Mystery of Unicellular and Multicellular Keratinous Tubercles of the Epidermal or Dermal Origin in Fish- Juniper Publishers
Juniper Publishers- Journal of Cell Science
Abstract
Multicellular keratinous tubercles in the form of
breeding tubercles (BTs) under a perplex variety of other terms have
long been known in the Ichthyology write-ups. These tubercles in fish
are most often found on the head, rostral cap or snout, around the eyes,
operculum, on the rays of the pectoral fins, pelvic fins, and even they
are arranged in fine patterns on scales usually be confused with
unicellular and multicellular non-BTs as there is no much variation in
the appearance of BTs and non-BTs morphology. Variety of non-BTs have
different purposes like minute tubercles may even used for observations
of larval development in some fish and most commonly the tubercles which
are non-breeding help to understand the functional significance in
relation to mechanical protection, friction and adhesion.
There is absence of very clear evidence about the
exact biological and evolutionary significance of BTs, however, there
are many possibilities. They may be significant for protection against
injury, weapons in intense pre-spawning behaviour or stimulators during
spawning, an indicator of health or dominance. In consequence, the
actual purpose of BTs could be some, or all, of the above things. The
review will make systematists, ethologists, other zoologists and
researchers more aware to look for the structures with clear concept of
differentiation in between BTs and non- BTs so as to use them in
understanding the adaptation, behaviour and evolution of fishes.
Keywords:
Contact organs; Epidermal appendages; Fish skin roughness; keratinised
skin nodules; Minute tubercles; Nuptial tubercles; Pearl organs
Abbreviations: BTs: Breeding Tubercles; LM: Light Microscope; SEM: Scanning Electron Microscope
Introduction
Sykes [1] was one of the first workers to describe and figure tubercles on Indian minnows in five new species that he placed in Cyprinus, Varicorhinus, and Barbus. Leydig [2] figured the tubercles of Cyprinus carpio, Rhodeus amarus (without a keratinized cap), and Discognathus (=Garra) lamta. Fowler [3] and Denoncourt [4], among others, examined tubercle distribution [5]. Branson [6] and Wiley & Collette [7] described tubercle anatomy. Wiley & Collette [7]
used differences in tubercle distribution and structure to support
evidence for phylogenetic relationships among ordinal groups of
cyprinids.
The literature review of the occurrence of breeding tubercles and contact organs was begun by Collette [8].
In 1969, Wiley compared the morphology and histology of the tubercles
and contact organs of representative fishes in an attempt to devise a
meaningful nomenclature for the nuptial structures. Here efforts are
made to elucidate not all of the references on tubercles and contact
organs but to include those most important ones which are biologically
and historically significant. Roberts [9] described horny projections arising from single epidermal cells as unculi; Egami & Nambu [10] stated that innervations of fin rays bearing tubercles of male Oryzias latipes
was greater than that in fin rays of females, which lack tubercles.
However, horny projections arising from multicellular cells, or
tubercles, of most teleosts are described and discussed regarding their
status as many indicators with special representative stimuli. The
well-known multicellular horny tubercles, especially prominent features
of portions of the epidermis are present on some species in at least
fifteen families of fishes in four orders:
1. Salmoniformes, suborder Salmonoidei (Salmonidae, Plecoglossidae, and Osmeridae) and suborder Galaxioidei (Retropinnidae)
2. Gonorynchiformes, suborder Chanoidei (Kneriidae, Phractolaemidae)
3. Cypriniformes, suborder Characoidei (Lebiasinidae
and Parodontidae), and suborder Cyprinoidei (Cyprinidae,
Gyrinocheilidae, Psilorhynchidae, Catostomidae, Homalopteridae,
Cobitidae)
Superficially similar structures that are not
sexually dimorphic have also been reported in an African family of
freshwater catfishes (Siluriformes, Mochokidae). Analogous dermal
structures, known as contact organs, are present on the scales or fin
rays of nine families in three orders:
a. Atheriniformes, suborders Exocoetoidei (Belonidae)
and Cyprinodontoidei (Oryziatidae, Cyprinodontidae, Anablepidae, and
Poecliidae);
b. Cypriniformes, suborder Characoidei (Characidae and Gasteropelecidae)
c. Scorpaeniformes (Cottidae and Cottocomephoridae), [7].
