature, reproductive phase, background patterns, UV exposure, ophthalmic system impairment, varied salinity, mechanical pressure, and maturation². Color patterns are the result of a complex process where pigment cells associate by attraction and repulsion⁹⁷. In many species of amphibians and reptiles, the pigment cell types congregate into orderly ‘dermal chromatophore units’; however in fish these are not as common⁹⁸.

Chromatophores are a diverse population of neural crest cells that generate membrane-bound organelles (arising from endoplasmic reticulum-ER) to absorb light, impart color, or reflect light. The pigmentation patterns in zebrafish have an embryologic and adult form⁹⁵. In the embryologic pattern chromatophores differentiate directly from the neural crest, migrating dorsolaterally along the body surface as well as ventromedially along nerves to the dorsal root ganglia^{99, 100}. Melanocyte progenitors are SOX10 positive, and express MITF (microphthalmia transcription factor), KIT, TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), and DCT (dopachrome tautomerase) along embryologic and adult melanocyte development pathways¹⁰¹. Other chromatophore types are regulated by different receptor-ligand pairs; iridophores by leukocyte tyrosine kinase, and xanthophores by colony stimulating factor⁹⁵. Embryonal chromatophores eventually die and are replaced by adult cells.

During metamorphosis, another set of chromatophores develop from stem cell intermediates that reside in the dorsal root ganglia, and migrate along nerves to the skin, where they first differentiate into bipotent chromatophores (melanocyte-iridophore or melanocyte-xanthophore) and then proliferate to become well differentiated cells^{100, 102}. While they arise from a similar origin, chromatophore types respond differently to hormone and nervous control⁹⁸.

Chromatophores are classically designated by the in vivo color/appearance (to the unaided eye) rather than by the chemical composition of the pigment organelles, which is a cause for some confusion since they are not strictly correlated. However, separation of cell types can also be made by the type of pigment contained; namely those that contain melanins (eumelanin, pheomelanin) and those that lack melanin but contain other substances, whether reflective or light absorbing (pteridines, carotenoids, purines, etc.)¹⁰³. However, the common origin for these cells is evident in that more than one active pigment type may be found in the same chromatophore. Pigmented cells are conspicuous by light microscopic evaluation and variation may denote subtle evidence of pathology. A thorough description of any chromatophore should include: cell color in vivo, chemical identification of pigments, and ultrastructural examination to verify organelle types since different species may achieve the same color through different pigment mechanisms¹⁰⁴. Chromatophores in fish skin are generally seen in the dermis; however, epidermal melanocytes (and extensions) and xanthophores may be seen, depending on species⁹⁸. In cases of skin injury, melanocyte extensions may be present in the epidermis along with pigment containing-macrophages¹⁰⁵. Within the dermis, chromatophores tend to congregate in two areas; at the interface of the epidermis and dermis, and the interface of the dermis with the subcutis, the stratum argenteum¹⁰⁶.

Melanin containing cells can be further subdivided into those that actively transfer melanosomes to other cells (typical mammalian melanocytes), or those that maintain melanosomes (non-transferring) within the cell, termed melanophores. Melanocytes are the only chromatophores that are known to transfer pigment⁹⁸. Melanosomes in fish have been reported to form via multivesicular premelanosomes, as well as fibrillar premelanosomes seen typically in mammals^{107, 108}. Premelanosomes are more common in epidermal melanocytes as compared to dermal melanocytes⁹⁸. The terminological distinction between ‘melanocyte’ and ‘melanophore’ will not be maintained throughout the text, and the term ‘melanocyte’ will refer to both, as suggested by others¹⁰⁹. In fish, the epidermal melanocytes can be of the actively transferring type, but all other chromatophores are of the non-transferring type^{98, 110}. Melanocytes are most common in the dermis just beneath the basal lamina (gadoids and siluroids) and in fish contain only eumelanin³⁷. They are also very common in the peritoneum and meninges in fish. The relationship between dermal and epidermal melanocytes has not been resolved; some have suggested that epidermal melanocytes originate from, or are a degenerate form of, dermal melanocytes^{111, 112}. Depending on the mutant or color variety of medaka (and presumably other species) melanocytes may be amelanotic, where melanosomes respond appropriately to nervous and hormonal stimuli, but lack pigmented content^113,114.

Aside from melanocytes, other light-absorbing cells include xanthophores (yellow), erythrophores (red), and cyanophores (blue). Xanthophores and erythrophores produce their color by a combination of pteridines (in pterinosomes) and carotenoid-filled vesicles and are reality the same basic pigment cell type¹⁰³. Yellow cells are considered xanthophores (containing xanthosomes), red cells are considered erythrophores (containing erythrosomes), and orange cells can be referred to as either⁹⁷. The only way reliable way to differentiate between them is by their color in vivo; they have no molecular or structural markers to distinguish them¹⁰⁹. Xanthophores and erythrophores produce water soluble pteridines in a manner similar to melanosome formation, producing pterinosomes that have a characteristic multivesicular configuration ultramicroscopically¹⁰³. In addition, these cells achieve their color with lipid-soluble carotenoid droplets associated with the endoplasmic reticulum that are obtained from the diet. Cyanophores are not commonly encountered.

Two types of reflecting pigment cells are recognized: iridophores and leucophores, which both appear white but are distinguished by organelle structure. Iridophores lack pigment organelles, but instead utilize stacked purine crystal platelets to reflect light, and provide the reflective shine seen in many species of fish. These stacked platelets are usually composed of guanine, but uric acid, hypoxanthine, and adenine may be present¹⁰³. There is evidence that pteridines also contribute to light reflection in iridophores and leucophores¹¹⁵. In zebrafish the iridophores in the skin are of two types; type S which have many small uniform platelets and type L, which have fewer large platelets¹¹⁶. Iridophores form a distinct layer in the dermis, the stratum argenteum, which is provides the characteristic reflective sheen of the skin.

Leucophores are much less common than iridophores (killifish Fundulus heteroclitus, Danio species including zebrafish, medaka Oryzias latipes, guppy Poecilia reticulata), and have organelles (leucosomes) that contain uric acid, among other substances, to produce a white color by scattering light rather than reflecting it^{98, 104, 117}. They are more closely related to xanthophores than iridophores.

Like melanocytes, other chromatophores are present at both cutaneous and non-cutaneous sites. Additionally, certain pigment cells may contain two or more prominent pigment compounds, termed the dichromatic chromatophores (erythro-iridophores, erythro-cyanophores, xantholeucophores, melanoleucophores)^{104, 109, 118}. Lastly, fluorescent chromatophores have recently been described in fish^{119, 120}. The presence of differentiated chromatophores with more than one pigment organelle type is not surprising, given their shared origin from the neural crest. Such “mosaic” chromatophores with intergrade organelles have been reported in fish, tumor cell lines and in neoplasms^{121,122,123,124,125,126}. Furthermore, chromatophore pigment composition can change to a limited degree with development; leucophores in larval medaka can appear orange due to pteridines¹²⁷. The medaka has four pigment cell types; melanocyte, xanthophore, iridophore, and leucophore⁹⁷. The zebrafish skin contains melanocytes, xanthophores, iridophores, and mosaic chromatophores including xantholeucophores and melanoleucophores^{104, 116, 128}.

Pigmentation patterns in fish have been intensively studied for many years. In zebrafish strains, variation in pigment pattern can be categorized into 4 broad classes according to changes in the chromatophores: 1) reduced chromatophore numbers, 2) abnormal chromatophore distribution, 3) reduced chromatophore pigment (incomplete melanogenesis, delayed melanogenesis, or melanocyte degeneration), 4) abnormal chromatophore morphology (spindly, small, stellate)¹²⁹. Such changes are also evident when fish are exposed to various compounds¹²⁸. Degenerate melanocytes are likely engulfed and eliminated by macrophages¹³⁰. Pigmentation defects, whether hypopigmentation/albinism or hyperpigmentation, are thought to arise from disturbances occurring during specific times in development, a ‘pigmentation window’. Diet, stress, and endocrine disorders have been implicated⁹³.

4. Physiology

Fish skin is hydrated, unkeratinized, and covered by mucus, making it particularly susceptible to waterborne chemicals and stressors. As a result, skin disease is relatively more common in fish than in terrestrial vertebrates. The high cutaneous permeability of fish provides an opportunity for the exchange of gases, nutrients, metabolic waste, and xenobiotics^{1, 4}. Pavement cells exhibit micropinocytosis at the surface, as well as the development of multiple cell-surface ring structures, which lead to the formation of macropinosomes¹³¹. Keratinocytes are also involved in the metabolism of xenobiotics, in part through cytochrome P450 enzymes¹³².

