Acta Zoologica (Stockholm) 92: 187–200 (April 2011) doi: 10.1111/j.1463-6395.2010.00469.

Histology, ultrastructure, and pigmentation in the horny

scales of growing crocodilians
Lorenzo Alibardi

Dipartimento di Biologia, via Selmi 3,
40126, University of Bologna, 40126
Bologna, Italy
Keywords:
crocodile, scale growth, cell differentiation,
pigmentation, immunocytochemistry.
Accepted for publication:
1 September 2010
Abstract
Alibardi L. 2011. Histology, ultrastructure, and pigmentation in the horny scales
of growing crocodilians. —Acta Zoologica (Stockholm) 92: 187–200.
The present morphological study describes the color of hatchling, juvenile, and
adult crocodilian skin and the origin of its pigmentation. In situ hybridization
and immunostaining indicate that crocodilian scales grow as an expansion of the
proliferating epidermis of the hinge region that form thin lateral rings. In more
central areas of growing scales, new epidermal layers contribute to increase the
thickness of the stratum corneum. The dark pigmentation and color pattern
derive from the different distribution of epidermal and dermal chromatophores.
The more intensely pigmented stripes, irregular patches and dot-like spots, espe-
cially numerous in dorsal scales, derive from the incorporation of the eumelano-
somes of epidermal melanocytes in differentiating beta cells of the epidermis.
Dermal melanophores, mainly localized in the loose upper part of the dermis,
also contribute to the formation of the dark or gray background of crocodilian
scales. The eumelanosomes of dermal melanophores determine the darkening of
the skin pattern in association with the epidermal melanocytes. Iridophores are
infrequent, while xantophores are present in the species analyzed with a sparse
distribution in the superficial dermis among melanophores. The presence of
xantophores and of the few iridophores in areas where epidermal melanocytes
are absent appear to determine the brown or the light yellow-orange background
observed among the darker regions of crocodilian scales.
Lorenzo Alibardi, Dipartimento di Biologia, via Selmi 3, 40126, University of
Bologna, 40126 Bologna, Italy. E-mail: lorenzo.alibardi@unibo.it

500 lm or thicker) (Maderson 1985; Landmann 1986;

Introduction
Crocodiles are large archosaurian reptiles adapted to the
aquatic environment, and their skin is generally devoid of
bright colors (Steel 1989; Webb and Manolis 1989). This
condition probably helps these predators to camouflage in this
environment. The skin of crocodilians presents variable pig-
mentation patterns including paler area located between dark
variably large stripes, or dark spots to broad, irregular dots
(Brazaitis 1987; Webb and Manolis 1989). Among the darkly
stained areas, the background color varies among different
species, from low yellow-orange in the saltwater crocodile, to
a pale brown in the Nile crocodile, to a white-gray color in the
alligator.
It is known that in adult crocodilians, a variably thick epi-
dermis (30–150 lm) rests upon a variably thick dermis (250–
Richardson et al. 2000; Alibardi 2003, 2005a). The latter is
mainly divided into a thinner loose dermis where different cell
types and collagen fibrils are irregularly distributed, and in a
deep dermis that mainly contains very large and organized col-
lagen bundles but few fibrocytes (Spearman and Riley 1968;
Alibardi and Thompson 2000; Richardson et al. 2000). Croc-
odilian scales vary in shape, thickness, size, and pigmentation
pattern, and the epidermis of young and adult crocodilians
contains resistant proteins indicated as beta-keratins (Sawyer
et al. 2000; Alibardi and Toni 2007; Dalla Valle et al. 2009;
Ye et al. 2010). The origin of the pigmentation pattern in
hatchlings of crocodilians has suggested that as scales grow,
they somehow tend to maintain the basic pigmentation pat-
tern that characterizes their scales (Alibardi and Thompson
2000, 2001). However, details in the variation of the