In addition to BTs, the various useful terms have
been given by Scientists such as in the form of "Contact organ" for the
dermal structures defined by Newman [11], "Small nodules" by Stokell [12], "Pearl organs" by Reighard [13]; Norman [14]; Jakubowski et al. [15]; "Hook shaped tubercles" by Bell-Cross & Jubb [16], "Keratinised epidermal appendages" by Fischer et al. [17], "Spike-like keratinized epidermal structures by McMillan et al. [18]. In 1970, Wiley and Collette described three different kinds of BTs. The 1st is composed of not obviously keratinised epidermal cells. In the 2nd type a hard conical keratinised epidermal cap is formed which resembles a plant thorn. The 3rd
type of tubercle is the contact organ which, although similar in
function, is quite different and is formed of calcified dermal
outgrowths from scales or fin rays. The conical epidermal cap like 2nd
type of BTs is most significant, here the cells become flattened as
they are keratinised and finally die, and the nuclei are either lost or
become parakeratotically shrunken. Evidence for keratinisation comes
from histological appearance and various staining reactions to detect
bound cysteine and bound phospholipids.
Spearman [19]
reported that cornification or keratinisation is uncommon in fish and
occurs only in restricted sites like in breeding or nuptial tubercles to
maintain close contact during spawning and probably around the sharp
jaws in herbivorous fish. On the basis of literatures on skin based on
morphological studies in several fish species such as, keratinocytes
remain metabolically active throughout, including the outermost layer,
and lack morphological signs of cornification, in contrast to the
organization of BTs, contact organs on the head and pectoral fin rays,
described as keratinized epidermal appendages covered by a layer of dead
cells with altered content, suggesting that in BTs, keratinocytes might
undergo more advanced, cornification-like differentiation processes [14].
Non-BTs
Non-BTs were also mentioned in old literatures with
various poorly descriptive, unspecific, or anatomically inappropriate
terms. In1841, Sykes reported "Spines" projecting from rostral cap,
lips, and tubercles (non-breeding) on mental adhesive disc.
Afterwards in 1901, Boulenger reported Horny projections from single
cells "Hornzahnchen" on rostral cap and mental adhesive organ and in
1921, Hora observed "Minute spines" on tubercles (non-breeding) of
rostral cap and mental adhesive disc. The term "minute tubercles"
defined by Suzuki & Hibiya [20,21], Suzuki et al. [22] on the skin surface of fish larvae. Singh et al. [23] reported the anterior region of the upper lip in Hill-stream fish Schizothorax richardsonii
(Cypriniformes: Cyprinidae) have much small and large number of
tubercles (non-breeding) with different shape and size such as
spherical, cylindrical and elongated, each tubercle bears numerous
glandular secretive device and possibly keratinized spine, help in
adhesion due to friction between tubercles and surface of substratum
making a firm hold in rapidly flowing waters.
Fish have a wide range of protective skin
adaptations, which enable them to occupy habitats ranging from rocky
bottom surface to turbulent water. The skin of general body, head and
snout is usually scale less and the epidermis is of both types i.e.
keratinized as well as mucogenic. The rough epidermis of snout bears
only epithelial cells. Surface of these epithelial cells are modified
into epidermal growth the unculi. These unculi are short and stumpy
structures. In these fish, the epidermis of middorsal part of snout
possesses epidermal tubercles. These type of structures are absent in
dorso-lateral part of general body and snout epidermis [18]. Hora [15] also suggested that the so-called anterior labial fold in Garra species is fringed and tuberculate and helps the fish adhere to rocks.
According to Roberts [24],
unculi are related morphologically to the relatively well-known
multi-cellular horny tubercles (including BTs) of Ostariophysi and other
fishes but differ from them in anatomical distribution and function.
Singh et al. [23] studied on a hill stream fish Schizothorax richardsonii
belongs to the family Cyprinidae and is predominantly adapted to life
in swift flowing waters. The anterior region of the upper lip had much
small and large number of tubercles (non-breeding) with different shape
and size such as spherical, cylindrical and elongated. Such as the
callus part drawn and a cavity was produced that surrounded by the
tuberculated borders. The lower lip on its ventral side was associated
with a specialized structure the tuberculated adhesive pad through a
thin fold of skin. These non-BTs were responsible for adhesion, resulted
due to friction between tubercles and surface of substratum.