The mucoid cuticle is a vital structure that is both mucous and proteinaceous, containing glycoproteins, mucopolysaccharides, and lipids that are continuously secreted and washed off the body¹³³. Production is highly dynamic in both quantity and quality; highly variable responses are reflected in alteration in composition. In addition to the reduction in friction and protection from toxicants afforded by mucus, osmotic gradients within the mucus help regulate hydration, salt, and ion needs⁴. Mucus is also an essential feature of the immune system; it functions to protect the skin from parasites, bacteria and predators by the secretion of mucins, enzymes, innate system components (complement, lectins, peptides), and immunoglobulins^61,134. Nevertheless, fish skin has its own bacterial and fungal microbiome¹³⁵.

Fish skin also has a limited capacity for respiration, which may provide oxygen for its own demands or for systemic needs in certain species. This capacity is pronounced in larvae and in eel, where skin may contribute the majority of the oxygen uptake⁴.

Changes in the coloration of fish skin may occur rapidly (physiologic change) or slowly (morphological change) and are often indicative of stress. Physiologic changes, under nervous and endocrine control, arise from changes in pigment granule motility. Morphologic change results from changes in the number and size of chromatophores, as well as the amount of pigment in a cell³⁸. Generalized or segmental pigment change in the skin may be associated with abnormal neurologic function.

II. Microscopic Findings – Epidermis, Integument

Table 1 shows nomenclature, diagnostic criteria, and differential diagnosis of epidermis.

Table 1. Nomenclature, diagnostic criteria, and differential diagnosis of epidermis.

Epidermis			Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	Apoptosis			X
	*Atrophy			X
	*Edema			X
	*Erosion/Ulcer		X
	*Increased cellularity, Rodlet cells			X
	*Infiltrate, inflammatory cell			X
	Inflammation			X
	*Necrosis			X
	*Nuptial tubercles/Dorsal nape pad, present /increased /decreased			X
	*Parasite		X
	*Pustule			X
	*Vesicle			X
Proliferative
	Non-neoplastic
		*Hyperplasia, epidermis	X
		*Hyperplasia, modified secretory cell	X
	Neoplastic
		*Papilloma		X
		*Carcinoma		X

*Terminology with diagnostic criteria or comments described in the main text.

Epidermis – Non-Proliferative

Atrophy, epidermis

(Figs. 7.3 and 7.16)

Fig. 7.3.

Channel catfish. Skin. Atrophy, epidermis. H&E, 100×. Image courtesy of Wes Baumgartner.

Fig. 7.16.

.Channel catfish. Skin. Atrophy, dermis (lateral line canal/dermal bone) and atrophy, epidermis. H&E, 40×. Image courtesy of Wes Baumgartner.

Modifiers

Keratinocyte, Modified Secretory, Sensory, Chloride Cell, Nuptial or breeding tubercles

Other Terms

• Epidermal thinning

Pathogenesis/Cell of Origin

• Result of reduced cell proliferation or increased turnover

Diagnostic Features

• Decreased number and/or thickness of epidermal layers

• Reduced number and size of cells whether epithelial, secretory, chemosensory, or chloride cell

• Changes in the pattern, number, and height of pavement cell microridges

Differential Diagnoses

• Erosion/ulcer: Loss of superficial layers (erosion) or all layers (ulcer).

• Necrosis, epidermal: Loss of cellular detail.

• Artifactual loss of epidermis post-mortem.

Comments

Atrophy may be associated with vitamin deficiencies, underlying expansile masses, increased temperature, chemicals and steroids¹⁰⁵. Guppies treated with thyroxine and testosterone developed epidermal cell thinning, increased cell interdigitation, and alterations in the glycocalyx¹³⁶. Partial ischemia or severe malnutrition are also possible etiologies. Exposure to acid water (pH 5.0) caused a transient (up to 3 days) epidermal atrophy due to loss of pavement cells and loss or expulsion of content by secretory cells at the surface, followed by compensatory hyperplasia over the next several days and weeks¹³⁷. Goblet cell numbers may be decreased in association with gonadotropin or 11-ketotestosterone exposure¹³⁸. If specific cell types within the epidermis are affected or are to be specifically described, then the modifiers may be applied.

Nuptial or breeding tubercles, normally present on the face of male fathead minnows, may atrophy in males upon exposure to exogenous estrogens^{2, 5}.

Edema, epidermis

(Figs. 7.4 and 7.5)

Fig. 7.4.

Channel catfish. Skin. Edema, epidermis, intercellular. H&E, 200×. Image courtesy of Wes. Baumgartner.

Fig. 7.5.

Channel catfish. Skin. Erosion, epidermis, with edema, epidermis, intracellular and necrosis, epithelial (upper layers). The sensory papilla is in the center of the image. H&E, 200×. Image courtesy of Wes Baumgartner.

Comments

Intercellular edema (spongiosis) results from the expansion of intercellular spaces by edema fluid, but the epidermal cells remain connected to one another by desmosomal attachments^{3, 10}. Severe edema may lead to rupture of these attachments, and vesicle formation¹³⁹. Example of intraepidermal edema presented in Fig. 7.4 with erosion.

Erosion/ulcer, epidermis

(Fig. 7.5)

Comments

Erosion and ulcer are a continuum: erosions comprise loss of epithelial layers down to the basal layer while ulcers include the loss of all epithelial layers. The skin itself has an oxygen demand that is supplied from the environment; hypoxic water will lead to keratinocyte necrosis and subsequent erosion/ulceration¹⁴⁰. While ulcers may derive from external causes, they often occur as a result of internal disease, particularly bacterial sepsis where organisms lodge in the skin vasculature¹⁴¹. Ulceration is often associated with inflammation, with early fibrin exudation and neutrophilic response that quickly changes to macrophage predominance. Secondary infections, as a result of environmental bacteria and/or fungi, are common in ulcers and may obscure the inciting cause. Chronic ulcers develop rounded hyperplastic epidermal margins and may include melanocyte hyperplasia¹⁴².

Abnormal temperatures, hypoxia, pH changes, UV exposure, toxin exposure, high water ammonia, nutritional deficiencies, and parasites are possible causes of ulcers^{2, 13, 143, 144}.

As mentioned in the introduction, the epidermis of fish is extremely delicate and artifacts are inevitable; this is a frequent source of misdiagnosis¹⁴⁵. Mild pressure to the tissue, even after euthanasia, may cause leakage of blood into the epidermis or around scales. The outer layers of the epidermis are typically removed by gentle contact which may result in misdiagnosis of erosion or ulceration when in fact this is a handling artifact.

Increased cellularity, rodlet cells, epidermis

Pathogenesis/Cell of Origin

• Focal accumulation of rodlet cells in the epidermis.

Diagnostic Features

• Cells contain fine, acicular, hyaline pink/red, evenly spaced, regimented crystalline structures in the cytoplasm and a basally located nucleus

• Rodlet cells may occur in small clusters

Differential Diagnoses

• Infiltrate, inflammatory cell

• Protozoan parasites

Comments

Rodlet cells are a cell type of uncertain or undetermined lineage, possibly with an inflammatory role, that is unique to fish; they are typically located in secretory epithelia (biliary, gills), endothelial surfaces (endocardium, small vessels in the cranium), renal hematopoietic tissues, and around neuromasts⁶⁵. Increased numbers may indicate a heightened immune response and/or stress. Their appearance in the skin is not typical, and has been reported in association with polluted water, heavy metal exposure, saline stress, temperature stress, and/or wounds^{6, 105, 146}.

Infiltrate, inflammatory cell, epidermis

(Figs. 7.6 and 7.7)

Fig. 7.6.

Largemouth bass. Skin. Infiltrate, lymphocyte, epidermis, due to intraepithelial myxozoan infestation. H&E, 200×. Image courtesy of Wes Baumgartner.

Fig. 7.7.

Channel catfish. Skin. Infiltrate, lymphocyte, epidermis. H&E, 200×. Image courtesy of Wes Baumgartner.

