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The nictitating membrane is an anatomic structure exclusively exhibited by Carcharhiniformes, the largest order among sharks. Here we present a detailed description of morphological characteristics of the nictitating membrane through light microscopy (LM) and scanning electron microscopy (SEM) in the following shark species: Carcharhinus limbatus, Galeocerdo cuvier, Prionace glauca, Rhizoprionodon lalandii, R. porosus, Sphyrna lewini and S. zygaena. Differences in the microscopic aspects of dermal denticles from the species studied were observed. P. glauca, a pelagic shark, showed a well-developed protection apparatus when compared with other pelagic species, while coastal sharks showed even higher structural complexity. In the blue shark the denticles are enameled, presenting an extensive pulp cavity and a base inserted in a connective tissue. Moreover, the species exhibits the higher number of ridges (up to nine) of varied size and shape and the muscular tissue is inserted in the ventral region of the connective tissue. Dermal denticles from C. limbatus, R. lalandii, R. porosus, S. zygaena and G. cuvier exhibit up to five ridges with hexagonal ornamentations in the crown. In S. lewini and S. zygaena, the denticles are rounded shaped and glandular cells are present. The patterns observed in the present study suggest a high level of specialization and evolutionary conservation shaped by the function of the structure. In addition, we hypothesize that the morphological simplification observed in the membrane when compared to the dermal denticles from the skin, is an evolutionary trait that evolved to improve the dynamic and biomechanics of this highly mobile structure allowing this way, a rapid and efficient protection against abrasion, mainly during predation events.
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Vol.:(0123456789)
1 3
Zoomorphology
DOI 10.1007/s00435-017-0351-1
ORIGINAL PAPER
Microscopic aspects ofthenictitating membrane
inCarcharhinidae andSphyrnidae sharks: apreliminary study
AlineNayaraPoscai1· Biancade SousaRangel1,2· AndréLuisda SilvaCasas3·
NataschaWosnick4· AlexandreRodrigues5· RoseEliGrassiRici6·
JoséRobertoKfouryJunior1
Received: 23 August 2016 / Revised: 6 March 2017 / Accepted: 7 March 2017
© Springer-Verlag Berlin Heidelberg 2017
tissue. Moreover, the species exhibits the higher number of
ridges (up to nine) of varied size and shape and the muscu-
lar tissue is inserted in the ventral region of the connective
tissue. Dermal denticles from C. limbatus, R. lalandii, R.
porosus, S. zygaena and G. cuvier exhibit up to five ridges
with hexagonal ornamentations in the crown. In S. lewini
and S. zygaena, the denticles are rounded shaped and glan-
dular cells are present. The patterns observed in the present
study suggest a high level of specialization and evolution-
ary conservation shaped by the function of the structure.
In addition, we hypothesize that the morphological simpli-
fication observed in the membrane when compared to the
dermal denticles from the skin, is an evolutionary trait that
evolved to improve the dynamic and biomechanics of this
highly mobile structure allowing this way, a rapid and effi-
cient protection against abrasion, mainly during predation
events.
Keywords Nictitating membrane· Dermal denticles·
Requiem sharks· Electron microscopy
Introduction
The Carcharhiniformes order is the largest among sharks,
with about 55% of all species currently described (~250)
(Compagno 2005). The presence of a nictitating membrane
distinguishes the sharks of the order (Compagno 1988;
Hueter etal. 2004). It is denominated the third eyelid, cov-
ering the shark eye completely (Gruber and Schneiderman
1975; Gruber and Myrberg 1977) during the feeding activi-
ties as observed in other vertebrates such as felines (Craw-
ford and Marc 1976), canines (Pires etal. 2008), equines
(Puff etal. 2008) and primates (Crawford and Marc 1976).
The structure protects against mechanic damages, being
Abstract The nictitating membrane is an anatomic struc-
ture exclusively exhibited by Carcharhiniformes, the largest
order among sharks. Here we present a detailed description
of morphological characteristics of the nictitating mem-
brane through light microscopy (LM) and scanning electron
microscopy (SEM) in the following shark species: Car-
charhinus limbatus, Galeocerdo cuvier, Prionace glauca,
Rhizoprionodon lalandii, R. porosus, Sphyrna lewini and S.
zygaena. Differences in the microscopic aspects of dermal
denticles from the species studied were observed. P. glauca,
a pelagic shark, showed a well-developed protection appa-
ratus when compared with other pelagic species, while
coastal sharks showed even higher structural complexity.
In the blue shark the denticles are enameled, presenting an
extensive pulp cavity and a base inserted in a connective
* Aline Nayara Poscai
aline.poscai@usp.br
1 Setor de Anatomia, Departamento de Cirurgia da Faculdade
de Medicina Veterinária e Zootecnia da Universidade de São
Paulo, SãoPaulo, SP, Brazil
2 Departamento de Fisiologia, Instituto de Biociências,
Universidade de São Paulo, Rua do Matão, travessa 14, no
321, Cidade Universitária, SãoPaulo, SP, Brazil
3 Laboratório de Biologia Animal, Universidade Federal
doAcre, Campus Floresta, CruzeirodoSul, AC, Brazil
4 Departamento de Fisiologia, Setor de Ciências Biológicas,
Universidade Federal doParaná, Centro Politécnico,
Curitiba, Paraná81531-990, Brazil
5 Instituto de Pesca /APTA/SAA/SP, Av. Bartolomeu de
Gusmão, 192, Santos, SP11030-500, Brazil
6 Central de Facilidades à Pesquisa da Faculdade de Medicina
Veterinária e Zootecnia da Universidade de São Paulo,
Rua Professor Orlando Marques Paiva, s/n, SãoPaulo,
SP05508-270, Brazil
Zoomorphology
1 3
activated in predation events or as response against changes
in pressure around the eyes or head (Bell and Satchell
1963; Gruber and Schneiderman 1975). Recently, the nicti-
tating membrane has been used as a stress indicator during
scientific capture by analyzing the imparity reflex activated
by external stimulation (Danylchuk et al. 2014; Gallagher
etal. 2014).
