Gross morphology and histology of the

olfactory organ of the Greenland shark

Somniosus microcephalus

S. Ferrando, L. Gallus, L. Ghigliotti,

M. Vacchi, J. Nielsen, J. S. Christiansen
& E. Pisano

Polar Biology

ISSN 0722-4060

Polar Biol
DOI 10.1007/s00300-015-1862-1

1 23
Your article is protected by copyright and
all rights are held exclusively by Springer-
Verlag Berlin Heidelberg. This e-offprint is
for personal use only and shall not be self-
archived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com”.

1 23
 Author's personal copy
Polar Biol
DOI 10.1007/s00300-015-1862-1
ORIGINAL PAPER
Gross morphology and histology of the olfactory organ
of the Greenland shark Somniosus microcephalus
S. Ferrando1 • L. Gallus1 • L. Ghigliotti2 • M. Vacchi2 • J. Nielsen3
J. S. Christiansen4 • E. Pisano1
Received: 28 July 2015 / Revised: 8 December 2015 / Accepted: 9 December 2015
Ó Springer-Verlag Berlin Heidelberg 2015
Abstract The Greenland shark (Somniosus microcephalus)
is the largest predatory fish in Arctic waters. Knowledge of
the fundamental biology and ecological role of the Greenland
shark is limited, and the sensory biology of the Greenland
shark has been poorly studied. Given the potential relevant
contribution of chemoreception to the sensory capability of
the Greenland shark to forage and navigate in low-light
environments, we examined the architecture of the peripheral
olfactory organ (the olfactory rosette) through morphological,
histological and immunohistochemical assays. We found that
each olfactory rosette consists of a small number of lamellae
(22) associated with a relatively high surface area of the
olfactory epithelium. The general organization of the
epithelium is similar to that described for other elasmo-
branchs. However, details that have emerged concerning the
cell type composition (absence of crypt neurons, presence of
unusually large cells along the olfactory fiber bundles)
deserve further investigation. Overall, the structure of the
olfactory rosette suggests a well-developed olfactory capa-
bility for the Greenland shark coherent with a bentho-pelagic
lifestyle.
Keywords Greenland shark
Somniosus microcephalus