BTs
Nuptial tubercles may be grouped into three general categories on the basis of their structure:
1. Tubercles consisting of aggregations of
non-keratinized epidermal cells (if keratinization is present, it is
confined to the most superficial layers of cells and may form a light
cuticle).
2. Tubercles containing substantial numbers of fully
keratinized cells that are organized to form a discrete, usually conical
cap, a major component of the tubercle.
3. Contact organs (a term proposed by [27])
composed of dermal bony outgrowths or spicules projecting from a fin
ray or scale margin and surrounded by the epidermis, through which the
bony outgrowths may protrude.
Cornification observed on the unculiferous multicellular BTs in a small number of cyprinoids and siluroids [7].
In histological sections of tubercles prepared with Mallory's Triple
Stain, the unculi are orange-red, a staining reaction presumably due to
keratin (Our personal observations). In our lab during my doctoral
studies, we observed BTs towards head region on rostral cap and
operculum in one of the six fish species that is a hill stream fish Garra lamta,
Its histological and histochemical properties were same as keratinised
region of rostral cap (Pinky et al. 2004) but the BTs vary in
appearance, projecting out from epithelial surface so as to look cone
like V-shaped outgrowth with light microscope (unpublished data).
All or most species of family Cyprinidae develop
multicellular horny tubercles or BTs on the head and other parts of the
body and a few develop enormous rostral projections heavily set with
large, bifid or trifid tubercles. The most heavily tuberculate
individuals tend to be sexually mature females [27,24]. Roberts [24] had various family observations on BTs such as Gyrinocheilidae is rheophilic bottom-dwelling family, adults of genus Gyrinocheilus
develop multicellular BTs on the snout and head which are sometimes
better developed in females than in males; Catostomidae is predominantly
bottom-dwelling cyprinoid family comprises large BTs on the head, body,
and fins of sexually mature adults; small multicellular BTs occur on
the head and on the dorsal surface of the pectoral fin in Psilorhynchus sucatio and P. balitora; mature males of genus Barilius, family Cyprinidae also exhibit extensive development of multicellular BTs on the head, spawning behaviour of Barilius bendelisis
is unknown. Roberts predicted that it involves substrate contact by the
unculiferous prepectoral groove and horny tubercles on the gular region
of the male; Cobitididae is the predominantly Asian cyprinoid
bottom-dwelling family in which the outermost branched pectoral fin-ray
is greatly enlarged or thickened, and its dorsal surface may bear horny
BTs. BTs are restricted to fishes belonging to the cohort Euteleostei.
To explain their presence, we must consider what the fishes with them
have in common. First, fishes that have breeding tubercles, or the
species within a group that have them, are fresh-water or inshore marine
inhabitants. The analogous dermal contact organs present in the
Cyprinodontoidei, Characidae, and Cottidae are also developed in
fresh-water and inshore marine species. No pelagic or epipelagic marine
fishes are known to develop the tubercles. Secondly, most families with
these tubercles belong to groups of fishes with cycloid scales. Only few
families (Percidae, Cottidae, and Cottocomephoridae) belong to the
great group of higher fishes-the Acanthopterygii- which usually have
ctenoid scales [7].
Histology of BTs
The multicellular keratinized epithelia are mainly
composed of the epithelial cells. The lymphocytes are infrequent and
could be located on careful search, confined in the intercellular spaces
in between the basal cells. The gland cells and the taste buds usually
occur in the mucogenic epithelia are absent. Epidermal cells are
produced by mitoses in a columnar to cuboidal stratum germinativum next
to the basement membrane. The cells in more superficial layers
hypertrophy and become polygonal. They are characterized by large
vesicular nuclei with one or more prominent nucleoli and often granular,
acidophilic cytoplasm. Well-developed intercellular spaces and many
intercellular bridges usually lie between the hypertrophied cells. The
transition between hypertrophied cells and the keratinized layer is so
abrupt that transitional stage is rarely observed. During
keratinization, the nuclei disappear or persist as pyknotic remnants in
the usually flattened, irregular cells of the keratinized layer. The
keratin becomes light orange to red, in Mallory's triple stain. In a
typical preparation, undifferentiated epidermal cells of the germinal
layer have light blue cytoplasm that becomes darker blue as cells
hypertrophy and accumulate prekeratin granules, and violet in the
largest cells before keratinization. The nuclei of hypertrophied cells
contain prominent, darkly stained nucleoli.