Comments

The epidermis is non-vascularized, and inflammatory cells derive from the dermis. It is important to recognize that low numbers of leukocytes, and in particular lymphocytes, are normally present in the epidermis as a part of immune system function. Inflammation as a result of exposure to microbial organisms and pathogens is a common occurrence, similar to mammals. Furthermore, non-specific injury to the skin, such as exposure to acidic or polluted water, will lead to increased numbers of inflammatory cells as well^{105, 137}. Increased cortisol, a response to stress, can cause necrosis of epidermal lymphocytes¹².

Necrosis, epidermis

(Figs. 7.8 and 7.9)

Fig. 7.8.

Channel Catfish. Skin. Necrosis, epidermis localized to the upper layers with karyorrhexis. H&E, 400×. Image courtesy of Wes Baumgartner.

Fig. 7.9.

Brown trout. Skin. Necrosis, epidermis, localized to the middle layers (ulcerative dermal necrosis syndrome). H&E, 200×. Image courtesy of Wes Baumgartner.

Modifiers

• Full Thickness

• Laminar

• Single cell

• Keratinocyte

• Modified Secretory

• Sensory

• Chloride Cell

Other Terms

• Necrolysis (full thickness)

• Sunburn

Pathogenesis/Cell of Origin

• Non-specific cell death of epidermal cells

Diagnostic Features

Full thickness/laminar

• Complete loss of cellular detail from the epidermis

• Often associated with epidermal separation with cell death of basilar layer and detachment from underlying dermis

• Cells may be affected in the outer, middle, or basal layers

Single cell

• Individual cells show hyaline hypereosinophilic cytoplasm and nuclear pyknosis

• Necrotic cells may be surrounded by leukocytes

• Non-degenerate, associated cells may have phagosomal bodies

• Intraepithelial lymphocytes may be misinterpreted as pyknotic keratinocyte nuclei

Differential Diagnoses

• Erosion/ulcer: Loss of epidermal layers, partial or complete

• Artifactual loss of epidermis post-mortem characterized by lack of inflammatory response.

Comments

Necrosis of epidermal cells occurs in spontaneous and experimentally induced lesions. The skin itself has an oxygen demand that is supplied from the environment; hypoxic water will lead to keratinocyte necrosis and subsequent erosion/ulceration¹⁴⁰. Exposure of carp to acid water (pH 5.0) led to necrosis primarily of the pavement cells and surface secretory cells¹³⁷. Necrosis of individual cells in ultraviolet light (UV) exposed epidermis have been described in some literature as ‘sunburn cells’¹⁴¹, although this term is no longer preferred. Higher doses of UV will lead to full thickness epidermal sloughing¹⁴⁴. Skin necrosis has been reported as a result of various stressors including hypoxia^146,147,148. Experimental exposure of cortisol to rainbow trout led to necrosis of the pavement cells (outer layer), secretory cells, and intra-epithelial lymphocytes¹². Similar changes were evident in trout exposed to polluted river water¹⁰⁵. If specific cell types within the epidermis are affected or are to be specifically described, then the modifiers may be applied. Exposure of trout to transient hyperthermia caused epidermal necrosis in the outer and inner layers, sparing the intermediate keratinocytes¹⁰⁵. Epithelial cells are phagocytic and will contain phagosomal bodies in association with necrotic cells nearby¹⁰⁵. Tissue handling artifacts may mimic epidermal necrosis¹⁴⁵. Use of the terms necrosis, apoptosis and/or the combination term of apoptosis/necrosis in the skin are aligned with the INHAND publication¹⁴⁹.

Nuptial tubercles/Dorsal nape pad, present/increased/decreased, epidermis

(Figs. 7.10, 7.11, and 7.12)

Fig. 7.10.

Fathead minnow, male. Dorsal nape pad, increased (Grade 3). Note the expansion of the underlying dermis by specialized mucoid-appearing collagenous matrix (mucochondroid). H&E, 100×. Image courtesy of Jeffrey Wolf.

Other Terms

• Nuptial tubercles

• Breeding tubercles

• Dorsal nape pad: “fat” pad

Pathogenesis/Cell of Origin

• The dorsal nape pad is an androgen-induced bilaterally-symmetrical spongy plaque that develops normally on dorsal necks of reproductively-active mature male fathead minnows during the breeding season. Although sometimes referred to as a “fat pad”, the underlying dermis is actually expanded by a specialized mucoid-appearing collagenous matrix (mucochondroid)

• Reductions in the size and/or number of these structures in male fathead minnows can be caused by exposure to exogenous estrogens

• While typically nuptial tubercles and dorsal nape pads are entirely absent in females, nuptial tubercles and/or dorsal nape pads in females can be induced by exposure to exogenous non-aromatizable androgens

Diagnostic Features

• Nuptial tubercles are androgen-induced cone-shaped exophytic keratinized epidermal proliferations that develop normally on the faces of reproductively-active mature male fathead minnows, Pimephales promelas, during the breeding season. The pattern of tubercle formation is bilaterally symmetrical

• Nuptial tubercles may be increased in size and/or number, decreased in size and/or number or present in a sex not otherwise expected to exhibit them, depending on exposure to exogenous estrogen or androgens (see Comments)

Differential Diagnoses

• Papilloma: although microscopic appearance of an individual tubercle is similar to that of a papilloma, the presence of multiple bilaterally symmetrical tubercles on the face of a male fathead minnow should allow these to be differentiated from papilloma

Comments

The fathead minnow is a commonly used research model for endocrine disruption studies intended for regulatory agency submission. In such studies, alteration in the size and/or number of nuptial tubercles and/or dorsal nape pads (both are essentially secondary sex characteristics) is used frequently as external morphologic indicators of exposure to substances with estrogenic or androgenic activity¹⁵⁰.

Parasite, epidermis

(Fig. 7.6)

Other Terms

• Infestation (if metazoan) and/or infection (if crustaceans, leeches, monogenea, ciliates, flagellates, amoebae, myxozoans, or hydroids)

Pathogenesis/Cell of Origin

• Dependent on the agent involved.

Diagnostic Features

• Identifiable protozoans or metazoans on or in the skin

• Erosion or ulceration of the epidermis

• Epidermal hyperplasia in chronic conditions

• Alteration in secretory cell numbers

• Alteration in secretory cell content

• Inflammation, often lymphocytes and macrophages

Differential Diagnoses

• None

Comments

A wide variety of organisms infect/infest fish skin, inducing varying degrees of inflammation, ulceration, and hyperplasia depending on the species and stage of infection^{151,152,153,154,155}. Parasitism of the dermis and deeper tissues by protists may develop into epidermal cell parasitism as a result of severe infection.

It is also important to be aware of “X cells”, recently verified to be xenoma-forming protists (Xcellia spp. and Gadixcellia spp.), which are known to produce tumor-like papillary epidermal nodules in various species¹⁵⁶. X cells appear very similar to hyperplastic keratinocytes or club cells and may be present in the dermis and epidermis; they have a large nucleus & nucleolus, lack intercellular bridges typical of keratinocytes, and occur in small groups¹⁵⁷.

Pustule, epidermis

Comments

Epidermal pustules are rare in fish but are characteristic of certain disease conditions where intercellular edema and discohesion allow the accumulation of leukocytes. Pustules are named for the predominant leukocyte type. Pustules may develop as a fistulous process from deep dermal disease².

Vesicle, epidermis

Comments

Vesicles can result from immune mediated injury, as a result of edema, trauma, parasitism, sunburn, or a combination of these factors¹⁵⁸. Intraepidermal vesicles may develop from severe edema with cell rupture, known as ‘reticular degeneration’¹⁵⁹. In ulcerative dermal necrosis syndrome of salmon, a pemphigoid pattern is produced^{139, 160}.