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Growth and pigmentation in crocodilian scales • Alibardi
pigmentation pattern from hatchlings to adult crocodilians are
not specifically known, and little information on the cytology
and ultrastructure of crocodile pigmentation is so far available.
It is generally known that the coloration of reptilian skin
derives from multiple sources, physical and cellular (Cooper
and Greenberg 1992). The structural or physical coloration
depends on the interaction of the incident light with ordered
epidermal microstructures or with collagenous fibrils in the
dermis. In particular, the iridescent coloration observed in
some reptilian species depends on the phenomenon of inter-
ference of the incident light on the regularly spaced microor-
namentation of the epidermal surface (Gans and Baic 1977).
Interference and diffraction of the light incident to the ordered
collagen fibrils of the dermis often produces a prevalent blue
background derived from the scattering of prevalent blue
wavelengths (Bagnara et al. 2007). In the presence of yellow
xantophores in the more external part of the dermis, the scat-
tered blue color gives rise to the green coloration of most rep-
tilian skin (Kuriyama et al. 2006; Bagnara et al. 2007).
Another type of coloration of the skin (morphological)
derives from the slow redistribution of pigment organelles
(melanosomes) and from the de-novo synthesis of pigment
granules within chromatophores located in the epidermis and
in the dermis. This process, however, takes days or weeks and
is related to the adaptation to a new environment. In some
lizards with rapid color changes (Anolis carolinensis and
Chamaleon sp), dermal-melanophore units with a vertical
organization are present. In most lizards and snakes, where no
rapid color changes occur, chromatophores have a different
vertical organization and bright colors are also present
(Gosner 1989; Kuriyama et al. 2006).
Finally, another process of color variation (physiological
change) is because of the release of neurotransmitters or hor-
mones (Bagnara 1983). These molecules determine a rapid
redistribution (within minutes or hours) of pigment granules
(melanosomes) within the terminals of pigment cells and of
their position in the dermis. Physiological changes occur in
few species of lizards, such as A. carolinensis (iguanids) and in
numerous species of chameleons. In other reptiles such as
crocodilians, color changes take much longer time, and the
histological distribution of pigment cells is not known. The
contribution of other chromatophores (xantophores and irido-
phores) for the origin of the brown, yellow, or pale-orange
background of crocodilian skin (among the dark areas) is not
known.
The goal of the present study is the description of the
microscopic distribution of the different pigment cells present
in the skin of juveniles and adult crocodilians to explain the
origin of colors in different areas of growing scales. Initially,
the localization of pigment cells in the epidermis (melano-
cytes) and in the dermis (melanophores) is studied. The histo-
logical and ultrastructural analysis shows the cytological
characteristics and the distribution of melanophores and other
chromatophores in the epidermis and dermis. The observa-
tions on growing scales have indicated that pigments are also
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Acta Zoologica (Stockholm) 92: 187–200 (April 2011)
passed to beta-keratin cells of the newly formed corneous
layer that expands the scale surface. The study allows under-
standing the origin of the basic pigmentation pattern of croc-
odilians skin.
Materials and Methods
The study was conducted over 6 juveniles (2, 3, and 4 years
old, 50 and 80 cm in length) and on two adults (longer than
2 m) of Nile crocodile, Crocodylus niloticus. Some samples
from the skin of the belly, back, and tail were fixed for 5–8 h
in 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.5,
rinsed in buffer, osmicated for 2 h (2% OsO4 in buffer). Tis-
sues were then rinsed in buffer, dehydrated in ethanol, and
finally embedded in Durcupan resin (see details in Alibardi
2003). Other skin samples were collected from the neck and
tail of two adults of Crocodylus porosus (see details in Alibardi
2003). Finally, other dorsal and ventral scales were sampled
from three hatchlings of Alligator missippippiensis (see details in
Alibardi and Thompson 2000, 2001). The samples from the
skin of the latter two species served for the morphological
comparison with the scales from C. niloticus. In the present
study, the tissues were mainly used for the electron micros-
copy analysis of epidermal and dermal chromatophores.
Other samples from C. niloticus were fixed as above but they
were not osmicated in order not to alter the pigmentation
pattern for the macroscopic and gross microscopic study. The
tissues were embedded at 4C in Bioacryl resin under UV light
(Scala et al. 1992). Finally, some other samples were fixed in
4% of paraformaldehyde, dehydrated, and embedded in either
wax (for in situ hybridization) or Bioacryl resin without osmica-
tion. The omission of the osmium allowed avoiding the intro-
duction of further stain in the skin and permitted carrying out
some immunocytochemistry (see later). The embedding into a
transparent resin (Bioacryl) of an entire group of scales allowed
the examination of the pigmentation pattern directly under the
stereomicroscope. In fact, after sectioning the tissue with a
microtome, it was possible to observe the pigmentation pattern
in specific areas of the dermis or the epidermis, and observe
how this specific tissue distribution produces the final pigmen-
tation pattern visible from the surface of the skin (see Results).
The adult skin derived from digit and head skin was fixed
in formaldehyde 10%, dehydrated in ethanol, and then
embedded in Bioacryl Resin for the light microscopy study.
Semithin sections, 1–3 lm in thickness, were collected over
glass slides using an ultramicrotome. The sections were
stained with 1% toluidine blue and they were studied under
an optical microscope. From pigmented areas of the skin, thin
sections (40–90 nm in thickness) were collected on copper or
nickel grids, stained with uranyl acetate and lead citrate (stan-
dard method), and observed under a transmission electron
microscope.
For immunocytochemistry, paraformaldehyde-fixed and
nonosmicated tissues were utilized as previously described
(Alibardi 2003). In brief, the sections were incubated
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Acta Zoologica (Stockholm) 92: 187–200 (April 2011)
overnight in the primary antibody (rabbit serum against
chicken beta-1 keratin, see Sawyer et al. 2000) at a dilution of
1 : 200 in 0.05 M Tris–HCl buffer containing 1% BSA at pH
7.6. In control sections, the antibody was omitted. The sec-
tions were rinsed three times in the buffer and incubated in an
anti-rabbit fluorescein-conjugated secondary antibody for one
hour (dilution 1 : 70) and observed under a fluorescent
microscope (the positive signal was green-yellow, see Results).
For the in situ hybridization detection, 5- to 7- lm-thick
wax sections of skin were obtained using a microtome. The
sections were dewaxed in xylene, hydrated in ethanol and
double distilled water, and incubated overnight at 65C in the
hybridizing medium containing a specific anti-sense RNA
probe for crocodilian beta-keratin conjugated with digoxige-
nin (see details in Dalla Valle et al. 2009). Control sections
were incubated with the sense-digoxigenin-tagged RNA probe
for the same crocodilian beta-keratin. Sections were treated
with ‘standard saline citrate solution’ at decreasing concen-
trations in order to reduce the nonspecific hybridization
products. Some sections were incubated for 1 h with an anti-
digoxigenin antibody that is fluorescein conjugated (Roche,
Basel, Switzerland) and diluted 1 : 20 in the phosphate-
buffered Tris solution. In this case, positive sites were revealed
by a green fluorescence (see Results).
Other sections utilized for in situ hybridization were incu-
bated for 3 h in a primary mouse antibody against digoxi-
genin (Roche), diluted 1 : 500 in the buffer as mentioned
previously. Control sections were incubated with the sense-
digoxigenin-tagged RNA probe for the same crocodilian beta-
keratin. After rinsing in the buffer, the sections were incubated
for 1 h in the anti-mouse secondary antibody conjugated with
alkaline phosphatase (diluted 1 : 500). After rinsing, detection
of the antigen–antibody complex was obtained using the
chromogens 4-nitroblue tetrazolium chloride and 5-bromo-4
chloro-3-indolyl phosphate as substrates, using the protocol
indicated by the manufacturer (Roche). The positive signal
appeared as a reddish coloration in the sections (see Results).
Results
Stereomicroscopic observations
The ventral scales of juveniles of C. niloticus were mainly
milky-pale and often the typical sensory pith was present
(Fig. 1A,B). In juveniles from 1 to 4 years, one or two thin
lines of growth were noted along the margins of the scales
(arrowheads in Fig. 1A,B). Similar concentric and narrow
lines of growth were also seen in the lateral scales of the trunk,
in tail scales (often grayish in color), and in the dark dorsal
scales (Fig. 1C). In the dark scales, a brownish to low orange
color formed the background present among the prevalent
dark color. One to three concentric growth lines were detected
in growing crocodiles from 1 to 4 years.
From the sectioned scales that were embedded in the trans-
parent resin for the histological study, it appeared that the
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Acta Zoologica  2010 The Royal Swedish Academy of Sciences
Alibardi • Growth and pigmentation in crocodilian scales
color of the skin derived from different combinations in the
distribution of pigment cells. In one case, large irregular or
dot-like dark-brown spots resulted from the accumulation of
melanocytes in the epidermis, while the dermis showed a dif-
fuse, low to absent pigmentation (Fig. 1D). The corneous
layer of the epidermis in ventral and tail scales (pale scales)
was mainly nonpigmented, acting as a transparent layer over
the pigmented keratinocytes of the epidermis. In the dark dor-
sal scales, also large parts of the corneous layer were pig-
mented (epidermal pigmentation).
In another case, the brownish to grayish background pres-
ent in the skin (as observed from the external surface) was
derived from the presence of melanocytes along the basal part
of the epidermis (Fig. 1E). In more darkly stained areas of dif-
ferent scales, ventral and dorsal, the pigmentation was located
in both the epidermis and the stratum corneum but not in the
dermis (epidermal pigmentation only, Fig. 1F). Another type
of intense-dark pigmentation was derived from the combina-
tion of numerous melanocytes in the epidermis, including
those within the stratum corneous, with melanophores mainly
localized in the superficial dermis (epidermal and dermal pig-
mentation). In these cases, the superficial loose dermis was
dark, while few melanophores were present in the dense col-
lagenous layers forming the inner part of the dermis in these
pigmented areas (Fig. 1G).
Finally, some of the long stripes that were present in the
ventral and tail scales were mainly derived from the intense
epidermal pigmentation (melanocytes), while the dermal pig-
mentation (melanophores) underneath was diffuse (Fig. 1H).
The presence of other chromatophores (xantophores and
iridophore) was not surely determined at this level of resolu-
tion (see the histology and ultrastructure sections later on).
Skin histology at different ages
The mature and compact corneous layer of crocodilian and
alligator scales contained beta-keratins, as indicated by the
immunofluorescence of the corneous and precorneous layers
after immunostaining with the Beta-1 antibody (Fig. 2A,B).
The synthesis of new beta-keratin during the growth of croco-
dilian scales was active underneath the mature corneous layer,
as indicated by in situ hybridization using a crocodilian beta-
keratin-specific probe (see Methods). The mRNAs for this
beta-keratin were present in differentiating beta cells localized
along the entire transitional layer localized between the living
epidermis and the corneous layer (Fig. 2C–F). No expression
was detected in control sections, using both the alkaline phos-
phatase and the immunofluorescence detection system
(Fig. 2E,G). The fusiform cells of the transitional layer (pre-
corneous) were therefore actively producing these proteins
before being incorporated into the corneous layer. One to
three layers of fusiform cells expressing mRNAs for crocodil-
ian beta-keratin were seen beneath the corneous layer from
the center of the scale to the hinge regions located along the
perimeter of scales.
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Growth and pigmentation in crocodilian scales • Alibardi Acta Zoologica (Stockholm) 92: 187–200 (April 2011)