Olfactory rosette
Chemoreception
& S. Ferrando
sara131274@gmail.com
1
DISTAV-University of Genoa, Viale Benedetto XV 5,
16132 Genoa, Italy
2
ISMAR – CNR, Via de Marini 6, 16149 Genoa, Italy
3
University of Copenhagen, Strandpromenaden 5,
3000 Helsingør, Denmark
4
UiT The Arctic University of Norway, 9037 Tromsø, Norway
•
Introduction
The Greenland shark Somniosus microcephalus (Bloch and
Schneider, 1801) is the largest elasmobranch native to
Arctic waters, with a maximum body size exceeding 6 and
possibly 7 m (Bigelow and Schroeder 1948; Compagno
1984). The geographical distribution includes the deep and
cold waters masses of the entire North Atlantic Ocean
(Yano et al. 2007; MacNeil et al. 2012). Because of its oil-
rich liver, the Greenland shark was historically subject to
massive targeted fishery across the northern North Atlantic
where thousands of Greenland sharks were harvested on an
annual basis (reviewed in MacNeil et al. 2012). Although
fishery statistics for the Greenland shark are poor, con-
temporary bycatch captures in trawl and long-line fisheries
indicate that the species is still abundant in Arctic regions
(e.g., Nielsen et al. 2014; Hussey et al. 2015). Arctic fishes,
elasmobranchs in particular, are little studied (Christiansen
et al. 2014). The Greenland shark is currently listed as near
threatened (NT) in the IUCN Red List of Threatened
Species (Kyne et al. 2006; Lynghammar et al. 2013) but
will be recategorized as data deficient (DD) in the
upcoming Norwegian Red List (unpublished 2015).
The life history of the Greenland shark is poorly
understood, and the acquisition of data is crucial to clarify
the biology and ecology of this elusive species. Recent
studies have revealed that the Greenland shark undertakes
large-scale migrations and occupies depths between sur-
face waters and at least down to about 1800 m (Fisk et al.
2012; Campana et al. 2013), 2200 m (Kiraly et al. 2003) or
possibly 2647 m (Ebert and Stehmann 2013). Vertical
movements (detected by acoustic tracking) suggest active
foraging on ice-bound seals, which corresponds well with
observations of live whales and seals with distinct bite
marks most likely caused by Greenland sharks (Idrobo and
123
 Author's personal copy
Berkes 2012; MacNeil et al. 2012). This contradicts their
apathetic and lethargic behavior when caught on long lines
or in trawls. Scavenging by Greenland sharks has been
documented (Leclerc et al. 2011), and there is little doubt
that they feed on carrion on the ocean floor whenever
possible. On the other hand, increasing evidence from
studies on stomach contents (Leclerc et al. 2012; Nielsen
et al. 2014), stable isotopes (Fisk et al. 2002) and con-
taminants (Corsolini et al. 2014) indicates that the Green-
land shark is an active top predator mainly feeding on seals
and benthic/epibenthic fishes. In general, there is a growing
belief that the Greenland shark is a top predator of the
Arctic marine ecosystem (e.g., Leclerc et al. 2012; Mac-
Neil et al. 2012; McMeans et al. 2013; Nielsen et al. 2014).
The sensory biology of the Greenland shark is little
known, and the structure and functioning of the sensory
organs sustaining its lifestyle and behavior have been scar-
cely studied. Indirect information on the relative importance
of vision versus olfaction can be inferred by the fact that the
cornea of the Greenland shark eye often is infected by the
ectoparasitic copepod, Ommatokoita elongata (Berland
1961). The parasite probably impairs vision, but its presence
does not appear to hamper the foraging success of Greenland
sharks (Borucinska et al. 1998; Benz et al. 2002). In addi-
tion, olfaction is likely a prime sense, especially in low-light
environments such as deep waters or ice-covered seas.
Considering the putative importance of olfaction in
chemoreception, feeding and other behavioral traits of the
Greenland shark, we examined the architecture of the
peripheral olfactory organ where olfactory cues are received
from the environment—the olfactory rosette. This was done
by (1) counting the number of olfactory lamellae that make
up the rosette and estimating the sensory epithelial surface
area and (2) characterizing the olfactory epithelium by
histological and immunohistochemical assays. The struc-
tural organization of the olfactory rosette was used as a first
proxy of the olfactory capability of the Greenland shark.
Materials and methods
Two sub-adult and sexually immature female Greenland
sharks were captured on scientific long lines from the RV
Helmer Hanssen (Christiansen 2012) in Kejser Franz
Josephs Fjord (NE Greenland) in August 2013. Specimen
no. 1 measured 330 cm (total length) and no. 2 230 cm.
The right olfactory rosette from specimen no. 2 was
excised and used for gross anatomical analysis, lamella
counts and epithelial surface area estimates. The term
‘‘lamellae’’ has been used to indicate different structures
throughout the literature on Chondrichthyes (Meng and Yin
1981a, b; Takami et al. 1994; Kajiura et al. 2005; Theiss
et al. 2009; Meredith and Kajiura 2010; Cox 2013). Here it
123
Polar Biol
is intended as the two wing-shaped structures that protrude
from the two sides of the raphe. This allows comparison of
our data to those available in the literature (Kajiura et al.
2005; Theiss et al. 2009; Schluessel et al. 2008).
The rosette was immediately immersion-fixed in 10 %
formalin, washed in PBS and stored in 70 % ethanol in
distilled water. The volume of the rosette was calculated
through immersion in a graduated cylinder (2 ml gradua-
tions). The lamellae were exposed and counted. All the
lamellae were photographed using a digital camera (Sam-
sung S630) and analyzed. The total epithelial surface area
for each lamella was determined from digital images using
the open source ImageJ software (Rasband 1997–2014) in a
single plane. The secondary fold was not accounted for in
the surface area calculations. The area was multiplied by
two to account for both sides of each lamella.
Olfactory lamellae were taken from specimen no. 1 and
processed for histological and immunohistochemical
investigations. Accordingly, the excised tissues were
immediately immersion-fixed in 4 % paraformaldehyde in
0.1 M saline phosphate buffer (PBS, pH 7.4), washed in
PBS and stored in ethanol 70 % in distilled water. Tissue
pieces were embedded in Paraplast (Bio-Optica, Italy) and
sectioned at 5 lm thickness. Histological analyses were
performed after hematoxylin and eosin staining (Bio-Op-
tica, Italy). Immunohistochemical reactions were carried
out to highlight three G-protein alpha subunits using the
following polyclonal antisera (Santa Cruz Biotechnology
CA, USA): anti-Gaao (cat. sc-387), anti-Gaas/olf (cat. Sc-
383) and anti-Gaai3 (cat. Sc-262). As the secondary anti-
serum, Alexa 488 conjugated anti-rabbit (1:800 in PBS,
Molecular Probes, Invitrogen, Carlsbad, CA) was used.
The use of antisera on shark tissues for different G protein
alpha subunits raised against mammalian proteins is a well-
documented and reliable application of these antisera
because of the high conservation of these proteins across
vertebrate classes (e.g., Hansen et al. 2004; Wakabayashi
and Ichikawa 2008; Ferrando et al. 2009; Jungblut et al.
2009; Ferrando et al. 2010b; Quintana-Urzainqui et al.
2014). DAPI was used in order to counterstain nuclei.
Results
Gross morphology
The olfactory rosette of the Greenland shark is a sac-like
structure enveloped by a connective capsule that is in con-
tinuity with the olfactory bulb and peduncle dorsally and
opens ventrally to leave the olfactory lamellae exposed to the
water within the nasal cavity (Fig. 1a–c). The olfactory
lamellae are arranged in parallel along a central support
(central raphe) (Fig. 1c, e, f, h). The lamellae are largest in
 Author's personal copy
Polar Biol