Epidermis – Proliferative Non-Neoplastic

Hyperplasia, epidermis

Modifiers

• With Cellular Atypia

• Keratinocyte

• Sensory

• Chloride Cell

Pathogenesis/Cell of Origin

• Derives from epidermal cells; increased number of epidermal-derived cells

Diagnostic Features

• Increased thickness of epidermis, either generalized or plaque-like

• Rete ridge/dermal peg formation may be present, but is generally minimal

• Small clusters of filament cells may bulge from the surface

• Increased mitotic figures, can occur at all levels

• Keratinocytes may be hypertrophied

• Megalocytic cells are seen in certain herpesvirus infections

• Spongiosis or cyst formation may be present

• The basal lamina is intact

• Gland cell numbers may be increased or decreased

• Gland cells may be hypertrophied

• Chromatophores, esp. melanocytes may be increased

• Subjacent vascular networks may become prominent or atypical with cellular atypia

• Increased atypia, multinucleate cells, and whorls/pearls can be associated with more clearly demarcated plaques/nodules

Differential Diagnoses

• Papilloma: Thickened epidermis with exophytic/papilliform growth, extensive dermal interaction/ridging, well differentiated

• Carcinoma: Invasive growth into the basal lamina, increased mitoses and atypia

• Myxozoan parasite infections

• Cytomegaly (Lymphocystis) characterized by nodules formed by enlarged fibroblasts that contain iridovirus inclusions

• Infection with Gadixcellia and Xcellia parasites: Xenomas composed of large pleomorphic epithelioid protozoans (see gill chapter)

Comments

Epidermal hyperplasia results from a variety of chronic insults including inflammation, toxic irritation, abrasions, parasitism, extreme pH shifts, and UV exposure^{8, 137, 154, 161, 162}. Similar insults often cause erosion/ulceration or atrophy in the early stages¹³⁷. It is important to recognize that fish skin can vary markedly in thickness depending on the site sampled, sex, and time of year¹⁶³. In white suckers from the Great Lakes, plaques of hyperplasia, bulging white mucoid lesions, papillomas, and carcinomas occurred on a continuum grossly and microscopically¹⁶⁴. Hyperplasia may also present as small tufts or nodules in fish skin, particularly when they are in polluted waters, are host to parasites, or are at the low end of their temperature range¹⁰.

Depending on the species, hormones (prolactin, thyroxine, androgens, estrogens, corticosteroids, etc.) and increased exercise can potentially induce skin hyperplasia, increased filament cell turnover, and even atrophy^{12, 138, 165,166,167}. Carp pox (cyprinid herpesvirus 1) is a well-known example of virus-induced epidermal hyperplasia, but other viruses have been reported^{168, 169}. Giant karyomegalic cells have been reported in certain herpesvirus infections in pike, turbot, and cod^{170, 171}. In some cases, gland cell changes are prominent¹³. Infection with myxozoan parasites, Myxobolus episquamalis for example, can mimic epidermal hyperplasia very well¹⁷². Infection of the skin by surface dwelling protozoans like Ichthyobodo sp. can induce hyperplasia of the pavement cell layer¹⁵⁴. Keratinocyte hypertrophy has been described secondary to herpesvirus infection¹⁷³. If specific cell types within the epidermis are affected or are to be specifically described, then the modifiers may be applied.

It is also important to be aware that when perusing the literature “X cells”, recently verified to be xenoma-forming protists (Xcellia spp. and Gadixcellia spp.), which are known to produce tumor-like papillary epidermal nodules¹⁵⁶. X cells appear very similar to hyperplastic keratinocytes and may be present in the dermis and epidermis; they have a large nucleus & nucleolus, lack intercellular bridges typical of keratinocytes, and occur in small groups¹⁵⁷.

Hyperplasia, modified secretory cell, epidermis

Modifiers

• Gland cells

• Goblet/mucous cell

• Sacciform cell,

• Club cell (alarm cell)

• Chloride cell

Pathogenesis/Cell of Origin

• Derives from epidermal cells; increased number of modified epidermal-derived cells

Diagnostic Features

• Increased numbers of epidermal gland cells

• Cells may develop/accumulate in the basal/mid layers, or in some cases near the skin surface

• May be associated with epidermal cell hyperplasia

• May be associated with secretory cell hypertrophy or hyperplasia

• Mitotic figures are uncommon

Differential Diagnoses

• Papilloma: Thickened epidermis with exophytic/papilliform growth, well differentiated

Comments

Modified secretory cells (various types-see Modifiers and introductory comments) are highly adaptive with various specialized functions; depending on the body region, sex, temperature, feeding regimen, exercise, type of skin insult and disease chronicity they may vary widely in number, cell size, distribution, and composition of secretion^{105, 137, 165, 174,175,176,177,178}. In some cases, abundant mucus is secreted rapidly, which should be taken into account when assessing secretory cell numbers as mucous cells may not be readily apparent due to content loss/discharge¹⁴⁶. After discharge, secretory cells are shed from the skin¹⁰⁵. In carp exposed to manure-laden water, mucous cells were no longer seen in the skin by day 3, only to re-appear by day 8; this potential window of mucus-production impairment is important to consider¹³⁷.

In general, situations that lead to epidermal hyperplasia can also cause gland cell hyperplasia, including hormones, changes in water parameters, chronic parasitism, etc^{2, 8}. These findings can be combined under the finding of epidermal hyperplasia with details narrated in the report or finding comment. Cortisol treated rainbow trout exhibited increased filament cell and secretory cell turnover, with increased differentiation of mucous cells¹².

Exposure of carp to acidified water and cadmium led to increased numbers of chloride cells, which were absent in controls^{137, 162}.

Exposure of fish to cadmium led to reduced mucous/goblet cell numbers and changes in mucus composition; this effect is seen with other toxicants/heavy metals and certain parasites^{15, 137, 154, 162, 179, 180}.

Epidermis – Proliferative Neoplastic

Papilloma (B), epidermis

(Figs. 7.13, 7.14, and 7.15)

Fig. 7.13.

Koi. Skin. Papilloma, epidermis (lip), demonstrating interdigitating folds on fine stroma. H&E, 40×. Image courtesy of Wes Baumgartner.

Fig. 7.14.

Koi. Skin. Papilloma, epidermis (lip), with atypia and edema, epidermis, intercellular (higher magnification of Fig. 7.13). H&E, 200×. Image courtesy of Wes Baumgartner.

Fig. 7.15.

Koi. Skin. Papilloma, epidermis (lip), with atypia, epithelial cell. H&E, 400×. Image courtesy of Wes Baumgartner.

Other Terms

• Epithelioma

• Angioepithelial nodule/polyp

• Cauliflower disease

Modifiers

• Exophytic

• Endophytic

• With cellular atypia

Pathogenesis/Cell of Origin

• Proliferative, exophytic mass derived from epidermal cells; generally induced by naturally-occurring and/or exogenous toxins but generally considered not viral-associated in fish

Diagnostic Features

• Well circumscribed papilliform exophytic or endophytic mass without a capsule

• Composed of epidermal cells on a well vascularized stroma

• Atypia or an increased nucleus-cytoplasm ratio is evident

• A minority of cells may be multinucleate or karyomegalic

• Mitotic figures may be common, but not necessarily so

• Interdigitating dermal cores/folds are characteristic

• Specialized secretory cells may be present in abundance

• Fluid filled cysts or cavities may develop in areas of necrosis

• Ulceration, inflammation (esp lymphocytes), entrapped scales (complete or partial), mineral, and bone (metaplasia) may be seen

• Entrapped teeth may be seen near the surface of lip lesions

• Melanocytes may be abundant in the epidermis and stroma

• Marked angiomatous or stromal proliferation may be evident

• Exophytic: a more or less distinct stalk at the base

• Endophytic: no evidence of a stalk; the mass is continuous with the epidermis and possibly craterous invagination

• Tumor stroma is distinct from the dermis

Differential Diagnoses

• Hyperplasia, epidermis: Thickened epidermis generally without atypia, may form a distinct plaque; without formation of a papilliform mass or distinct dermal folding/interaction (see Comments)

• Lymphocystis (nodules formed by enlarged fibroblasts that contain iridovirus inclusions)

• Carcinoma: Invasive growth through basal lamina, increased mitotic figures and atypia.

• Infection with Gadixcellia and Xcellia parasites: Xenomas composed of large pleomorphic epithelioid protozoans (see gill chapter)

• Dental neoplasms: Cells produce dental hard tissues which are unequivocally neoplastic

• Fibrosis/fibroplasia: scar/repair tissue lined by normal to hyperplastic epidermis

• Fibroma: a spindle cell neoplasm that may be lined by hyperplastic epidermis

• Melanoma: melanocyte forming neoplasm

• Nuptial (or breeding) tubercles in male fathead minnows (faces of male fathead minnows) – see atrophy or increased

Comments

Papillomas are the most common skin neoplasms and vary from slightly raised to bulging, smooth to verrucose, tan to red, and sessile to pedunculated^{8, 181}. In most reported cases, keratinocytes predominate, however, ethylnitrosurea treated zebrafish developed papillomas with admixtures of keratinocytes, mucous cells, and club cells¹⁸². They have been reported in response to chronic toxin exposure where hyperplasia was an early change^{161, 164}. Virus infections have also been implicated in papillomata with seasonal variation^{10, 168, 183,184,185,186}. Papillomas on the face of eels in Northern Europe are a well-known entity; lesions are markedly affected by salinity¹⁸⁷. Those associated with salmon are notable in that they are shed, or resolve in association with seasons but can present with anaplasia and bizarre mitoses^188,189,190.