In sharks, the membrane is thin and opaque, composed
by connective tissue and covered externally by dermal den-
ticles. The denticles are also called placoid scales which are
also observed across the shark’s body (Bell and Satchell
1963; Raschi and Tabit 1992; Kemp 1999). Dermal denti-
cles exhibit a variety of shapes closely related with func-
tion, including protection against predators and ectopara-
sites (denticles with thorns and mucus), reduction of the
hydrodynamic drag (denticles with furrows in the crown),
and accommodation of sensory and bioluminescent struc-
tures (Reif 1978; Raschi and Tabit 1992). For that reason,
the denticles are composed of calcified dentine and covered
with a thin layer of enamel (Gravendeel etal. 2002), being
more resistant than the cartilaginous skeleton and an effec-
tive tool for fossils identification (Gravendeel etal. 2002)
and phylogenetic assessment (Cappetta 1987; Marshall
2011).
However, studies describing the detailed morphology
of the dermal denticles in a comparative level are scarce
(Marshall 2011; Mello etal. 2013; Ciena etal. 2015; Ran-
gel etal. 2016). Since the morphological structure differs
between body regions, it is imperative to perform a detailed
study in order to identify the unique characteristics, allow-
ing its use as a taxonomic tool (Marshall 2011; Mello etal.
2013). In this context, the present study used light (LM)
and scanning electron microscopy (SEM) to describe in
details the microstructure and distribution of dermal den-
ticles in the nictitating membrane of seven Carcharhinidae
and Sphyrnidae species.
Materials andmethods
Animals
Samples of ten sharks were obtained from commercial fish-
ing in Southern and South Brazil. Carcharhinus limbatus
(n = 2), Prionace glauca (embryos n = 3 and adults n = 3),
Rhizoprionodon lalandii (n = 2), R. porosus (n = 2), Sphy-
rna lewini (n = 2), and S. zygaena (n = 2) were captured
from December 2014 to November 2015. Samples from
Galeocerdo cuvier (n = 2) were obtained in the fish col-
lection from the Laboratory of Ichthyology located in the
Institution of Bioscience of University of São Paulo. The
samples were obtained using tweezers and a scissor to
separate the membrane from the ventral preorbital and the
elevating musculatures (Fig.1).
The work was approved by the Brazilian Ministry
of Environment and IBAMA through SISBIO (Sistema
de Autorização e Informação em Biodiversidade), per-
mit number 48348-7 and by the Ethics Committee from
FMVZ-USP, Protocol Number 4245050214.
Light microscopy (LM)
The nictitating membrane of one blue shark adult (P.
glauca) was fixed in 10% formaldehyde solution. The sam-
ple was rinsed for 15min and stored in 70°GL alcohol and
then dehydrated in ascending ethanol series (from 70 to
100°GL) and thereafter cleared in xylene for subsequent
embedding in paraplast. Paraplast blocks were sectioned
using a microtome (Leica, German) and stained with hema-
toxylin and eosin (HE) for analysis.
Scanning electron microscopy (SEM)
Nictitating membranes from P. glauca, C. limbatus, R.
lalandii, R. porosus, S. lewini, S. zygaena and G. cuvier
were separated, fixed in 4% formaldehyde solution (over-
night) and stored in 70°GL alcohol. The samples were
then dehydrated in a series of increasing ethanol density.
After dehydration, the samples were dried (overnight) in a
Balzers CPD 020 critical-point device mounted onto metal
stubs with carbon adhesive and sputtered with gold in an
Emitech K550 sputter apparatus and read in scanning elec-
tron microscope Leo 435 VP (FMVZ-USP).
Results
The morphological analysis showed that there are relevant
differences in the microscopic aspects of dermal denticles
Fig. 1 Nictitating membrane of Rhizoprionodon porosus
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1 3
from the species studied. It was not possible to define the
orientation of the denticles according to the body axis
(head–tail).
Prionace glauca
In SEM, it was possible to observe the dermal denticles
covering the entire external surface of the nictitating mem-
brane. The denticles vary in shape and size (Fig.2a, b). The
denticle crown is rounded shaped, exhibiting between 3 and
9 ridges, which extend from the crown base to a third or
half of the upper region of the crown (Fig.2a–c).
In the lateral extremities it was possible to observe
the presence of hexagonal ornamentations (Fig. <link
rid="fig2">2</link>b, c). Close to the ridges, the orna-
mentations were bigger, reaching the base of the denticle,
while the structures in the upper region were smaller.
Under LM, it was possible to observe the dermal denti-
cles in a transversal angle. The denticles are composed of
a thin layer of dentine and enameloid (upper region) pre-
senting a wide pulp cavity and a denticle base inserted in
the connective tissue. The musculature is located below the
connective tissue (Fig.2d).
Carcharhinus limbatus
The dermal denticles in C. limbatus are equally distrib-
uted in the external surface, exhibiting differentiated
crown structure (Fig.3a). The anterior denticle margin is
monocuspid and rounded shaped (Fig. 3b, c). In the dor-
sal surface, it was possible to observe 3–5 well-marked
ridges, which extend from the denticle base until the upper
half region of the crown. Hexagonal microstructures are
observed, being bigger in the posterior region (Fig.3b, c).
The denticles overlap in the species (Fig.3b, c).
Rhizoprionodon lalandii
The dermal denticles in R. lalandii are oval/flattened-
shaped with uniform distribution, presenting similar struc-
tural characteristics along the crown (Fig. 3d, e). The
anterior margin is monocuspid and rounded shaped, as
observed in C. limbatus (Fig.3f). It presents 3–5 slightly
marked ridges in the crown, which extend from the base
to the upper half region of the structure (Fig. 3e, f). The
hexagonal microstructures are observed across the surface,
being bigger in the anterior region, where the ridges are
located (Fig.3e, f).
Rhizoprionodon porosus
The dermal denticles of R. porosus are distinct from each
other, due to the area analyzed (Fig. 3g, h). Some are
monocuspid with a rounded-shaped anterior margin, while
others are flattened-shaped (Fig. 3g) or oval-flattened-
shaped (Fig.3h). The monocuspid denticles exhibit uneven
distribution, covering all the blank spaces in the surface
(Fig. 3G), while the oval-shaped denticles exhibit spaced
distribution in some regions (Fig.3h).