A B C

D E

F G

Fig. 1—Macroscopic image of whole scales (A–C) and stereomicroscope view of the surface of sectioned scales embedded in the transparent
Bioacryl resin (D–H) of Crocodylus niloticus. —A. lower belly square-shaped scale from 2 years old juvenile showing the concentric growth rings
(arrowheads) and the characteristic hole of integumentary sensory organ (double arrowhead). Bar, 1 mm. —B. more lateral ventral rectangular-
shaped scale in 2- year-old juveniles also showing thin growing rings in the hinge region among scales (arrowheads). Bar, 1 mm. —C. dark dorsal
scale of 2 years old juvenile. Background, orange-yellow coloration (arrowhead) among the dark areas (double arrow) is light orange. The arrow
indicates thin growing rings on the periphery of the scale. Bar, 1 mm. —D. surface of sectioned scale (embedded in transparent resin) from the
ventral tail in a 2- year-old juvenile. The arrowhead indicates a dark, pigmented spot seen by transparency on the surface of the scale. The arrow
points to the epidermis, the double arrowheads to the pale corneous layer. Bar, 0.5 mm. —E. pale digital adult scale with sectioned surface show-
ing the thick stratum corneum (double arrowheads). Few epidermal keratinocytes are located at the base of the epidermis (arrowhead). Bar,
0.5 mm. —F. dark band of pigmentation in a dorsal scale of 2- year-old juvenile as seen from the sectioned surface. Both the basal keratinocytes
(arrow) and the epidermis are blackened by pigment deposition. Bar, 0.5 mm. —G. other dorsal scale showing on the sectioned surface that both
epidermis (arrow) and mainly the superficial dermis are pigmented. The arrowhead indicates the dark pigmented areas on the scale surface that is
visible through the transparent resin. Bar, 0.5 mm. —H. surface of sectioned dorsal scale in a 4- year-old juvenile showing through transparency
the extension of dark areas (double arrows). The epidermis and the corneous layer are pigmented (arrows), while the superficial dermis shows a
lighter pigmentation (arrowheads). Double arrowheads indicate osteoderms. Bar, 0.5 mm. c, corneous layer; d, dermis; e, epidermis; h, hinge
region; kl, keel; sc, scale (outer view through the resin).

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190 Acta Zoologica  2010 The Royal Swedish Academy of Sciences
Acta Zoologica (Stockholm) 92: 187–200 (April 2011) Alibardi • Growth and pigmentation in crocodilian scales

A C D E

B F G

Fig. 2—Immunocytochemistry for beta-keratin (A, B) and in situ hybridization for beta-keratin mRNA (C–G) in growing (2 year old) crocodilian
scales. —A. beta-1 immunolabeled corneous layer (arrow) in Crocodylus niloticus adult ventral scale. —B. beta-1 immunolabeled corneous layer
(arrow) in hatchling of Alligator missippippiensis epidermis. —C. phosphatase-alkaline detection of the expression (red color, arrowheads) in a ven-
tral scale of C. niloticus. —D. expression in a thin layer of keratinocytes (red in color, arrow) in the central part of a ventral scale (alkaline phospha-
tase detection) in C. niloticus. —E. absence of expression in a sense control section of a ventral scale (alkaline phosphatase detection) in
C. niloticus. —F. immunofluorescent detection of the expression (arrow) in precorneous, fusiform beta-keratinocytes of ventral scale of C. niloti-
cus. —G. control section (sense) with no expression in C. niloticus. Bar in all figures is 10 lm. aR, anti-sense RNA probe; b1, beta-1 antibody;
c, corneous layer; d, dermis; sR, sense RNA probe control; dashes indicate the limit of the basal layer of the epidermis.