In some ulcerative or traumatic lesions free-floating dermal fibers may become re-epithelialized to form a papilliform non-neoplastic mass^{14, 191}.

In masses associated with the lips or fins, complex infoldings or continuity with the dental lamina may present with pseudoglandular formations and mineralized concretions^{192,193,194,195}. During tumor development the associated vasculature may become markedly hyperplastic and atypical^{157, 196}.

It is also important to be aware that when perusing the literature “X cells”, recently verified to be xenoma-forming protists (Xcellia spp. and Gadixcellia spp.), have been described in association with epidermal tumors in various species¹⁵⁶. X cells may be present in the dermis and epidermis, increasing in size and number along with tumor development; they have a large nucleus & nucleolus, lack intercellular bridges typical of keratinocytes, and occur in small groups¹⁵⁷.

Carcinoma (M), epidermis

Other Terms

• Epidermoid carcinoma

• Epithelioma

Modifiers

• Well-differentiated

• Poorly-differentiated

• Squamous cell

Pathogenesis/Cell of Origin

• Neoplastic mass derived from epidermal cells

Diagnostic Features

• Poorly demarcated mass

• A desmoplastic reaction is evident

• Cells are arranged in islands/cords that invade the basal lamina, extending into the dermis or deeper

• Pearl-like formations (concentrically packed cells) may be evident, but keratin cores are exceptional/not formed

• Cells are polygonal to pleomorphic with anisokaryosis, prominent nucleoli, and increased mitoses

• Ulcers, necrosis, and inflammation are common

• Fluid filled cysts or cavities may develop in areas of necrosis

• A glandular appearance may develop from complex infoldings, necrosis, or entrapment of secretory cells

• Some tumors may contain entrapped secretory cells, entrapped teeth near the surface of lip lesions, fragments of scales, mineral, or bone

• Tumor emboli, metastasis may be present, but they are exceptional

• Well differentiated characterized by mild atypica, keratinocyte features are readily evident and/or confluent growth/loss of papilliform arrangement

• Poorly differentiated characterized by cells in many areas do not readily resemble keratinocytes, due to atypia, pleomorphism, spindle cell morphology

Differential Diagnoses

• Papilloma: Thickened epidermis with exophytic/papilliform growth, well differentiated; evidence of invasion is lacking

• Dental neoplasms: Cells produce dental hard tissues which are unequivocally neoplastic

• Infection with Gadixcellia and Xcellia parasites: Xenomas composed of large pleomorphic epithelioid protozoans (see gill chapter)

• Lymphocystis (nodules formed by enlarged fibroblasts that contain iridovirus inclusions)

• Chromatophore tumors- see entry under dermis/subcutis

Comments

Carcinomas may originate from papillomas as part of a progressive change, from many proposed etiologies^164,190. They have been reported from various species^{186, 196,197,198,199,200}. In normal fish epidermis the most mitotically active cells are in the more basal layers, but with cancerous change the outermost layers become the most active²⁰¹. In the brown bullhead, a distinctive hyperemic pattern developed prior to epidermal hyperplasia, which developed into metastatic carcinomas^{8, 202}.

It is also important to be aware that when perusing the literature “X cells”, recently verified to be xenoma-forming protists (Xcellia spp. and Gadixcellia spp.), have been described in association with epidermal tumors in various species and may appear to be anaplastic epidermal cells¹⁵⁶. X cells may be present in the dermis and epidermis, increasing in size and number along with tumor development; they have a large nucleus & nucleolus, lack intercellular bridges typical of keratinocytes, and occur in small groups¹⁵⁷.

Non-proliferative lesions in the dermis/subcutis/adipose/muscle such as inflammation, necrosis, fibrosis, metaplasia, mineralization etc are all described in the soft tissue chapter. For other changes associated with scales which are applicable to bony tissues, please consult the ‘musculoskeletal’ chapter for terminology.

III. Microscopic Findings – Dermis and Subcutis, Integument

Table 2 shows nomenclature, diagnostic criteria, and differential diagnosis of dermis and subcutis.

Table 2. Nomenclature, diagnostic criteria, and differential diagnosis of dermis and subcutis.

Dermis & Subcutis			Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	*Atrophy			X
	*Chromatophores, decreased			X
	*Edema		X
	*Increased cellularity, Pigmented macrophage aggregates		X
	*Malformation/dysplasia, scale			X
	*Parasite			X
Proliferative
	Non-neoplastic
		*Cytomegaly, stromal cell	X
		*Hyperplasia, chromatophore, melanosis		X
	Neoplastic
		*Chromatophoroma, benign		X
		*Chromatophoroma, malignant		X
		*Melanoma, benign		X
		*Melanoma, malignant		X
		*Lepidosarcoma/malignant scale neoplasm		X

*Terminology with diagnostic criteria or comments described in the main text.

Dermis and Subcutis – Non-Proliferative

Atrophy, dermis or scale, dermis and/or subcutis

(Fig. 7.16)

Pathogenesis/Cell of Origin

• Reduced metabolic activity of dermal fibroblasts or scleroblasts (scales), or increased osteoclast activity

Diagnostic Features

• Loss of collagen fibers and extracellular matrix

• Fibroblasts or scleroblasts are smaller and more ovoid

• Osteoclasts may be present

• Inflammation may be present

Differential Diagnoses

• Edema, dermal: separation of collagen fibers by clear fluid or pale amorphous/granular substance

• Necrosis: loss of cell detail and nuclei

Comments

Scales may be resorbed preferentially as a source of calcium and would be expected to affect many and possibly all scales; pattern loss may preferentially affect scales above the lateral line²⁰³. Fish utilize scale minerals preferentially for physiologic mobilization, which may occur in calcium-poor water, with exposure to 17-beta estradiol, glucocorticoid exposure, during starvation, and during vitellogenesis^{72, 204}.

Scale resorption in precocious salmon parr begins on the posterolateral margins and progresses anteriorly^{205, 206}. Alternatively, resorption has been reported to occur at or near the scale focus^{72, 203}. As mentioned in the musculoskeletal chapter, fish can remove mineral from bone without removing the collagenous matrix (halastasis) and thus, true measure of mineral mobilization cannot strictly rely on evidence of osteoclastic resorption.

Regional scale loss can be associated with dermal inflammation or dermal atrophy secondary to space occupying masses²⁰⁷. Pollutant exposure is associated with scale breaks and abnormal scale formation^{208, 209}. Scale resorption as a consequence of increased osteoclast activity has been reported in an obesity model in zebrafish²¹⁰.

Loss of scale matrix may preferentially affect certain layers (limiting, external, or basal)^{203, 210,211,212,213}. In a scale regeneration study in zebrafish, osteoclasts were only evident on the episquamal surface of the scales, indicating a preferential site for physiologic calcium/phosphate mobilization⁷³.

Chromatophores, decreased, dermis and/or subcutis

Other Terms

• Hypomelanosis

Pathogenesis/Cell of Origin

• Loss of pigmented cells in the epidermis or dermis

Diagnostic Features

• Reduced numbers of pigmented cells

• Pigment cells may be necrotic, with karyorrhexis, karyolysis, and dispersion of pigment into the stroma

• Pigment containing macrophages are often increased

• Pigmented materials may be present in the epidermis due to expulsion through the skin

• Chromatophore location, pigmentation, or morphology may also be affected

Differential Diagnoses

• Edema: Excessive fluid in/between epidermal cells or soft tissue components

• Epidermal hyperplasia: Increased numbers of epidermal cells

• Increased mucus production

Comments

Chromatophore numbers in fish are a result of a balance between differentiation of the resident chromatoblasts and apoptosis of cells^{94, 214, 215}. Such changes may be a manifestation of “morphological color change”⁹⁶. Changes in environmental color can induce apoptosis/necrosis of melanocytes and leucophores in medaka skin²¹⁶. Macrophages and epidermal cells can ingest chromatophore fragments and facilitate elimination through the epidermis^{112, 216}. In flatfish hypomelanosis has been associated with inadequate nutrition or light exposure, a condition noted in other species^2,111. A recent study described various chromatophore changes in zebrafish skin when exposed to various compounds relevant to toxicologic studies; chromatophore numbers, location, pigmentation, and morphology were noted¹²⁸.