Slightly marked and well-marked ridges are observed
(2–5) extending from the denticle base to the upper half
region of the crown (Fig.3g–i). The hexagonal structures
are observed across the crown surface, being bigger in the
anterior region, where the ridges are located (Fig.3i).
Sphyrna lewini
The denticles are distributed evenly across the membrane,
with scarce spaces in the epithelium and an oval-shape
(Fig. 4a). Hexagonal ornamentations in the anterior and
posterior extremities are observed. It was possible to iden-
tify glandular cells between the denticles (Fig.4b).
Fig. 2 Nictitating membrane of an adult Prionace glauca. a SEM
showing distribution of denticles (dd) in the membrane; b, c SEM
of denticles (dd) in the membrane, highlighting the hexagonal orna-
mentations (ho) and arrows indicating the longitudinal demarcations;
d LM showing denticles (d) with wide pulp cavity (pc) a thin layer
composed by dentine (d),enamel (e) with connective tissue (ct) and
musculature (m); e denticles (dd) overlapped; f a single denticle with
hexagonal ornamentations (ho) and arrows indicating the demarca-
tions
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1 3
Sphyrna zygaena
The denticles are distributed evenly, arranged side-by-
side, with little or no space between them (Fig.4c, d). The
structures are oval-shaped, with hexagonal ornamentations
restricted to the margins and small ridges in the posterior
region of the crown (3–6) (Fig.4e, f). The denticles exhibit
differentiated shape, size and position (flat; without longi-
tudinal demarcations) (Fig.4e, f). Secretory cells are pre-
sent in the epithelium adjacent to the denticles (Fig.4e, f).
Galeocerdo cuvier
The denticles are similar to S. lewini and S. zygaena: oval-
shaped, evenly distributed (Fig.5a, b) with hexagonal orna-
mentations restricted to the crown margins (Fig.5c). The
bigger denticles exhibited up to five ridges, restricted to the
posterior margin (Fig.5c).
Discussion
This is the first description of the microscopic aspects of
the nictitating membrane of Carcharhinidae and Sphyrni-
dae sharks. Through the technique, it was possible describe
in details the morphological features of the structure, thus
allowing a discussion to better understand the functional
and evolutionary importance of the membrane.
As observed in this preliminary study, the denticles
found in the membrane are covered by a thin layer com-
posed of dentine and enameloid (apical region), being
denser than denticles from other body regions. It has
been hypothesized that the presence of denser denticles in
the nictitating membrane is related to a higher protection
needed during feeding events or stressful situations. Cor-
roborating that, it has been described that during feeding,
Carcharhiniformes close the nictitating membrane and
open the jaw at the same time (Ritter and Godknecht 2000),
showing a close relation between prey capture and eye
protection. In Negaprion brevirostris, the nictitating mem-
brane response was tested facing luminous and electric
stimulation, showing the conditioning facing uncomforta-
ble/stressful events (Gruber and Schneiderman 1975). This
protective function is also corroborated by the morphologi-
cal conformation of the denticles around the eyes, charac-
terized by thickened, knob-like crowns highly sculpted
(Raschi and Tabit 1992).
Among the analyzed species, C. limbatus, P. glauca
and Rhizoprionodon spp. presented the most generalized
conditions, compared to features exclusively observed
Fig. 3 SEM from nictitating membrane of several shark species.
a dermal denticles of nictitating membrane in C. limbatus, b oval-
shaped denticles (dd); c oval-shaped denticles with longitudinal
demarcations, hexagonal ornamentations (ho) and arrows indicat-
ing the demarcations; d dermal denticles distribution in R. lalandii;
e oval-shaped denticles (dd) with well-marked demarcations; f den-
ticles with ornamentations (ho) across the surface and demarcations
(arrows) restricted to the posterior margin; g dermal denticles in R.
porosus of a variety of formats; h oval/rounded-shaped denticles (dd)
with restricted demarcations in the posterior margin; i oval-shaped
denticles with hexagonal ornamentations (ho) restricted to the poste-
rior margin with arrows indicating the demarcations
Zoomorphology
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in S. lewini, S. zygaena and G. cuvier (oval-shaped denti-
cles distributed evenly, with a lower amount of hexagonal
ornamentations and crowns with up to six ridges). Thus,
it is possible that the generalized condition in C. limba-
tus, P. glauca and Rhizoprionodon spp. species is related
to a higher eye protection needed during feeding. Since all
feature similar head-shape (diamond) and eye angle, it is
possible that those morphological conditions are more sus-
ceptible to damages during prey capture.
On the other hand, the similarity between hammerheads
and tiger sharks (simplified) could be related to a reduced
need for protection but for distinct reasons. While hammer-
head sharks exhibit eyes at the end of the cephalofoil, the
chances of eye damage during capture are reduced, allow-
ing an evolutionary decrease in energetic expenditure for
shaping and maintenance of the structure. In G. cuvier,
the triangular and robust shape of the head may be a mor-
phological advantage that protects the eyes from direct
angles during capture, also reducing the evolutionary cost
of shaping and maintenance. Finally, the glandular struc-
ture observed only in hammerhead species may be related
to a higher need for eye lubrication (Klećkowska-Nawrot
and Dziegiel 2007) and increase in water flow through eyes
during swimming as a way to reduce the impacts caused by
quick maneuvers against the water current during hunting.
The main structural differences between denticles from
body and from the nictitating membrane are the ridges
that vary in shape, size and density. The ridges are closely
related to hydrodynamic adaptations in some species (Mar-
shall 2011), being proposed that the ornamentations are
responsible for increase the resistance of the crown, result-
ing in weight reduction and better performance (Marshall
2011). Study performed with R. lalandii described the skin
denticles as specialized/complex structures (three cusps,
and well-defined crests and ridges) (Laranjeira etal. 2015),
being different from the denticles in the membrane, which
are less complex. The same pattern is observed in C. lim-
batus (Motta etal. 2012), S. lewini, S. zygaena (Mello etal.
2013) and G. cuvier (Dillon etal. 2017), where the com-
plexity of denticles of the body is greater when compared
to denticles in the membrane.