Hatchlings of C. niloticus were not available in the present
study for an initial comparison with juveniles of 1–4 years.
However, a relatively thin corneous layer was present in
hatchlings of both alligator (A. missippippiensis, 10–15 lm in
the central part of ventral scales, Fig. 3A–B) and C. porosus
(10–12 lm, data from Richardson et al. 2000). The thickness
of the stratum corneum thins out moving toward the hinge
regions (Fig. 3C).
In the available samples of growing individuals of C. niloti-
cus (1–4 years), three skin regions were analyzed for determin-
ing the histological variations of the epidermis during growth:
scales from the belly, scales from the tail, and scales from the
dorsal midtrunk. Our indicative measurements have shown
that the thickness of the living part of the epidermis was simi-
lar in crocodiles of different ages (thickness variations were
probably because of seasonal fluctuation of keratinocytes pro-
liferation). Differently, the thickness of the corneous layer of
the epidermis tended to increase from 1-year-old juveniles up
to 4 years in all the three regions analyzed, and even more in
adults (Fig. 3).
In ventral scales (but also in other types of scales) of 1-year-
old juveniles, the stratum corneum was thicker in the central
part of scales (20–40 lm in the central parts of ventral scales,
Fig. 3D–H). The stratum corneum was thicker in juveniles of
2 years (40–60 lm, Fig. 3I–M), and even thicker in juveniles
of 4 years (60–110 lm, Fig. 3N–S). In the adult head and
digit scales, the thickness of the corneous layer of scales was
even higher, 120–160 lm (Fig. 3T).
Histology of pigmentation
As previously indicated (Alibardi and Thompson 2000,
2001), embryonic and hatchling pigmentation mainly
derives from epidermal melanocytes (Fig. 3A). In 1-year-old
specimens of C. niloticus, the ventral scales were paler on aver-
age in comparison with tail scales (gray on average). The pig-
mentation because of epidermal melanocytes was less intense
in ventral and tail scales than in dorsal scales. Dorsal scales
appeared dark on average but a closer inspection revealed a
brown to orange color among the dark areas (Fig. 1C).
In ventral and tail scales, pigment cells were mainly local-
ized beneath the epidermis (melanocytes), while they
appeared sparse in the superficial, loose part of the dermis
(melanophores, see Fig. 3D–F). In the central and hinge
regions of the dark dorsal scales, pigment cells were present in
both the dermis and epidermis, and also the corneous layer
was heavily pigmented (Fig. 3B,C,G,H). In dorsal scales, a
thick layer of melanocytes was present beneath or among
basal epidermal cells, pigments were sparse in suprabasal cells,
while the transitional and corneous layers were heavily pig-
mented. This histological condition was observed in the
skin of hatchlings of the alligator (Fig. 3B,C), in juveniles of
C. niloticus at 2 and 4 years, and in the adult epidermis of
C. niloticus and C. porosus (Fig. 3K,L,Q–S).
In the pale ventral scales and in scales of the tail in 2–
4 years old juveniles of C. niloticus and in adult individuals of
all species analyzed, the number of melanocytes was lower
than in dorsal scales, and generally limited to the base of the
epidermis. Melanophores were sparse in the upper part of the
dermis (Fig. 3I,J,N–P,T). In areas of ventral or tail scales
where the dark pigmentation was intense (dots or stripes of
different sizes, as observed from the embedded tissue in the
transparent resin before sectioning, see Fig. 1D,F), a thick
stratum of melanocytes was present beneath the epidermis.
Dark pigmented cells were also present in the superficial, pre-
corneous and corneous layers of pale and gray ventral and tail
scales (Fig. 3M). This observation indicated that also in the
dark spots or stripes of ventral and tail scales, pigments

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B C

E F

H I

J K L M

N O P

Q R S T

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Acta Zoologica (Stockholm) 92: 187–200 (April 2011) Alibardi • Growth and pigmentation in crocodilian scales

Fig. 3—Sequence of light microscopic sections stained with toluidine blue showing scales of different body areas collected at different ages from
Alligator missippippiensis (A–C), growing juveniles (D–S) and adult (T) of Crocodylus niloticus. —A. ventral scale epidermis in hatchling alligator,
which is lightly pigmented from epidermal melanocytes only (arrows). Bar, 10 lm. —B. intensely pigmented dorsal scale epidermis in hatchling
alligator. The pigmentation of differentiating beta cells (double arrow) is mainly because of pigment transfer from epidermal melanocytes
(arrows). Sparse dermal melanophores (arrowhead) are present. Bar, 10 lm. —C. hinge region of dorsal scale epidermis in hatchling alligator
showing pigmented corneous layers (arrows). Epidermal melanocytes are sparse as well as dermal melanophores. Bar, 20 lm. —D. pale ventral
scale epidermis (the arrowhead indicates the transitional layer) in an 1- year-old crocodile where few dermal melanophores are present (arrow).
Bar, 20 lm. —E. hinge region of ventral pale scale with few dermal melanophores (arrowheads). Bar, 20 lm. —F. tail scale epidermis of gray
color showing diffuse dermal melanophores (arrowheads). Bar, 20 lm. —G. hinge region in dorsal scale of 1- year-old crocodile. Both the sparse
epidermal melanocytes and the numerous melanophores (arrows) contribute to the pigmentation in this zone. Bar, 20 lm. —H. darkly pigmented
beta cells of dorsal scale epidermis showing sparse epidermal melanocytes and numerous dermal melanophores (arrow) at the base of the epider-
mis. Bar, 20 lm. —I. tail scale of gray color in crocodile of 2 years. An accumulation of mainly sub-epidermal (dermal) melanophores (arrow) is
determining the color of this scale (gray-colored). Bar, 20 lm. —J. pale ventral scale epidermis in 2- year-old crocodile showing sparse dermal
melanophores (arrows). Bar, 20 lm. —K. dorsal scale epidermis intensely pigmented in the transitional and corneous by epidermal melanocytes
(arrows). Few dermal melanophores are seen (arrowhead). Bar, 10 lm. —L. hinge region of dorsal scale in 2- year-old crocodile. Sparse melano-
cytes and dermal melanophores (arrows) are present in this zone. Bar, 20 lm. —M. detail on intense pigmentation of the superficial dermal layer,
while the outer corneous layer is also pigmented (arrows). Bar, 10 lm. —N. pale epidermis of ventral scale in a 4- year-old individual. Only sparse
dermal melanophores (arrowhead) are present. Bar, 20 lm. —O. other epidermis from ventral scale of a 4-year-old crocodile containing sparse
dermal melanophores (arrowhead). Note the thicker stratum corneum (the arrow indicates the transitional layer) with respect to the living epider-
mis. Bar, 20 lm. —P. thick epidermis of tail scale in a 4- year-old individual. Pigmentation is diffuse in the superficial dermis (melanophores) of
this gray scale. Bar, 20 lm. —Q. pigmented beta cells (arrow) forming the corneous layer of a dorsal scale epidermis in a 4-year-old crocodile.
Numerous dermal melanophores are also seen in the superficial dermis. Bar, 10 lm. —R. pigmented beta cells (arrows) are present in the transi-
tional layer of a dorsal scale in a 4- year-old crocodile. Bar, 10 lm. —S. hinge region of dorsal scale in a 4- year-old crocodile. Sparse epidermal
and dermal (arrowheads) melanophores are present. Bar, 20 lm. —T. thick pale digital scale of adult crocodilian. Sparse melanocytes are present
in the thin living epidermis and in the superficial dermis. Bar, 20 lm. c, corneous layer; d, dermis; e, epidermis; h, hinge region.