Edema, dermis and/or subcutis

(Figs. 7.17 and 7.18)

Fig. 7.17.

Zebrafish. Skin. Edema (protein rich), scale pocket, with hemorrhage. H&E, 100×. Image courtesy of Wes Baumgartner.

Fig. 7.18.

Goldfish. Skin. Dermis, edema. H&E, 200×. Image courtesy of Wes Baumgartner.

Other Terms

• Lepidorthosis

Modifiers

• Dermal

• Scale pocket

Pathogenesis/Cell of Origin

• Accumulation of interstitial fluid

Diagnostic Features

• Separation of collagen fibers and displacement of scales by clear fluid or pale amorphous/granular fluid

Differential Diagnoses

• Atrophy, dermal: Loss of both collagen fibers and extracellular matrix

Comments

Edema often accompanies dermal inflammation and ulceration^{159, 207}. Generalized hypoalbuminemia can also cause dermal edema.

Increased cellularity, pigmented macrophage aggregates, dermis and/or subcutis

(Fig. 7.19)

Fig. 7.19.

Channel catfish. Skin. Increased cellularity, pigmented macrophage aggregates and infiltrate, mononuclear cell. H&E, 400×. Image courtesy of Wes Baumgartner.

Other Terms

• Macrophage aggregates

• Increased pigmented macrophages

Pathogenesis/Cell of Origin

• Macrophages accumulated in dermis or subcutis containing a variety of pigment

Diagnostic Features

• Accumulation of pigmented round cells within the dermis or epidermis, which lack convincing features of chromatophores

• Pigment granules may be clumped or irregular, and a mixture of chromatosomes may be evident

• Often associated with skin inflammation or alterations in chromatophore numbers

Differential Diagnoses

• Chromatophores, hyperplasia/melanosis: A non-neoplastic, non-tumorous accumulation of well differentiated pigmented cells in dermis

• Melanoma/chromatophoroma, benign

• Melanoma/chromatophoroma, malignant

Comments

A result of altered chromatophore numbers, chromatophore injury, or chromatophore neoplasia is the escape of chromatosomes into the tissues. Macrophages accumulate this pigment and may form loose aggregates, more discrete nodules, exit through the epidermis, or possibly circulate to other organs where melanomacrophage centers occur²¹⁷. Non-specific injury to the skin may lead to pigment dispersion and increased numbers of macrophages filled with pigment in the epidermis^{105, 137}.

Malformation/dysplasia, scale, dermis and/or subcutis

(Fig. 7.20)

Fig. 7.20.

Betta splendens. Dermis. Dysplasia, scale, in association with chromatophoroma. Melanin bleach, 100×. Image courtesy of Wes Baumgartner.

Pathogenesis/Cell of Origin

• Scleroblasts

• Alteration in typical morphology as a result of trauma or other impact on typical development

Diagnostic Features

• Scales that distinctly vary in shape/size relative to normal scales

• Scleroblasts may be hyperplastic

• Altered scale matrix production or mineralization

• Osteoclasts/scleroclasts may be evident

Differential Diagnoses

• Decreased scale matrix or mineral (Decreased bone, trabeculae, cortex, or mineral): reduced quantity of scale matrix or mineral, which may include increased osteoclasts and eroded surfaces

Comments

Trauma to the scale pocket as a result of loss or inflammation can lead to abnormal scale growth or regeneration. Glucocorticoid exposure to zebrafish resulted in malformed and fused scales²⁰⁵.

Parasite, dermis and/or subcutis

(Figs. 7.21, 7.22, 7.23,7.24 and 7.25)

Fig. 7.21.

Zebrafish. Subcutis of vent. Algae. H&E, 200×. Image courtesy of Jan Spitsbergen.

Fig. 7.22.

Zebrafish. Subcutis of vent. Algae (higher magnification of Fig. 7.21). H&E. 400×. Image courtesy of Jan Spitsbergen.

Fig. 7.23.

Crappie. Dermis (fin). Cytomegaly, stromal cell, due to lymphocystis. H&E, 20×. Image courtesy of Wes Baumgartner.

Fig. 7.24.

Crappie. Dermis (fin). Cytomegaly, stromal cell, due to lymphocystis (higher magnification of Fig. 7.23). H&E, 100×. Image courtesy of Wes Baumgartner.

Fig. 7.25.

Bluegill. Dermis. Cytomegaly, stromal cell, due to lymphocystis. H&E.

Dermis and Subcutis –Proliferative Non-Neoplastic

Proliferative lesions in the dermis/subcutis/adipose/muscle are described in the soft tissue chapter (Chapter 13).

Cytomegaly, stromal cell, dermis and subcutis

(Figs. 7.26, 7.27, and 7.28)

Fig. 7.26.

Betta splendens. Dermis/subcutis. Chromatophoroma, benign, mixed, iridophore and melanocyte. H&E, 400×. Image courtesy of Wes Baumgartner.

Fig. 7.27.

Betta splendens. Dermis/subcutis. Chromatophoroma, benign, mixed, iridophore and melanocyte under polarized light to accentuate iridophores. H&E, 400×. Image courtesy of Wes Baumgartner.

Fig. 7.28.

Koi. Dermis. Melanoma, benign. H&E, 100×. Image courtesy of Wes Baumgartner.

Other Terms

• Lymphocystis

Pathogenesis/Cell of Origin

• Extraordinary fibroblast hypertrophy associated with iridovirus (Lymphocystivirus) infection

Diagnostic Features

• Megalocytic fibroblasts (up to 2 mm wide) forming exophytic clusters in the dermis

• Karyomegaly with abundant basophilic cytoplasmic inclusion material (lacy, corded, or globular)

• Cells are surrounded by a 5–20 micron thick hyaline capsule

• Cell necrosis occurs, with subsequent inflammation and fibrosis

• Possible retention of hyaline capsules, occasionally with mineralization/ossification

Differential Diagnoses

• Papilloma: Thickened epidermis with exophytic/papilliform growth, well differentiated; evidence of invasion is lacking

• Infection with Gadixcellia and Xcellia parasites: Xenomas composed of large pleomorphic epithelioid protozoans (see respiratory chapter)

• Encysted metazoan parasite – fibroplasia with or without inflammation, encircling a parasite

• Myxozoan or microsporidian cysts - thin-walled cysts/cells filled with monomorphic protozoans

Comments

Lymphocystis disease is an old, well described viral infection that occurs in many fish species^{218,219,220,221,222}. The virus infects mesenchymal cells throughout the skin and fins, and less commonly other organs (including mouth, gastrointestinal tract, heart, kidney, mesentery, spleen, liver, and ovary)²²³. Each infected cell enlarges up to 2 mm wide, forming botryoid nodules and coalescent proliferative lesions. Cells become apparent as soon as 5 days post-infection, and eventually slough¹⁷². Macroscopically, the epidermal proliferation associated with Icthyophthirius multifilis or Cryptocaryon irritans infections may be mistaken for lymphocystis²²⁴.

Hyperplasia, chromatophore, dermis and subcutis

Other Terms

• Hypermelanosis

• Hyperpigmentation

• Malpigmentation

• Black pigmented lesions

Pathogenesis/Cell of Origin

• Chromatophores

Diagnostic Features

• Accumulation of pigmented cells, individually or in clusters, in the dermis or epidermis

• Melanocytes may exhibit marked cytoplasmic extension/spindle conformation

• Lesions may be raised (associated with epidermal hyperplasia) or flat

• Often associated with inflammation or parasite encystment

Differential Diagnoses

• Increased cellularity, Pigmented macrophages

• Chromatophoroma/melanoma: Dense nodular proliferation of neoplastic pigmented cells that exhibit atypia, vacuolation, spindle shapes

• Parasite-related granuloma (trematodes, Ichthyophonus)

• Papilloma with increased melanocytes

Comments

Chromatophore numbers in fish are a result of a balance between resident chromatoblast differentiation (leading to increased numbers) and apoptosis, both of which are largely regulated by hormones¹¹⁴. Melanocyte numbers in fish increase due to maturation of precursor cells, not by division of mature cells²¹⁴. On the other hand, xanthophores and iridophores maintain the ability to proliferate and spread as fully mature cells⁹⁵. Such changes may be a manifestation of “morphological color change”⁹⁶. In zebrafish models of inflammation, melanoblasts appear within 3 hours, and melanocytes can appear as soon as 18 hours post injury²²⁵. Hyperplasia of melanocytes and/or iridophores, without apparent infectious cause, has been reported in wild bullhead and dab^{8, 226}.