Changes in denticle complexity was reviewed by Raschi
and Tabit (1992), bringing evidence that ecological aspects
such as habits (e.g. benthic species vs. active species) mold
Fig. 4 a, b Nictitating membranes of juvenile Sphyrna lewini (cf)
and adult S. zygaena. a Denticles (dd) distribution in the membrane
of S. lewini; b glandular structure, wrapped by denticles with hexago-
nal ornamentations (ho) and epithelium (e) in the structure center; c
denticles (dd) distribution in the membrane of S. zygaena, the yellow
arrows indicating the empty spaces between the denticles; d close-up
in the denticles and spaces (yellow arrows) between them; e close-up
in the spaces, showing the epithelium (e) present, the denticles that
surrounded this glandular structure and white arrows indicating the
glandular cell; f close-up into the denticle epithelium (e) with hex-
agonal ornamentations (ho)
Fig. 5 Nictitating membrane of
Galeocerdo cuvier. a Distribu-
tion of denticles (dd) in the
membrane; b oval-shaped den-
ticles (dd); c bigger structures
with up to 5 ridges in the crown,
with hexagonal ornamentations
(ho)
Zoomorphology
1 3
the structure of the denticles. In fact, denticles can be sepa-
rated according to their function (e.g. drag reduction; abra-
sion strength; defense), being scattered according to the
region of the body compatible with its function (Dillon
etal. 2017). By comparing our findings with the structures
molded for protection against abrasion, it is possible to con-
firm that in fact the less complex morphology is compatible
to a protective function (Dillon etal. 2017).
The sharks analyzed in the present study share an active
hunting behavior, which could explain by the highly-pre-
served morphology shaped over millions of years of selec-
tive pressures. However, differences among life habits and
environment occupation could explain the specializations
observed. We conclude that in terms of function, the der-
mal denticles in the nictitating membrane are not only
related to protection during hunting, but also responsible to
facilitate water flow during swimming, as a possible way
to reduce the drag in an evolutionary attempt to reduce
energy cost by modifying morphological features. Also, the
lower complexity when compared to the denticles of the
body, may be related to the greater need of mobility of the
structure, thus improving the biomechanical versatility of
the membrane. Lastly, since the structure seems to vary in
details among species, a deeper analysis relating membrane
features, head shape and eye angle could elucidate points
raised in the present study such as the use of this new char-
acters complex to understanding the evolution of the eye
protection during the feeding and predation events in the
Carcharhiniformes.
Acknowledgements We would like to thank CAPES (granted ANP
and NW) for the support, the postgraduate program of Department
of Surgery, Faculty of the Veterinary Medicine and Animal Sci-
ence, University of São Paulo and the funding provided by FAPESP
through contract number 2016/09095-2 (Granted to BRS).
References
Bell JP, Satchell GH (1963) An undescribed unilateral ocular reflex
in the dogfish Squalus acanthias. Austr J Exp Biol 41:221–234
Cappetta H (1987) Chondrichthyes II. Mesozoic and Cenozoic Elas-
mobranchii. Gustav Fischer, New York, pp1–193
Ciena AP, Rangel BS, Bruno CEM, Miglino MA, Amorim AF, Rici
REG, Watanabe I (2015) Morphological aspects of oral denti-
cles in the Sharpnose shark Rhizoprionodon lalandii (Muller
and Henle, 1839) (Elasmobranchii, Carcharhinidae). Anat Histol
Embryol 45:109–114. DOI:10.1111/ahe.12178
Compagno LJV (1988) Sharks of the order carcharhiniformes. Prince-
ton University Press, New Jersey, p572
COMPAGNO LJV (2005) Checklist of living Chondrichthyes. In:
Hamlett WC (eds) Reproductive biology and phylogeny of Chon-
drichthyes: sharks, batoids and chimaeras. Science Publishes,
Inc, United States, pp501–548
Crawford MLJ, Marc RE (1976) Light transmission of cat and mon-
key eyelids. Vision Res 16:323–324
Danylchuk AJ, Suski CD, Mandelman JW, Murchie KJ, Haak CR,
Brooks AML, Cook SJ (2014) Hooking injury, physiological
status and short-term mortality of juvenile lemon sharks (Negap-
rion bevirostris) following catch-and-release recreational
angling. Conser Physiol. doi:10.1093/conphys/cot036
Dillon EM, Norris RD, Dea AO (2017) Dermal denticles as a tool to
reconstruct shark communities. Mar Ecol Prog Ser 566:117–134
Gallagher AJ, Serafy JE, Cooke SJ, Hammerschlag N (2014) Physi-
ological stress response, reflex impairment, and survival of five
sympatric shark species following experimental capture and
release. Mar Ecol Prog Ser 496:207–218
Gravendeel R, Neer WV, Brinkhuizen D (2002) An identification key
for dermal denticles of Rajidae from the North Sea. Int J Osteo-
archaeol 12:420–441. doi:10.1002/oa.645
Gruber SH, Myrberg AA (1977) Approaches to the study of the
behavior of sharks. Am Zool 17:471–486
Gruber SH, Schneiderman N (1975) Classical conditioning of the nic-
titating membrane response of the lemon shark (Negaprion brev-
irostris). Behav Res Methods Instrum 7:430–434. doi:10.3758/
BF03201554
Hueter RE, Mann DA, Maruska KP, Sisneros JA, Demski LS (2004)
Sensory biology of elasmobranchs. In: Carrier JC, Musick JA,
Heithaus MR (eds) Biology of sharks and their relatives. CRC,
Boca Raton, pp325–368
Kemp NE (1999) Integumentary system and teeth. In: Hamlett WC
(ed) Sharks, skates and rays: the biology of elasmobranch fishes.