Wiley & Collette [7] reported two species, Moxostoma erythrurum and Erimyzon sucetta, both are distinctly different from each other in tubercle morphology. The tubercles of M. erythrurum
were simply mounds of cells formed by epithelial hypertrophy and
hyperplasia with keratinization of the tissue above a plane parallel to
the surface. In contrast, the snout tubercle of E. sucetta was a
solid keratinized cone supported by vascularized hypertrophied
epithelium and closely resembles some of the larger cyprinid tubercles.
Many of the larger cyprinid tubercles develop vascularised dermal
papillae which extend into the hypertrophied epithelium of the tubercle
core, while, in some tubercles, the dermal papillae are accompanied by a
layer of the stratum germinativum and probably serve the dual function
of providing nutrition to the rapidly growing epidermal cells of the
tubercle and increasing the number of germinative cells required for the
rapid growth that is characteristic of developing tubercles Fischer et
al. [17]
identified the zebrafish BTs as sites with higher keratinocyte
proliferation in basal layers as well as more advanced,
cornification-like keratinocyte differentiation in upper layers,
including transglutaminase expression and stronger keratinization,
exclusive presence of tight junctions in second-tier keratinocytes, and
rudimentary lipid envelope formation and constant desquamation and
renewal of surface keratinocytes.
Histochemistry of BTs
In the older literature, however, the unculi, in
general, are described as cornified or horny without any histochemical
characterisation. Strong eosinophilia with Papanicolaou stain and strong
fluorescence with the different fluorochromes, with or without prior
DNase/RNase treatment further confirms the keratinized nature of the BTs
in Garra lamta. (Personal observations). Cysteine bound -SH
groups elaborated by the epithelial cells during keratinization
gradually increase in concentration in the cells towards the surface.
These, in the major portions of the unculi, do not seem to be strongly
converted into cystine bound -SS groups at the thin peripheral regions
of the unculi [28] and cone like projections of BTs (unpublished data) in Garra lamta.
The absence of cystine bound -SS groups, which are reported to hold
together adjacent keratin side chains (Alexander et al., 1963),
indicates that keratin in the BTs in Garra lamta is not so strongly bonded. Schwerdtferger & Bereiter-Hahn [29] reported the presence of cystine bound -SS groups in addition to cysteine bound -SH groups in the pearl organs (=BTs) of Abramis brama, Rutilus rutilus.
Presence of GPs with oxidizable vicinal diols in the BTs (personal
observation), suggests further studies, necessary to understand its role
in relation to keratinization in BTs of fish. The GPs synthesised in
the epithelial cells are secreted at the surface forming an
extracellular coat. It varies considerably in thickness and consistency
in different groups of fishes. It is thick and even appears fibrous on
epidermal tubercles of Agonus cataphractus Whitear & Mittal (25).
Discussion
Leydig [2]
remarked on the similarities in morphology and development of
tubercles, mammalian hair, saurian femoral pores, and skin sense organs.
Newman [26]
long ago suggested, ctenoid scales may have evolved in higher fishes to
replace permanently the temporary contact organs and BTs found during
the breeding season in lower fishes. The BTs, or pearl organs, presumed
to be secondary sexual structures are composed of substantial amounts of
keratin. They are common in several families of cypriniform fishes and
they are also known in a few gonorynchiform and salmonoid fishes. The
correlation between contact organ location and behaviour suggests that
they may be tactile, enabling the male to determine his exact position
relative to the female [30]. Wiley & Collette [7] and Collette [31]
discussed the reproductive role of multicellular horny tubercles (BTs)
of fishes. In many species the head and especially the snout are heavily
tuberculate, and this does not appear to be sexually dimorphic: the
tubercles are already well developed in very small juveniles and are
equally well developed in large mature specimens of both sexes [24].