Darkening of the skin is a non-specific change in fish caused by melanosome dispersion or increased chromatophore numbers; it is associated with asphyxia, spinal disease, ophthalmic impairment, social interactions, water quality issues, disease, other sources of stress, and aging^{2, 6, 12, 105}.

Pigmented spots are reported to develop in various species and like chromatophore neoplasia, they may be associated with chemical, UV radiation, or even viral exposure^{8, 227}.

Dermis and Subcutis – Proliferative Neoplastic

Neoplasms in the dermis & subcutis that derive from mesenchymal cells (except scales) are all described in the soft tissue chapter. Scale neoplasms are described below. Neoplasms originating from nervous tissue (nerve sheath tumor), vascular tissue (hemangioma, hemangiosarcoma), and lymphoid tissue (lymphosarcoma) are all described in the corresponding chapters of this publication. Chromatophores, including melanocytes, predominantly reside in the dermis, and neoplasms of these cells are described herein.

Chromatophoroma, benign (B), dermis and/or subcutis

(Figs. 7.20, 7.26 and 7.27)

Modifiers

• Mixed type

• Mosaic type

Other Terms

• Chromatoblastoma

• Iridophoroma

• Guanophoroma

• Erythrocytoma

• Xanthocytoma

• Leucocytoma

• Cyanophoroma

Pathogenesis/Cell of Origin

• Chromatophores; pigmented cells from the neural crest, including melanocytes, xanthophores, iridophores, erythrophores, leucophores, and mixed/mosaic cell types

Diagnostic Features

• Dense nodular proliferation in the dermis, with or without association towards the epidermis

• Tumor cells are polygonal, spindled, or epithelioid with variable degrees of cytoplasmic pigment production, which may include pteridine, purine, melanin, and carotenoid derivatives

• Cells may palisade similar to that in schwannomas/nerve sheath tumors

• Pigment granules are usually present to variable degrees

• Mitoses and nuclear atypia are not prominent

• Pigment laden macrophages or non-neoplastic melanocytes may also be present

• Mixed type: More than one well differentiated neoplastic chromatophore cell type is present, each in abundance; mosaic cells may be a minor component

• Mosaic type: The majority of the neoplastic cells produce an abundance of more than one pigment type which is inconsistent with normal organelle differentiation in that cell population

Differential Diagnoses

• Melanoma, benign: Non-invasive, fewer mitoses, well differentiated, cells produce only melanin

• Chromatophoroma, malignant: Invasive or metastatic, increased mitoses and nuclear atypia

• Lipogranulomatous inflammation: pigment laden macrophages, multinucleated giant cells, pigment is histochemically consistent with ceroid/lipofuscin

Comments

Neoplasms of pigment cells have been reported in many species and have been given a wide variety of names, depending on color (e.g. erythrocytoma) or pigment chemistry (e.g. pterinophoromas). Classically the color of the tumor, and therefore the diagnosis, is determined by their natural appearance in vivo; this is problematic since different chromatophore types can exhibit similar colors by different means (see introductory comments). Definitive characterization may require in vivo color documentation, pigment compound analysis, and ultramicroscopy.

Chromatophores naturally have an affinity for one another, and it should not be surprising to find, in addition to a neoplastic chromatophore population, a population of non-neoplastic chromatophores in or around the mass; this is admittedly problematic when trying to discriminate mixed-type tumors. Non-neoplastic chromatophores should be well differentiated and lack atypia or alteration in pigment organelle abundance/morphology.

In mixed tumors, well differentiated chromatophores of more than one type are present and tend to congregate with one another. On the other hand, the presence of many poorly differentiated chromatophores with an abnormal mixture of pigment organelles is supportive of a mosaic tumor. The occurrence of mosaic cells (chromatophores producing more than one pigment type) has been diagnosed in reptile chromatophoromas; this is consistent with the understanding that chromatophore types originate from common stem cells, and when neoplastic may acquire the ability to produce variable pigment types.

Chromatophores are known to exhibit short and long range interactions, leading to attraction and repulsion of certain cell types⁹⁴; this tendency may be manifested in the propensity for certain chromatophore types to aggregate within masses. Mixed chromatophoromas have been named according to pigment involved (irido-melanophoroma, etc.)²²⁸. The term ‘chromatoblastoma’ was coined to emphasize the common cell origin. Neither of these nomenclatures are preferred. A benign mixed tumor of iridophores and melanocytes is best diagnosed as: chromatophoroma, benign, mixed, iridophore and melanocyte. See additional references of interest^{122, 229,230,231,232}.

Chromatophoroma, malignant (M), dermis and/or subcutis

Modifiers

• Mixed type

• Mosaic type

Other Terms

• Malignant chromatoblastoma

• Malignant iridophoroma

• Malignant guanophoroma

• Malignant erythrocytoma

• Malignant xanthocytoma

• Malignant leucocytoma

• Malignant cyanophoroma

Pathogenesis/Cell of Origin

• Malignant neoplastic proliferation of chromatophores, which are pigmented cells from the neural crest

Diagnostic Features

• Dense nodular proliferation in the dermis, with or without association towards the epidermis

• Invasion or metastasis may be evident

• Tumor cells are polygonal, spindled, or epithelioid with variable degrees of cytoplasmic pigment production, which may include pteridine, purine, or carotenoid derivatives

• Pigment granules are usually present to variable degrees

• Cells may palisade similar to that in schwannomas

• Mitoses and nuclear atypia are increased

• Multinucleate cells may be present

• Pigment laden macrophages or non-neoplastic melanocytes may also be present

• Mixed type: More than one neoplastic chromatophore cell type is present

• Mosaic type: The neoplastic cell population produces more than one pigment type within the cells

Differential Diagnoses

• Melanoma, malignant: Poorly differentiated cells that produce only melanin

• Chromatophoroma, benign: Expansile, well differentiated cells, minimal mitoses or nuclear atypia

• Fibrosarcoma: Fibroblastic cells that lack pigment and form dense fascicles

• Schwannoma: Spindle cell neoplasm with storiform arrays, distinct whorling, Verocay bodies, and exhibit immunohistochemical and ultrastructural features of nerve sheath cells

Comments

Neoplasms of pigment cells have been reported in many species and have been given a wide variety of names, depending on color (e.g. erythrocytoma) or pigment chemistry (e.g. pterinophoromas). Classically the color of the tumor, and therefore the diagnosis, is determined by their natural appearance in vivo; this is problematic since different chromatophore types can exhibit similar colors by different means (see introductory comments). Definitive characterization may require in vivo color documentation, pigment compound analysis, and ultramicroscopy.

Chromatophores naturally have an affinity for one another, and it should not be surprising to find, in addition to a neoplastic chromatophore population, a population of non-neoplastic chromatophores in or around the mass; this is admittedly problematic when trying to discriminate mixed-type tumors. Non-neoplastic chromatophores should be well differentiated and lack atypia or alteration in pigment organelle abundance/morphology.

In mixed tumors, well differentiated chromatophores of more than one type are present and tend to congregate with one another. On the other hand, the presence of many poorly differentiated chromatophores with an abnormal mixture of pigment organelles is supportive of a mosaic tumor. The occurrence of mosaic cells (chromatophores producing more than one pigment type) has been diagnosed in reptile chromatophoromas; this is consistent with the understanding that chromatophore types originate from common stem cells, and when neoplastic may acquire the ability to produce variable pigment types.

Malignant chromatophoromas with metastases have been reported, albeit rarely²³³. Interestingly, the development of melanomas within existing chromatophoromas has been reported in Xiphiphorus hybrids, a well-known melanoma model^{234, 235}.

Chromatophores are known to exhibit short- and long-range interactions, leading to attraction and repulsion of certain cell types⁹⁴; this tendency may be manifested in the propensity for chromatophore types to aggregate within masses. Mixed chromatophoromas have been named according to pigment involved (irido-melanophoroma, etc.)²²⁸. The term ‘chromatoblastoma’ was coined to emphasize the common cell origin. Neither of these nomenclatures are preferred. A malignant mixed tumor of iridophores and melanocytes is best diagnosed as: chromatophoroma, malignant, mixed, iridophore and melanocyte. See additional references of interest^{122, 229,230,231,232}.