John Hopkins University Press, Baltimore, pp43–68
Klećkowska-Nawrot J, Dzięgiel P (2007) Morphology of
the third eyelid and superficial gland of the third eye-
lid on pig fetuses. Anat Histol Embryol 36:428–432.
doi:10.1111/j.1439-0264.2007.00780.x
Laranjeira ME, Guimarães JP, Amorim AF, Rotundo M, Rici REG,
Mari RB (2015) Ultrastructure of dermal denticles in sharp-
nose shark (Rhizoprionodon lalandii)(Elasmobranchii, Car-
charhinidae). Microsc Res Tech 78(10):859–864. doi:10.1002/
jemt.22546
Marshall LJ (2011) The fin blue line: Quantifying fishing mortal-
ity using shark fin morphology. Dissertation, University of
Tasmania
Mello WC, De Carvalho JJ, Brito PMM (2013) Microstructural mor-
phology in early dermal denticles of hammerhead sharks (Elas-
mobranchii: Sphyrnidae) and related taxa. Acta Zool-Stockholm
94:147–153. doi:10.1111/j.1463-6395.2011.00547.x
Motta P, Habegger ML, Lang A, Hueter R, Davis J (2012) Scale
morphology and flexibility in the shortfin mako Isurus oxyrin-
chus and the blacktip shark Carcharhinus limbatus. J Morphol
273(10):1096–1110
Pires AG, Algueró MC, Mendes JL, Trindade H, Correia M (2008)
Immunophenotyping of lymphocyte subsets in the third eyelid
tissue in dogs (Canis familiaris): Morphological, microvascular,
and secretory aspects of this ocular adnexa. Microsc Res Tech
7:521–528
Puff C, Herder V, Philipp A, Baumgartner W (2008) Lymphangio-
sarcoma in the nictitating membrane of a horse. J Veter Diagn
Invest 20:108–110
Rangel BS, Ciena AP, Wosnick N, Amorim AF, Kfoury-Junior JR,
Rici REG (2016) Ecomorphology of oral papillae and denticles
ofZapteryx brevirostris (Chondrichthyes, Rhinobatidae). Zoo-
morph 135:189–195. doi:10.1007/s00435-016-0304-0
Raschi W, Tabit C (1992) Functional aspects of placoid scales: a
review and update. Aust J Mar Fresh Res 43:123–147
Reif WE (1978) Protective and hydrodynamic function of the dermal
skeleton of elasmobranchs. Neues Jahrbuch Für Geologie Und
Paläontologie 157:33–141
Ritter EK, Godknecht AJ (2000) Agonistic displays in the blacktip
shark (Carcharhinus limbatus). Copeia 2000:282–284
... Scale bar = a, b, c, 100 μm; d, 30 μm ecological and sensory roles (Kemp, 1999;Raschi & Tabit, 1992;Reif, 1985). Most studies addressing these dermal derivatives focused on the external (head, trunk, fins and eyes), taxonomic, hydrodynamic, paleontological and bioecological issues (Compagno, 1977;Dillon et al., 2017;Feld et al., 2019;Meyer & Seegers, 2012;Poscai et al., 2017;Serra-Pereira et al., 2008;Tomita et al., 2020). ...
... Regarding the analysis of dermal denticles of elasmobranchs, recent studies have been employing micro CT scan to describe their morphological aspects (e.g., Tomita et al., 2020) or to investigate their biomimetic properties (Ankhelyi et al., 2018;Domel et al., 2018;Wen et al., 2015). Although histology and SEM are the most usual tools for this kind of work (Compagno, 1988;Dillon et al., 2017Dillon et al., , 2021Popp et al., 2020;Poscai et al., 2017Poscai et al., , 2021Rangel et al., 2016Rangel et al., , 2017Rangel et al., , 2018Reif, 1974Reif, , 1985, CT scan method works as a complement to elucidate the morphology of dermal denticles, which is not possible with the use of traditional techniques. It is worth mentioning that the same tissue sections (dorsal and ventral) used in CT scan were analysed under SEM. ...
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Oral denticles of sharks are composed by a crown, dentine covered by a layer of enameloid and pulp cavity, the same structure of the dermal denticles found across the body surface of most elasmobranchs. In addition, oral papillae and taste buds are distributed among denticles within the oropharyngeal cavity, playing a fundamental role for tasting as part of the chemosensory system of fishes. Scanning electron microscopy (SEM) has been employed as an important tool for the study of dermal denticles and other structures, as well as histology and more recently CT scan analysis. Herein, we used two methods for the study of the morphology of the oropharyngeal cavity of Lamna nasus (Lamniformes), an oceanic and pelagic shark: SEM and CT scan. The general morphology of oral denticles studied herein is related to abrasion strength since they are diamond‐shaped, lacks lateral cusps and presented less pronounced ridges. In addition, smooth ridges and broad rounded denticles could be related to prevent abrasion during food consumption and manipulation. Oral papillae had a round shape and were only observed under SEM. The densities of papillae were estimated in 100 per cm2, whereas denticles were 1760 and 1230 cm2 over the dorsal and ventral regions, respectively. The high numbers of denticles are inversely proportional to papillae density; denticles seem to restrict papillae distribution. Regarding the differences between methodologies, under SEM, only the crown was visualized, as well the papillae, allowing the estimation of size and density of both structures. However, under CT scan, the whole components of denticles were clearly visualized: different views of the crown, peduncle, basal plate, and pulp cavity. On the other hand, oral papillae were not visualized under CT due to the tissue preparation. Furthermore, both methods are complementary and were important to extract as much information as possible from denticles and papillae.
... The development and forms of elasmobranch placoid scales and teeth provide insights into the evolution of the regulatory networks that control the occurrence of such structures within chondrichthyans and among vertebrates. Batoids (Figure 1.1) and sharks display placoid scales (Figure 1.3) and eventually fin spines on the head and body skin, inside the mouth and sometimes on nictitating membrane and eye [Poscai et al., 2017;de Sousa Rangel et al., 2019a;Tomita et al., 2020]. As opposed to elasmobranchs (Figure 1.2), mature chimaeras retain a very limited number of dermal denticles, notably next to the dorsal spine and to the lateral line canals [Didier and Stehmann, 1996;Didier, 2004;Møller et al., 2004]. ...
... Le développement et les formes des écailles placoïdes et des dents des élasmobranches fournissent des indications quant à l'évolution des voies de régulation contrôlant la présence de ces structures au sein des chondrichtyens et des vertébrés. Les batoïdes (Figure 1.2) et les requins ont des écailles placoïdes sur la tête et le corps (Figure 1.3), dans la cavité orale et parfois sur la membrane nictitante de l'oeil et certaines espèces ont également des épines dorsales [Poscai et al., 2017;de Sousa Rangel et al., 2019a;Tomita et al., 2020]. Contrairement aux élasmobranches (Figure 1.2), les chimères adultes conservent un nombre réduit de denticules dermiques, notamment à proximité de l'épine dorsale et de la ligne latérale [Didier and Stehmann, 1996;Didier, 2004;Møller et al., 2004]. ...