(identified as melanosomes by the ultrastructural observation)
were injected into the keratinocytes of the epidermis.
Ultrastructural observations on pigment cells
Unless otherwise specified, the following cytological descrip-
tion is valid for both the epidermis and dermis of alligator and
crocodile skin.
The normal and un-pigmented epidermis of hatchlings and
juvenile crocodilians contained sparse epidermal melanocytes
among keratinocytes (Fig. 4A). Melanosomes originated in
the Golgi apparatus of melanocytes, initially as pale vesicles
that rapidly darkened as they accumulated melanin (Fig. 4B
and the inset). The ultrastructure of crocodilian melanosomes
identified these organelles as eumelanosomes (Jimbow et al.
1979). These organelles contained parallel rows of electron-
dense material, previously indicated as ‘helical threads’
(Breathnach and Pointz, 1966) where melanin is deposited.
The other type of melanosomes, pheomelanosomes, derived
from the accumulation of vesicles that later produce pheomel-
anin, were not observed in crocodilian tissues. As previously
observed, under the stereomicroscope, the amount of melano-
cytes and of melanosomes that were donated to keratinocytes
in the dark areas of the epidermis was much higher with
respect to melanocytes observed in the paler areas of the epi-
dermis (Fig. 4C–F). While melanosomes were dispersed
among keratin bundles in keratinocytes of the spinosus layers
(Fig. 4C), melanosomes became condensed and formed pig-
mented rows inside the flattening keratinocytes of the corne-
ous layer (Fig. 4D,F).
In the dermis of the three species here analyzed (inset of
Fig. 5A), most of the extracellular space was occupied by
bundles of collagen among which amorphous ground sub-
stance (proteoglycans) was present (Fig. 5A). The collagen
bundles located around the fibroblasts in the superficial and
loose dermis were of relative small dimension (1–5 lm). The
presence of enlarged cisternae of rough endoplasmic reticu-
lum in fibroblasts of the dermis indicated that the synthesis of
collagen was continued during scale enlargement and contrib-
uted to the maintenance of collagen density in the growing
dermis. The dimension of collagen bundles increased to 10–
25 lm in the deep, dense dermis of juveniles and adult skin
(about 120–150 lm beneath the epidermis), which extended
for more than 500 lm underneath the epidermis in juveniles
and adult crocodilians. In the deep dermis, banded collagen
bundles closely surrounded the few cells present in this tissue,
and collagen fibrils occupied most of the intercellular matrix,
while little or no ground material was seen (Fig. 5B). These
large bundles determined a characteristic, large meshwork
texture of the dense dermal layer (inset in Fig. 5A). Few or
rare pigment cells were seen within the dense dermis, while
they were more frequent in the superficial, loose dermis.
In the loose dermis of C. niloticus only xantophores (lipo-
phores) were present, while no iridophores (guanophores)
containing the typical reflecting platelets (Jackson and Butler
1996; Richardson et al. 2000) were found. In our samples of
skin from C. porosus and A. missippippiensis, cells recognizable
as xantophores were commonly encountered in the loose der-
mis and sometimes they were located very close to the basal
cells of the epidermis (Fig. 6A). Their pterinosomes measured

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Acta Zoologica  2010 The Royal Swedish Academy of Sciences 193
Growth and pigmentation in crocodilian scales • Alibardi Acta Zoologica (Stockholm) 92: 187–200 (April 2011)

A B

C D

E F

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194 Acta Zoologica  2010 The Royal Swedish Academy of Sciences
Acta Zoologica (Stockholm) 92: 187–200 (April 2011) Alibardi • Growth and pigmentation in crocodilian scales

Fig. 4—Ultrastructural features of the pigmentation in the epidermis of hatchlings of Alligator missippippiensis (A, B, D) and in adult epidermis of
Crocodylus niloticus (C, E, F), —A. ventral epidermis showing melanocytes (arrowheads) among epidermal cells. The double arrowhead indicates
the section of a melanocyte arm. The arrows indicate melanosomes incorporated within epidermal cells. Bar, 1 lm. —B. detail on the Golgi appa-
ratus of melanocyte with blebbing vesicles (arrows), premelanosomes containing dark rods (arrowheads), and mature melanosomes (double
arrowheads), 200 nm. The inset at higher magnification shows the dark rods where melanin is deposited, Bar, 100 nm. —C. spinosus keratino-
cytes of the intermediate layer of tail scale containing numerous melanosomes (arrows) among keratin bundles (double arrows). Bar, 2 lm.
—D. pigmented corneous layer of dorsal scale containing parallel rows of melanosomes (arrows). Bar, 0.5 lm. —E. in paler areas of ventral scale,
few melanosomes (arrows) are present and they are not organized in parallel rows do. Bar, 2 lm. —F. juvenile dorsal scale showing the numerous
rows of melanosome aggregates (arrows) in corneocytes. Bar, 2.5 lm. c, corneous layer; Go, Golgi apparatus; k, keratinocyte; nu; nucleus.