Melanoma, benign (B), dermis and/or subcutis

(Figs. 7.28 and 7.29)

Fig. 7.29.

Koi. Skin. Melanoma, benign. H&E, 400×. Image courtesy of Wes Baumgartner.

Other Terms

• Melanocytoma

Pathogenesis/Cell of Origin

• Melanocytes; neuroectodermal cells from the neural crest

Diagnostic Features

• Dense nodular proliferation in the dermis, with or without association towards the epidermis

• Tumor cells are dendritic, polygonal, spindled, or epithelioid with variable degrees of melanin pigmentation

• Very large cells (macromelanocytes) may be abundant

• Cells may palisade similar to those in schwannomas/nerve sheath tumors

• Dark brown/black pigment granules are usually present in abundance

• Pigment laden macrophages may also be present

• They may be multicentric

• Amelanotic type: tumor cells do not contain pigment granules that can be identified by H&E standard light microscopy

Differential Diagnoses

• Hyperplasia, chromatophore: well differentiated cells, abundant pigment, no atypia

• Melanoma, malignant: Invasive growth, higher mitotic activity, metastasis

• Chromatophoroma: Alternate or additional pigment types present in neoplastic cells

• Fibrosarcoma: Fibroblastic cells that lack pigment and form dense fascicles

• Schwannoma/nerve sheath tumor: Spindle cell neoplasm with storiform arrays, distinct whorling, Verocay bodies, and immunohistochemical and ultrastructural features of nerve sheath cells

Comments

Xiphiphorus spp. (platyfish/swordtail crosses) have been used to study melanomas extensively; neoplasms in this species can arise spontaneously, by UV exposure or chemical agents²³⁶. Xiphiphorus have two mature melanocyte types, micromelanocytes and macromelanocytes which are terminally differentiated. A grading scheme has been published^237,238,239. A hybrid Xiphiphorus strain developed concurrent melanoma and chromatophoroma, and in some cases the former neoplasm invaded and partially replaced the latter²³⁵. Poorly melanized tumors may occur as a result of reduced melanosome production²⁴⁰, however the presence of additional pigment organelles should be considered (mosaic chromatophores) and may alter the diagnosis.

Several melanocyte selective antibodies have been used to delineate melanotic lesions. HMB-45 and Melan-A are associated with melanocyte activation (premelanosomal membranes), while tyrosinase is involved in melanin production. S-100 is highly sensitive but relatively non-specific, yet remains a valuable stain as it will detect most melanocytic cells. The utility of these stains in other chromatophore tumors is poorly understood²⁴¹.

Chromatophore neoplasia may be associated with chemical, UV radiation, or even viral exposure; epizootics have been reported^{8, 227}.

Melanoma, malignant (M), dermis and/or subcutis

Modifiers

• Amelanotic type

Other Terms

• Melanosarcoma

• Melanocytic tumor

Pathogenesis/Cell of Origin

• Malignant neoplastic proliferation of melanocytes which are neuroectodermal cells derived from the neural crest

Diagnostic Features

• Dense nodular proliferation in the dermis, with or without association towards the epidermis

• Tumor cells are polygonal, spindled, or epithelioid with variable degrees of melanin pigmentation

• There is invasive growth or metastasis

• Necrosis may be present

• Cells may palisade similar to that in schwannomas

• Dark brown/black pigment granules are usually present to variable degrees

• May show frequent mitoses and nuclear atypia

• Very large cells (macromelanocytes) may be abundant

• They may be multicentric

• Inflammation may be present

• Amelanotic type: Tumor cells do not contain pigment granules that can be identified by H&E standard light microscopy

Special techniques for diagnosis

• Histochemistry: Masson-Fontana stain for melanosome detection

• Transmission electron microscopy: demonstration of melanosomes

• Immunohistochemistry: staining for Melan-A (MART1), HMB-45, S-100, tyrosinase

Differential Diagnoses

• Hyperplasia, chromatophore: non-tumorous, well differentiated cells, abundant pigment, little or no atypia

• Melanoma, benign: Non-invasive, fewer mitoses, well differentiated

• Chromatophoroma, malignant: Alternate or additional pigment types present in neoplastic cells

• Fibrosarcoma: Fibroblastic cells that lack pigment and form dense fascicles

• Schwannoma/nerve sheath tumor: Spindle cell neoplasm with storiform arrays, distinct whorling, Verocay bodies, and immunohistochemical and ultrastructural features of nerve sheath cells

Comments

Xiphiphorus spp. (platyfish/swordtail crosses) have been used to study melanomas extensively; neoplasms in these fish can arise spontaneously, by UV exposure or chemical agents²³⁶. Xiphiphorus have two mature melanocyte types, micromelanocytes and macromelanocytes which are terminally differentiated. A grading scheme has been published^237,238,239. Tumor development is attributed to the activity of a dominant-acting sex-linked oncogene, Xmrk, an ortholog of human EGFR gene. It appears that full expression of Xmrk occurs when it is no longer inhibited by the suppressor gene Diff. A hybrid Xiphiphorus strain developed concurrent melanoma and chromatophoroma, and in some cases the former neoplasm invaded and partially replaced the latter²³⁵. Poorly melanized tumors may occur as a result of reduced melanosome production²⁴⁰, however the presence of additional pigment organelles should be considered (mosaic chromatophores) and may alter the diagnosis.

Several melanocyte selective antibodies have been used to delineate melanotic lesions. HMB-45 and Melan-A are associated with melanocyte activation (premelanosomal membranes), while tyrosinase is involved in melanin production. S-100 is highly sensitive but relatively non-specific yet remains a valuable stain as it will detect most melanocytic cells. The utility of these stains in other chromatophore tumors is poorly understood²⁴¹.

Chromatophore neoplasia may be associated with chemical, UV radiation, or even viral exposure; epizootics have been reported^{8, 227}.

Lepidosarcoma, dermis and/or subcutis

(Fig. 7.30)

Fig. 7.30.

Medaka. Skin. Lepidosarcoma. H&E. Image courtesy of Jeffrey Wolf.

Other Terms

• Scale osteosarcoma

• Scale osteoma

• Lepidoma

• Lepidocytoma

Pathogenesis/Cell of Origin

• Derives from scale-forming cells

Diagnostic Features

• Mesenchymal cells are variably differentiated, polygonal to spindled

• Cells are arranged in fascicles and storiform arrays

• Cell clusters around small scale spicules may be prominent or appear as multinucleate cells

• Mitoses are variable from rare to frequent

• Atypia may be marked but is variable.

• Scale hard tissues may or may not be abundant; osteoid may predominate

• More than one tumor may be present

Differential Diagnoses

• Lepidocytoma: Benign scale forming neoplasm

• Malformation/dysplasia, scale: Abnormal scale developing from non-neoplastic cells

• Nodular reactive bone: Non-neoplastic proliferation of well differentiated irregular bone

• Fibrosarcoma: Fibroblast neoplasm with entrapped or metaplastic bone matrix

• Osteosarcoma: Malignant osteoblast tumor producing bone, lacking scale or dental hard tissues

• Odontogenic neoplasm: Mesenchymal neoplasm producing dental hard tissues

• Lymphocystis: Non-neoplastic cytomegalic transformation of virus infected fibroblasts rimmed by hyaline capsules

• Osteochondroma: Benign mesenchymal neoplasm with bone and cartilage admixed

Comments

Neoplasms that arise from the scale may develop hard tissues resembling bone or dental elements; they are rare. Special histochemical staining of large matrix foci may reveal layering consistent with scale development²⁴².

Tumors described in the literature as lepidocytomas (preferred terminology lepidosarcomas) occurred in medaka exposed to MNNG; the amount of scale matrix varied considerably²⁴³. In some cases, cells exhibit marked anaplasia with a high mitotic count.

Macroscopically, lepidosarcoma must be differentiated from the walleye dermal sarcoma, which is a benign, multifocal seasonal neoplasm associated with a retrovirus; neoplastic cells arise from superficial scale surfaces and can produce bony matrices^{168, 244, 245}.

The bone surrounding the lateral line canals (canal tubes) is distinct from the scale matrix; bone producing neoplasms arising in the skin may therefore develop from the tissues that produce scales or canal tubes, lepidosarcomas or canal osteosarcomas, respectively.

Scleroblasts/scale forming cells, similar to endoskeletal osteoblasts have desmosome connections²⁴⁶.