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Teeth are serial structures whose evolutionary and developmental history is intricately linked with the emergence of mineralised tissues in vertebrates. Teeth display a broad range of forms and differ in developmental patterns in extant vertebrates, making them remarkable elements to study species diversification. Selachian teeth renew permanently and display morphologies that are correlated with mating and trophic behaviours.This work first assesses the variation of tooth forms in two scyliorhinids by using 3D geometric morphometrics and machine learning. The emergence of gynandric heterodonty is detailed for the first time along the ontogeny of sharks and it is demonstrated that this natural variation should be first assessed before performing species discrimination.This work also questions the role of specific proteins on the acquisition of a shark tooth form over development. Functional tests suggest an impact of Shh and Fgf3 in the cusp morphogenesis and in the mineralisation process. These proteins are promising explanatory variables to the inter- and intraspecific tooth differences observed, leading to hypotheses on their role in the evolution of structures with speciation and trophic and mating behaviours.Histological data on extant chondrichthyan vertebrae finally highlight the unsuspected proportion of extant elasmobranchs exhibiting fibrous mineralisation in the neural arches, a bone-like tissue which occurrence had long been refuted in this group. Evolutionary considerations are discussed in the light of the evolution of jawed vertebrates and question on the ecological factors that led particular tissues to be restricted to specific shark and batoid groups.
... In vertebrates, this mobile component of the ocular adnexa assists in spreading the tear film across the cornea, thereby removing any debris and acting as a barrier in the protection of the cornea and eye (Sivak and Glover 1986). Nictitating membranes have been described in many vertebrates including sharks (Gruber and Cohen 1978;Poscai et al. 2017;Collin 2018), amphibians (Lande and Zadunaisky 1970), reptiles and birds (Sivak and Glover 1986;Kühnel and Schramm 1989;Schobert et al. 2013), and mammals (Stibbe 1928;Barasa 2003). In birds, the "membrana nictitans" or third eyelid is pulled temporally from the medial canthus of the eye by the pyramidalis muscle located behind the globe (Friedmann 1932;Sivak and Glover 1986). ...
... For example, in ungulates, the nictitating membrane protects the cornea from grass spikes (Blogg 1980), although it is also important that some vision is maintained. Nictitating membranes have been described in many vertebrates including sharks (Poscai et al. 2017;Collin 2018), amphibians, birds, reptiles, mammals, including domestic animals such as ungulates (Stibbe 1928;Blogg 1980;Schramm et al. 1994;Schlegel et al. 2001), most non-human primates (Arao and Perkins 1968) and occurs, in a vestigial form, as the plica semilunaris in humans. ...
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Synopsis The ultrastructure of the nictitating membrane in the little penguin Eudyptula minor was studied using both scanning and transmission electron microscopy to improve our understanding of the function of ocular adnexa in diving birds. Following euthanasia, eyes were enucleated and immersion fixed in Karnovsky’s fixative. The nictitating membrane and conjunctiva were embedded in araldite and semi- or ultra-thin sections were stained and photographed using compound and transmission electron microscopes, respectively. Ultrastructural dimensions were measured directly from digital photographs. Surface ultrastructure was examined using scanning electron microscopy. The transparent nictitating membrane consists of a dense stroma surrounded by epithelia on both the external (conjunctival) and internal (bulbar) surfaces. The conjunctival surface of the membrane near the leading edge is covered by microvilli, which transition to microplicae and finally to microridges in the periphery. Beneath the epithelial cells, there is a well-developed basement membrane. Scattered throughout this epithelium are a few goblet cells. The surface of the bulbar epithelium is covered by microvilli near the leading edge, which become denser peripherally. The stroma consists of densely-packed collagen fibrils, which are randomly oriented in bundles near the leading edge but are aligned in the same direction parallel with the epithelial and corneal surfaces and with the leading edge, when the membrane is extended. The ultrastructure of the nictitating membrane in the little penguin differs from other birds and its function is predominantly protective, while preserving clear vision in both water and air.
... For example, carcharhinid and sphyrnid sharks have nictitating membranes or "third eyelids", which cover their eyes completely during their feeding activities [2]. The outer surface of this membrane is covered with dermal denticles, which likely increases its protective ability [3]. In contrast, many other elasmobranchs that are not equipped with nictitating membranes have to protect their eyes in different ways, such as retracting the eyeballs into the head (e.g., electric ray [4]; guitarfish [5]), or rotating the eyeballs back into the orbit (e.g., white shark [6]). ...
... The denticles on the rest of the whale shark's body, such as the head, trunk and fins, are characterized by parallel, triple ridges on the upper surface, presenting a drag-reduction type morphology (Fig 3E). Interestingly, the denticles that cover the nictitating membranes of carcharhinid and sphyrnid sharks have also been thought to play a role in eye protection [3]. Though the overall morphologies of the denticles in the nictitating membranes of carcharhinid and sphyrnid sharks are different from those of whale sharks, they both have especially thick denticle crowns, which suggests that they have a similar function of mechanical protection. ...
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This report elaborates on adaptations of the eyes of the whale shark Rhincodon typus (Elasmobranchii, Rhincodontidae), including the discovery that they are covered with dermal denticles, which is a novel mechanism of eye protection in vertebrates. The eye denticle differs in morphology from that of the dermal denticles distributed over the rest of the body, consistent with a different function (abrasion resistance). We also demonstrate that the whale shark has a strong ability to retract the eyeball into the eye socket. The retraction distance was calculated to be approximately half the diameter of the eye, which is comparable to those of other vertebrates that are known to have highly retractable eyes. These highly protective features of the whale shark eye seem to emphasize the importance of vision for environmental perception, which contradicts the general, though poorly established, notion of low reliance on vision in this species.
... Dermal denticles are found in the oropharyngeal cavity (Cook and Neil, 1921;Nelson 1970;Atkinson & Collin 2012;Rangel et al., 2018), as well as on the body surface (Reif 1985;Raschi & Tabit 1992;Dillon et al., 2017), and nictitating membrane (Poscai et al., 2017). The morphology of the dermal denticles has been explored for systematic and morphological purposes (Compagno 1977;Marshall 2011;Mello et al., 2013;Sullivan & Regan 2011;Laranjeira et al., 2015;Dillon et al., 2017;Rangel et al., 2018) According to Reif (1985), Raschi & Tabit (1992) and Ferr on & Botella (2017) they can be classified in five functional groups: (1) drag reduction, (2) abrasion strength, (3) defence, (4) luminescence, and (5) generalized functions. ...