melanophores with no apparent vertical organization. Also,

iridophores did not display the characteristic rectangular-
shaped reflecting platelets previously described in the skin of
other crocodilians (Jackson and Butler 1996; Richardson et al.
2000). In fact, electron-pale reflecting platelets of 0.05–
0.2 lm in size were rounder or more irregular than in previ-
ously described crocodilian samples.
These organelles appeared very likely to derive from the
rough endoplasmic reticulum and were often close to the
Golgi apparatus as well. At the beginning of platelet forma-
tion, small and pale flat or round pale organelles (vesicles)
were seen as sections of the rough endoplasmic reticulum, as
indicated from the presence of ribosomes on the membrane
(Fig. 6B). These vesicles were often located near the Golgi
A apparatus. Also denser bodies with some paler areas were seen
near the Golgi apparatus but these dark organelles were not
clearly identified (Fig. 6B). In other more peripheral areas of
the cytoplasm of iridophores, relatively far from the nucleus, a
similar pale material was present within parallel cisternae of
the rough endoplasmic reticulum, and the membrane of these
organelles were also surrounded by ribosomes (Fig. 6C and
inset). This suggested that the material content in the reflect-
ing platelets, likely electron-lucent guanine, derived from the
synthesis in the rough endoplasmic reticulum. In some occa-
sional cases, the copresence of pterinosomes with reflecting
platelets was observed in the same cells (data not shown).

B
Fig. 5—Cytological details of the dermis in Crocodylus niloticus. —A.
detail of fibroblast surrounded by numerous bundles of banded colla-
gen fibrils, located in the superficial dermis. Bar, 1 lm. The inset
(Bar, 20 lm) shows the aspect of the skin with the superficial pig-
mented dermis (arrow) and the reticulate dense dermis. —B. detail of
banded collagen fibrils (arrows) surrounding a deep fibroblast with
actively synthesizing ergastoplasm (arrowheads). Bar, 0.5 lm. co, col-
lagen; e, epidermis; dd, deep (dense) dermis); fi, fibroblast; gr, ground
(extracellular) substance; sd, superficial (looser) dermis.
0.15–0.4 lm in size and were often dark and contained circu-
lar lamellae. Most of the remaining cytoplasm was occupied
by free ribosomes, and few mitochondria were seen.
Cells recognizable as iridophores were occasionally encoun-
tered in the superficial, loose dermis of A. missippippiensis and
C. porosus with a random distribution below the epidermis.
The relatively few iridophores were mixed with dermal
Discussion
Scale growth
In the present study, the information on the cellular sites
involved in scale growth has been obtained through in situ
hybridization, histology, and immunocytochemistry for beta-
keratin coupled with the stereomicroscopic observations on
scale growth. The detection of thin growth rings in juvenile
scales coupled with the microscopic analysis of scales has indi-
cated that growth of crocodilian scales is symmetric (Fig. 7A).
In the dorsal scale, surface growth occurs through the addition
of new beta cells underneath the corneous layer that increases
in thickness, at least during the first 4–5 years, but slightly also
later on (Fig. 7B). In the hinge regions, growth occurs by a
lateral expansion of the epidermis with the formation of a
completely new and initially thin corneous layer. As a result of
this process, the corneous layer of the dorsal scale surface is

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Growth and pigmentation in crocodilian scales • Alibardi Acta Zoologica (Stockholm) 92: 187–200 (April 2011)

A B

Fig. 6—Ultrastructure of dermal chromatophores in the dorsal scale of Crocodylus porosus. —A. part of a cytoplasmic arm of a xantophore contain-
ing dark pterinosomes (arrows). Bar, 0.5 lm. —B. detail of cytoplasmic area of a chromatophore containing sparse ribosomes, pale vesicle
(reflecting platelets at different stage of maturation, arrowheads), and dark vesicle (forming pterinosomes, arrows). Bar, 250 nm. —C. detail of
iridophore cytoplasm featuring irregularly shaped reflecting platelets apparently forming from the endoplasmic reticulum (arrows), 250 nm.
The inset (Bar, 150 nm) shows a detail of a reflecting platelet). Go, Golgi apparatus; m, mitochondria; nu, nucleus.

thicker than the corneous layer near the hinge regions. The
increase in thickness of the stratum corneous with the age of
crocodilians is probably because of the net cell incorporation
versus a less intense process of superficial wearing. In general
terms, the process of cellular addition in crocodilian scales
resembles that of turtles and tortoises (Alibardi 2005b, 2006)
although it appears more gradual and continuous as the rings
are less evident in juvenile scales and almost undetectable in
older scales. It is not known whether the growth is continuous
throughout the year or present peaks followed by slow or even
resting periods. As new beta-keratin cells of growing areas
receive an intense pigmentation from epidermal melanocytes,
it is likely that the symmetric growth of the scale epidermis
also determines the symmetric expansion of the initial pig-
mentation pattern present in the scales of hatchlings.
Cytological characteristics of chromatophores
While melanocytes, melanophores, and xantophores (lipo-
phores) are always present in the skin of crocodilians, the

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196 Acta Zoologica  2010 The Royal Swedish Academy of Sciences
Acta Zoologica (Stockholm) 92: 187–200 (April 2011) Alibardi • Growth and pigmentation in crocodilian scales

Fig. 7—Schematic representation of the growth of crocodilian scales as indicated from the present observations. —A. dorsal view with the external
areas (net surface addition) indicated in green (progressive stages from 0 to 8). Dashes indicate the initial surface incorporated in the outer scale
surface (in red) during growth. —B. schematic longitudinal sections showing the formation of successive corneous layers (1–3, stained in different
colors) from an early stage through successive B1 and B2 stages. The curved arrows indicate the net addition of cells from the hinge region (h) to
create new epidermis (e) and the corneous layer toward the more central part of the scale. B3 indicates the layers as observed in cross-section.
The asterisks indicate position of main areas where cell proliferation takes place.