Initially there was little evidence to support the suggestion made by Reighard [32-34]
that tubercles protect body and fin surfaces in nest building. However,
later on after seven- eight decades, suggested roles of BTs include
defence against mechanical injury, microorganisms and parasites,
protection of nests and territories [35-38]. Branson [6]
suggested that tubercles also function in holding to the substrate
during oviposition or in tending the nest and eggs or that made by
Branson [39] that anterior head tubercles are used for scooping.
The importance of epidermal cornification in the
evolution of tetrapod vertebrates is common knowledge. The diversity of
terrestrial vertebrate life as we know it would be impossible without
the "boundary layer" of cornified epidermis, so vital for maintenance of
the internal milieu. If the presence of horny teeth on the mouth parts
of both living groups of primitive jawless fishes or Agnatha can be
taken as an indication, then horny structures may have played a
significant role in the evolution of early vertebrates before true teeth
evolved [24].
Males and females of Kneria develop numerous
small multicellular horny tubercles on the dorsal and lateral surface of
the head and body, but tubercles are absent on the paired fins.
Phractolaemidae family comprises a single species, air-breathing Phractolaemus ansorgei.
Sexually mature males bear large, spike-like horny tubercles
immediately beneath the minute eyes and on the caudal peduncle, rows of
microbranchiospines or unicellular tubercles on the gill rakers which
may or may not be BTs [40].
BTs occur in many higher taxa of generic or family level for which no
behavioural observations are available. Numerous well-developed
multicellular horny tubercles observed in both sexes in Homalopteridae
and Labeo long before they are capable of breeding, and that in catfishes these tubercles have not been linked to breeding behaviour [24].
According to [41],
structures resembling unculi were found when using SEM, but not with LM
studies. The unculus-like structures (seen with SEM) were identified
with LM as either nucleate squamate surface epidermal cells or
neuromasts. However, during my doctoral studies on many fish species we
only observed BTs on rostral cap and operculum of Garra lamta
towards head region with LM in histological sections only (unpublished
data). The tubercles are keratin based nodules which are induced by male
androgens and detach shortly after spawning [7,37].
The positive relationship between testosterone, elaborate breeding
tubercle ornamentation and papillomatosis was demonstrated by Kortet et
al. [37].
In Kortet et al. [38]. found that the BTs of the Roach (Rutilus rutilus)
serve as a status badge, with more dominant males possessing more
tubercles than less dominant males. It was also found that the tubercles
serve as an indicator of male quality, meaning that males with more
tubercles have greater reproductive success and are healthier (have
fewer parasites). This means that the BTs serve as a marker to help the
female fish pick mates that are healthier and have a better chance at
fertilizing their eggs during a spawn.
Fischer et al. [17]
identified the zebrafish BTs, epidermal appendages as specific
epidermal structures and unravel essential roles of transcription
factors TAp63 and p53 to promote both keratinocyte proliferation and
their terminal differentiation by promoting Notch signalling and caspase
3 activity, ensured formation and proper homeostasis of the
self-renewing stratified epithelium. Further they observed that Regular
epidermis and BTs display different patterns of superficial cell
renewal. BTs are formed during metamorphosis and undergo regular
desquamation and renewal of superficial layers, however, BTs of adult
zebrafish display more advanced stratification and keratinocyte
differentiation. Epidermal tubercles are usually sex-limited to males or
are better developed in males. They form during the breeding season and
are afterwards moulted. McMillan et al. [18]
reported that BTs are sexually dimorphic structures found in clusters
on the dorsal surface of the anterior rays of zebrafish male pectoral
fins, appear during sexual maturation and are maintained through regular
shedding and renewal of the keratinized surface. They analysed that
androgens induce and estrogens inhibit BT cluster formation [42].
Conclusion
This study provides new insight after the evaluation
of review on tubercles mystery. Finally, it is concluded that the
non-BTs which have no any relation with breeding and on the other hand
BTs which are directly or indirectly anyhow related to breeding or
spawning, often confused macroscopically with each other. On the basis
of BTs morphology there are two general decisive remarks, first it is of
great value to the distinguishing non-BTs and its various morphological
status as a key adaptive features. Secondarily the BTs morphology has
potential use as a character in studies of systematic and phylogenetic
relationships in fish. After these two general concluding remarks, there
is an extensive description offered, as some fish have tubercles and
some do not is still questioned that is why experimental studies are
needed to investigate the relation between presence and absence of BTs.
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