Article
Oropharyngeal dermal denticles and oral papillae are present throughout the oropharyngeal cavity, and incorporate the use of taste buds to orally process and evaluate the food items, whereas oral denticles are thought to provide a form of protection against abrasion during food consumption and improve ventilation efficiency. Herein, are compared the microstructure of the oropharyngeal denticles and papillae of large predatory requiem sharks (Carcharhinidae) (Carcharhinus brevipinna, Carcharhinus leucas, C. limbatus, Carcharhinus obscurus, Carcharhinus signatus, and Galeocerdo cuvier), under scanning electron microscopy. The results revealed that the largest oral denticles were found in adults of C. signatus, followed by juveniles of G. cuvier, C. leucas, and C. obscurus, respectively. Oral papillae were found to be larger in G. cuvier, C. signatus, and in C. leucas, and all these specimens presented round-shaped papillae. The higher denticles densities were found in the oral cavity of C. signatus, however, this species presented the lowest density of papillae. Carcharhinus limbatus presented the second highest rate of denticles density, followed by G. cuvier, C. obscurus, C. leucas, and C. brevipinna. The highest density of papillae was found in C. brevipinna, indicating that the density of denticles is inversely proportional to the papillae distribution, the same as we observed in C. signatus. The denticles density seems to be higher as the animal increases in size, as we observed in adult specimens of C. signatus, and this shark presented two different morphologies of denticles, different from the other species studied here. This may suggest that densities and sizes of these structures differ as the animals grow, expressed by the prey spectrum availability and the dietary shifts due to the distinct habitat which the species are associated during their life cycles.
... Some particularities of the denticles that make them interesting tools include: (a) their morphological aspects (i.e., shape, size, and arrangement) are highly variable intra and interspecifically; (b) they are correlated with shark ecology; (c) are very abundant across the body of sharks, including oral regions and nictitating membrane; and (d) are continually exchanged (e.g., Dillon et al., 2017;Poscai et al., 2017;Rangel et al., 2017). Although recent studies have addressed extensively about morphology, taxonomy, and function of denticles (e.g., Dillon et al., 2017), more studies are needed considering both the high shark diversity (~500 living species; Weigmann, 2016) and intra and interspecifically variability of denticles across the body and oral cavity (e.g., Ankhelyi, Wainwright, & Lauder, 2018;Dillon et al., 2017;Rangel, Salmon, Poscai, Kfoury Jr., & Rici, 2019). ...
Article
The dermal denticles are among the unique morphological adaptations of sharks, which have been acquired throughout their long evolutionary process of more than 400 million years. Species-specific morphological characteristics of these structures has been applied specially as tools for functional and taxonomic (family-level) studies. Nevertheless, few studies have explored the diversity of denticle structure in different around the body and oral cavity. In the present study, we described the morphological differences observed in skin and oral cavity of sharpnose sevengill shark Heptranchias perlo, using scanning electron microscopy. Our findings demonstrate substantial variation in morphological structure of the denticles of the body and oral cavity. Overall, the dermal denticles observed across body surface were overlapped, tricuspid, with the central cuspid being more pronounced, pointed, and triangular in shape compared with lateral ones. Unlike, the denticles on the tip of the nose had a smooth crown, with rounded edges, being compact, and overlapped. The oral denti-cles were found in the ventral and dorsal region of the oral cavity. They also were tricuspid, but with differences in arrangement and ridges. These results suggest a strict functional relationship with the morphological characteristics observed. Such morphological diversity body-region-dependent highlights the need for comparative studies that include oral denticles, since this structure has an important functional role in sharks and can be found in fossil and recent records.
... Inter-specific differences in the hexagonal arrangement of dermal denticles over the membrane suggest dynamic and biomechanical adaptation of this highly mobile structure to rapidly and efficiently protect against abrasion, mainly during predation events. 88 ...
Article
The eyes of apex predators, such as the shark, have fascinated comparative visual neuroscientists for hundreds of years with respect to how they perceive the dark depths of their ocean realm or the visual scene in search of prey. As the earliest representatives of the first stage in the evolution of jawed vertebrates, sharks have an important role to play in our understanding of the evolution of the vertebrate eye, including that of humans. This comprehensive review covers the structure and function of all the major ocular components in sharks and how they are adapted to a range of underwater light environments. A comparative approach is used to identify: species‐specific diversity in the perception of clear optical images; photoreception for various visual behaviours; the trade‐off between image resolution and sensitivity; and visual processing under a range of levels of illumination. The application of this knowledge is also discussed with respect to the conservation of this important group of cartilaginous fishes.
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The last 50 yr of fisheries catch statistics and ecological surveys have reported significant decreases in shark populations, which have largely been attributed to human activities. However, sharks are challenging to census, and this decline likely pre-dated even the longest fishery-dependent time series. Here we present the first use of dermal denticles preserved in reef sediments as a novel tool to reconstruct shark communities. We first built a dermal denticle reference collection and conducted a morphometric analysis of denticle characters to relate denticle form to taxonomy, shark ecology, and denticle function. Denticle morphology was highly variable across the body of an individual shark and between taxa, preventing species- or genus-level identification of isolated denticles. However, we found that denticle morphology was strongly correlated with shark ecology, and morphometric analysis corroborated existing functional classifications. In a proof of concept, we extracted 330 denticles from modern and fossil reef sediments in Bocas del Toro, Panama and found them to be morphologically diverse and sufficiently well-preserved to allow classification. We observed a high degree of correspondence between the denticles found in the sediments and the sharks documented in the region. We therefore propose that (1) denticle assemblages in the recent fossil record can help establish quantitative pre-human shark baselines and (2) time-averaged denticle assemblages on modern reefs can supplement traditional surveys, which may prove especially valuable in areas where rigorous surveys of sharks are difficult to perform.