iridophores (guanophores) are occasionally found, and these
cells are even absent in C. niloticus. This observation confirms
a previous light microscopic study on the skin in this species
(Spearman and Riley 1968). Occasionally, some chromato-
phores contain sparse melanosomes together with other
organelles such as pterinosomes, indicating cells with mixed
characteristics like those described for other vertebrates
(Bagnara 1983).
The general cytology of pigment cells observed in the pres-
ent study is similar to that of previous studies on the skin of
crocodilians although the shape of the reflecting platelets is
rounder (Jackson and Butler 1996; Richardson et al. 2000). It
is unlikely that the osmolarity utilized in our fixative might
have affected the shape and preservation of reflecting platelets
as the other organelles, such as mitochondria and pterino-
somes in xantophores, did not show any alteration. In both
C. porosus and A. missippippiensis, the observed reflecting
platelets appeared rounder and not bright white as those
shown in previous reports. Variation in the shape of reflecting
platelets can therefore be expected in other species of crocod-
ilians, perhaps derived from a different fixation, from the diet
or from variable environmental conditions.
As regards the origin of melanosomes in crocodilian mela-
nocytes, like in other species, it appears that the Golgi appara-
tus is involved and these organelles rapidly become dark,
probably after the activation of the specific tyrosinase (Bagn-
ara 1983). The ultrastructural characteristics of the pigment
organelles observed in the present study identify these organ-
elles as eumelanosomes. As the ultrastructural characteristics
are clearly differentiative for avian and mammalian melano-
phores (Jimbow et al. 1979, 1986) and the sampled material
included different body areas, the present study indicates that
organelles with the pheomelanosomes ultrastructure are
absent in the skin of crocodilians. However, the presence of
pheo- in conjunction with eu-melanin in crocodilians
awaits further data on the chemical composition of these
organelles using biochemical analysis or histochemical or
immunocytochemical localization of cysteine or enzymes
involved in eu- versus pheo-melanogenesis (Jimbow et al.
1986). Therefore, the brown, yellow-gold, or the pale orange
colors seen among the dark areas of the skin in different croco-
dilian species appear to derive from a chromatic combination
from dermal xanthophores, iridophore (when present), and
melanophores.
The reason for the difference in size of epidermal versus
dermal melanosomes is not known (Breathnach and Pointz,
1966). In lizards, where a shedding cycle is present in the epi-
dermis, the longer process of melanin synthesis in dermal mel-
anosomes versus the periodic production of melanosomes in
epidermal melanocytes may account for the different dimen-
sion. During the shedding cycle, epidermal melanocytes
become activated and synthesize melanin only for a brief per-
iod during the late renewal phase of the epidermis, at stages
4–5 before the epidermis enters the resting phase (Szabo et al.
1973). Under this condition, melanosomes might only reach
a certain dimension before terminating their maturation in the
resting stage. Melanosomes are injected into beta-keratin cells
only during the early stage of the following renewal phase
when beta cells are differentiating from the germinal layer.
Although a shedding cycle is absent in crocodilian epidermis,
the lack of pigmentation in the intermediate epidermal layers
of otherwise pigmented scales suggests that melanocytes with
their melanosomes are not continuously produced in crocodil-
ian epidermis.
In both lizards and crocodilians, the activity of dermal mel-
anosomes does not depend on the activity of the epidermis,
and dermal melanophores may have a potentially continuous
production of melanin granules. This is also indicated from
the larger number of intermediate stages of melanogenesis
detectable in dermal melanophores with respect to epidermal