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Elasmobranchs have an impressive range of highly specialized sensory systems shaped over 400 million years of evolution. The morphological analysis of oral papillae and denticle in elasmobranchs elucidates the biological role that these structures play during feeding and ventilation, bringing important descriptive information about ecological implications in an evolutionary context. The present study provides descriptions of the distribution patterns, histological characteristics and three-dimensional aspects of oral papillae and denticles in the lesser guitarfish Zapteryx brevirostris, through light microscopy and scanning electron microscopy. The presence of oral denticles in the oropharyngeal cavity suggests that this structure may have the following functions: protect against abrasion and parasites, increase the ability to grasp and hold prey and assist in reduction in hydrodynamic drag. The denticles in Z. brevirostris are similar to those found in pelagic sharks with forced ventilation (RAM). The structural conformity of denticles observed in the gill slits may facilitate water flow during prey grasp and food processing. This study supports the hypothesis that these structures may be an adaptive reflection shaped by feeding habits, capture strategies and processing prey.
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Sport fishing for sharks, including fishing with the intent to release, is becoming more prevalent within the recreational angling community. Common targets of recreational anglers are juvenile lemon sharks (Negaprion brevirostris) that frequent shallow tropical nearshore habitats. In this study, we captured 32 juvenile lemon sharks (530–875 mm total length) with conventional angling gear (i.e. spinning rods, dead fish bait and 5/0 barbed circle hooks) from the coastal waters of Eleuthera, The Bahamas, to determine the consequences of capture for individual sharks. Each shark was examined for hooking injuries, blood sampled to quantify physiological disturbance, assessed for reflex impairment and then monitored to assess postrelease behaviour and mortality. Four sharks (12.5%) died following release during the 15 min tracking period. Principal components (PC) analysis revealed four axes describing 66.5% of the variance for blood physiology parameters, total length and water temperature. The PC1 and PC3 scores, characterized by positive factor loadings for indicators of exercise-induced stress and blood ion concentrations, respectively, were significantly related to fight time but were not associated with short-term mortality. Short-term mortality was significantly related to factor scores for PC4 that loaded heavily for water temperature and total length. Ten sharks (31%) exhibited impaired reflexes, with loss of bite reflex being most prevalent. Sharks that died had the following characteristics: (i) they had two or more impaired reflexes; (ii) they were hooked in the basihyal; (iii) they exhibited no movement after the initial bout of directional swimming; and (iv) they experienced high water temperatures (i.e. >31°C). Collectively, these results indicate that for juvenile lemon sharks inhabiting tropical flats, fight time can influence the degree of physiological disturbance, while water temperature contributes to the likelihood of survival following release.
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The presence of denticles in the external surface, oral cavity, fins, and clasper of Elasmobranchii has been widely reported. These structures, called body denticles, may be observed on the body surface of sharks. Dermal and oral denticles are made up by a basal plate that is embedded in the dermis, forming a peduncle that grows from the base to the crown. These denticles may protect the skin against abrasion, and improve hydrodynamics and gill arches function. Rhizoprionodon lalandii is a widely distributed and very common species in Brazilian coastal areas. The aims of this study was to compare the morphology of oral and body denticles of R. lalandii to understand the implications of these structures in the behavior of these animals. Morphological analysis showed that there are differences between dermal and oral denticles, which are related to their role in different body regions. Body denticles have three cusps, and well-defined crests and ridges, and literature data suggest that suggest that hydrodynamics is their main function. Most of the oral denticles have only one cusp, and their morphology and distribution showed that their main functions are preparing food to be swallowed and protecting the oral cavity against abrasion. Microsc. Res. Tech., 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
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The oral denticles of some elasmobranchs are found on the surface of the oral cavity and are homologous to those on the body surface, being well developed, independent and non-growing, with varying morphology and distribution depending on the species. The structural and three-dimensional characteristics of oral denticles from the rostro-ventral surface of the sharpnose shark Rhizoprionodon lalandii were described following imaging by both light and scanning electron microscopy. The light microscopy results showed that the triangular shape of the denticles consisted of a base and an apex. Picrosirius staining showed the arrangement of collagen fibres and oral denticles, and a predominance of type-I collagen was found in both structures under polarized light. There was a broad homogeneous distribution of denticles on the ventral surface, forming a leaf-like shape with the cusp facing the caudal region. Interlocking, hexagonal, geometric structures on its rostral side and ridges on the rostral side of the oral denticles were observed under increased magnification. We concluded that the denticle morphology found in R. lalandii differ of others analysed species, and the descriptions of these structures therefore provide important information for the classification of the species. In this species, the main functions can be assigned to help reduce hydrodynamic drag, particularly by this being a species that uses ram ventilation, and to protect the epithelium of the oropharynx of abrasion and parasites. © 2015 Blackwell Verlag GmbH.
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Mello, W.C., de Carvalho, J.J., Brito, P.M.M. 2011. Microstructural morphology in early dermal denticles of hammerhead sharks (Elasmobranchii: Sphyrnidae) and related taxa. —Acta Zoologica (Stockholm) 00: 1–7. This study uses scanning electron microscopies to investigate and describe the microstructural diversity of dermal denticles in the family Sphyrnidae, which comprises all living hammerhead shark species, comparing them to other related taxa (i.e. Carcharhinus dussumieri, Carcharhinus plumbeus, Carcharhinus acronotus, Rhizoprionodon acutus, Negaprion brevirostris and Hemigaleus microstoma). The results reveal that sphyrnids present noticeable microstructures in the dermal denticles, distinguishing them from the other related species investigated. Additionally, scale patterns are the same in three distinct body regions (i.e. cephalic, branchial and dorsal fin). Species of Sphyrnidae that reach bigger total lengths and that are widely distributed (i.e. Sphyrna lewini and Sphyrna mokarran) presented more, smaller and nearly hexagonal microstructures that do not cover the entire scale surface, unlike species reaching smaller sizes and restricted to coastal habits (i.e. Sphyrna tiburo, Sphyrna tudes, Sphyrna media and Eusphyra blochii). The sphyrnid scales are similar to R. acutus and C. dussumieri rather than to the other species, but it is not possible to identify the sphyrnid species only by scale features. It is clear that a similar morphology of scales is not necessarily related to similar life habits, and that they are candidates to provide new characters in phylogenetical studies among sphyrnids.