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Acta Zoologica  2010 The Royal Swedish Academy of Sciences 197
Growth and pigmentation in crocodilian scales • Alibardi
melanocytes (present study). This condition may allow the
production of numerous and larger melanosomes in melano-
phores than in epidermal melanocytes. Furthermore, dermal
melanophores can increase the production of melanosomes
according to the need of darkening or lightening the skin.
The pterinosomes present in crocodilian xantophores
seem to derive from the accumulation of lipids or lipid lamel-
lae in vesicles of the smooth endoplasmic reticulum, often
associated with the secretory vesicle from the Golgi appara-
tus (Bagnara 1983). Cells storing pterinosomes and reflect-
ing platelets were occasionally recorded, indicating that
chromatophores with mixed characteristics are also present
in crocodilian skin, like in the skin of other vertebrates
(Bagnara 1983; Sherbrooke and Frost 1989). The origin of
the reflecting platelets is not clear from the present study.
These organelles seem to be initially derived from the accu-
mulation of a pale material in the endoplasmic reticulum,
presumably containing guanine ⁄ purines (Bagnara 1983;
Morrison and Frost-Mason 1991). The participation of the
Golgi apparatus in the elaboration of the material accumu-
lated in the endoplasmic reticulum of iridophores remains
uncertain. The present observations have indicated that the
reflecting platelets may also derive from the fragmentation of
the endoplasmic reticulum.
Pigmentation pattern
How is the pattern of pigmentation initially obtained in the
embryo and then maintained in juvenile and adult crocodil-
ian scales? What is the specific role of epidermal melanocytes
versus that of dermal melanophores in the formation of the
combined chromatic pattern? In the embryo, the initial pig-
mentation is mainly epidermal (Alibardi and Thompson
2000, 2001). However, in the adult skin, also the dermis
shows broad areas with intense pigmentation (Spearman and
Riley 1968; present study). Figure 8 summarizes the informa-
tion on the localization of chromatophores in the skin of adult
crocodilians, as deduced from the present observations. The
darkest areas forming small or large dots and stripes result
from the heavy pigmentation of beta cells of the epidermis
operated by epidermal melanocytes (epidermal pigmentation,
Fig. 8, areas indicated as 2, 3 and 5). Dermal melanophores
can be diffuse (Fig. 8, number 3) or more concentrated,
increasing the intensity of the dark spots or stripes already
present in these epidermal areas (Fig. 8, numbers 3, epider-
mal-dermal pigmentation). This superimposition of melano-
phores and melanocytes also determines the formation of the
dark, round, or more irregular spots observed in the skin of
crocodilians. Dark areas of the skin therefore derive from the
specific localization of epidermal or dermal melanophores
(epidermal or dermal pigmentation, see Fig. 8, number 1, 2
and 4).
Crocodilians do not generally show bright colors in the skin
and the distribution of their xantophores, iridophores (when
present), and melanophores is not vertical as in the dermal
198
Acta Zoologica (Stockholm) 92: 187–200 (April 2011)
Fig. 8—Schematic drawing showing the position of the different
chromatophores in relation to the formation of the external pattern of
pigmentation as indicated in the present study. 1, small dot derived
from dermal melanophores only (arrow). 2, other dot-like spot
derived by epidermal melanocytes only (arrow). 3, stripe formed by
epidermal melanocytes superimposed to dermal melanophores.
4, large dot-like area derived from dermal melanophores only. 5, large
dot-like area derived from epidermal melanocytes only. Iridophores
and xantophores are localized in the superficial dermis among the
dark, melanized areas of the skin (arrows). c, corneous layer; dd,
dense deep dermis; dm, dermal melanophores; e, epidermis (basal
layer and suprabasal layer); em, epidermal melanocytes; ld, loose
superficial dermis.
chromatophoric units of amphibians or chameleons. Sparse
iridophores are present in the dermis of both C. porosus and
A. missippippiensis, in agreement with recent ultrastructural
studies (Jackson and Butler 1996; Richardson et al. 2000).
The presence of highly pigmented melanocytes and mela-
nosomes-rich beta cells in the dorsal scales and in the dark
spots ⁄ stripes of the other scales indicates that most of the
darkest color of crocodilian skin is because of the epidermal
melanocytes (Fig. 8, areas indicated as 2, 3 and 5). The pres-
ence of darker spots or stripes on an otherwise pale-gray back-
ground in ventral and tail scales suggests that melanosomes
are injected into a limited number of keratinocytes (the ‘epi-
dermal unit’, see Jimbow et al. 1986) during a particular per-
iod of scale expansion.
During embryogenesis, melanin-rich areas (epidermal
units) form most of the initial pigmentation pattern of the
skin, from stage 22 onward and dermal melanophores are less
frequent and present a diffuse distribution (Alibardi and
Thompson 2000, 2001; Richardson et al. 2000). How mela-
nocytes become confined in these specific regions responsible
for producing the dark pigmentation pattern is not known.
After hatching and during the following growth, the initial pat-
tern of pigmentation is basically maintained through the
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Acta Zoologica  2010 The Royal Swedish Academy of Sciences
Acta Zoologica (Stockholm) 92: 187–200 (April 2011)
progressive enlargement of the epidermal units by melanocyte
expansion and, probably, by local proliferation of melano-
cytes. The maintenance of the initial pigmentation pattern, as
seen in growing individuals, is conserved through the expan-
sion of the fixed epidermal units already present in the grow-
ing epidermis. The number of dermal melanophores increases
during scale growth and determines the expansion of the dark
pigmented areas (Fig. 8).
The previous hypothesis could be checked only by a histo-
logical study on the variation of the color pattern during some
years of growth. Although such a study is not available, some
support to the above hypothesis comes from a study on the
color adaptation to the environment in growing crocodilians
(Kirschner 1985). In the latter study, during the 3 months of
growth of juvenile of C. porosus on a dark or on a light back-
ground, the position and rough size of the darkest spots does
not change while the animals grow (Fig. 4 in Kirschner
1985). As the darkest spots are likely derived from epidermal
melanocytes (present observations), the darkening or lighten-
ing effect on the skin within the 3-month period appears
mainly because of the dermal melanophores that slowly
expand among the darkest spots. It is also likely that, aside the
movement of pigments along the cells elongation of melano-
phores, these cells can also migrate or even proliferate during
the darkening process in order to colonize broader areas of the
dermis. This migration produces the darkening or the lighten-
ing effect on the skin. Therefore, dermal units of crocodilian
skin are horizontally and not vertically organized, and they
take much longer time (1–3 months) to produce a color
change than in the skin of A. carolinensis and chameleons
(Woolley 1957; Kirschner 1985; Cooper and Greenberg
1992). The same process probably occurs for the skin of tur-
tles, many snakes, and numerous lizards as well.
In the underlying dermis, outside the dark areas, the sparse
melanophores appear connected to the gray color, while the
combination of xantophores (and iridophores when present)
gives rise to the pale yellow-orange, brown or olive green color
present among the dark areas of crocodile epidermis (Fig. 8).
This diffuse coloration appears through the pale epidermis
located in correspondence of these dermal areas.
The present histological study on the localization of mela-
nophores in the dermis supports the idea for a dynamic role of
these cells in the low background color variation in crocodilian
skin. When the background where crocodilians (or turtles) live
changes for a period of 1 month or longer, the skin undergoes
a darkening process with a dark background or becomes pale
with a white background (Woolley 1957; Kirschner 1985).
This effect is likely because of the synthesis and the centrifugal
movement of melanosomes along the expanding arms of der-
mal melanophores. The centripetal movement of melano-
somes determines a lightening effect on the skin pigmentation.
The activation of melanocytes in the epidermis and the re-dis-
tribution of dermal melanophores is probably the result of a
hormonal or neuro-humoral stimulation (Bagnara 1983; Coo-
per and Greenberg 1992). The expansion of dermal
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Acta Zoologica  2010 The Royal Swedish Academy of Sciences
Alibardi • Growth and pigmentation in crocodilian scales
melanophores or their horizontal migration ⁄ proliferation
determines most of the darkening or lightening effect on the
skin background observed in a period of 1–3 months.
Acknowledgements
Prof. Matthias Starck (University of Munich II, Germany)
and Prof. Y. Sire (Universite’ de Paris II, France) kindly
supplied the skin samples from juvenile crocodiles of
C. niloticus. Adult crocodile skin was supplied by Dr. R.
Gattelli, Centro Didattico e Scientifico Aquae Mundi, Russi,
Ravenna, Italy, 1 sample), and Dr. E. Ferrandez (Le Farme
de Crocodiles, Pierralatte, France. 1 sample). Aside the elec-
tron microscope facility (University of Bologna), the present
study was completely self-supported (Comparative Histolab,
Padova, Italy).
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