Fig. 1 The olfactory rosette of

the Greenland shark. Olfactory
rosette (fixed by immersion in
10 % formalin, washed in PBS,
stored in 70 % ethanol):
photographs and diagrams.
a Dorsal view of the intact
rosette. The olfactory bulb (OB)
is visible, enveloped by the
connective capsule. Numbers 1
and 2 indicate the same zones in
b. Number 3 indicates the same
zone in e. Dashed line indicates
the dorsal edge of the raphe,
which is visible throughout the
connective capsule. b Ventral
view of the intact rosette. The
olfactory bulb (OB) is visible,
enveloped by the connective
capsule. Numbers 1 and 2
indicate the same zones in
a. Dashed line indicates the
dorsal edge of the raphe, which
is visible throughout the
connective capsule. c Ventral
view of the intact rosette. The
connective capsule ends and the
ventral edges of the lamellae
and raphe are free. d The raphe
is bent dorsoventrally and
anteroposteriorly. This diagram
represents the raphe, without the
lamellae and connective
capsule, from different points of
view. e Anterior view of the
dissected rosette. Number 3
indicates the same zone in
a. f Ventral view of the
dissected rosette. g A single
olfactory lamella. h Scheme of
the lamellar array. ant anterior
part, c canal, ic inlet chamber,
OB olfactory bulb, post
posterior part

the central part of the organ and gradually decrease in size
toward both ends (Fig. 1, 2b). The raphe is bent dorsoven-
trally and anteroposteriorly to follow the rounded shape of
the capsule (Fig. 1d), and, accordingly, the two wings of
each lamella are not symmetrical in shape and size, partic-
ularly at the more peripheral level of the rosette, showing a
larger and a smaller wing (Fig. 1g, 2a). Due to the
organization of the raphe, the inlet chamber reveals a two-
canal arrangement (Fig. 1c, g, 2a). The lamellae are attached
to the connective capsule, and the two free edges have
melanized flaps, which form a sort of imbricated structure,
with a possible implication in water flow regulation (Fig. 1c,
f, g, 2a). Furthermore, lamellae show secondary folding
(Fig. 1g). The central raphe is visible in the intact rosette

123
 Author's personal copy
Polar Biol

Fig. 2 a Drawings of four

lamellae from different
positions along the central raphe
(R) in the olfactory rosette; the
darker zones are melanized
areas. c canal, ic inlet chamber.
b Surface area estimates for the
22 lamellae composing a single
olfactory rosette of the
Greenland shark. For each
lamella, both sides were
considered to estimate the
lamellar surface area

(Fig. 1c), but it is more evident in the dissected one (Fig. 1e,
f). The free edges of the lamellae on opposite sides of the
raphe approach each other but do not meet. Such lamellar
morphology corresponds to the ‘‘type II parallel lamellae
array,’’ according to the classification by Cox (2013).
The olfactory rosette had a volume of 22 cm3, and 22
primary lamellae were counted. The surface area of each
lamella varied according to the size and position along the
raphe (Fig. 2b), giving a total surface area of 117 cm2.
Histology
The olfactory lamellae are lined by a pseudo-stratified,
columnar sensory epithelium of about 80-100 lm
thickness. According to the light microscopy resolution
after the hematoxylin-eosin staining (Fig. 3a–d) and Mas-
son’s trichrome staining (Fig. 3e–f), the sensory neuroep-
ithelium (E) appears to be characterized by basal cells
(BCs), supporting ciliated cells (SCCs), olfactory receptor
cells (ORCs), ionocytes (ICs) and goblet mucous cells
(MCs). The underlying lamina propria contains fila olfac-
toria, collagen fibers, fibroblast-like cells and blood ves-
sels. The BCs comprise the first cellular layer, adjacent to
the basal lamina. They are proliferating cells presenting at
least two different nuclear morphologies, thus suggesting
the presence of two populations of BCs (Fig. 3b, e,f). The
SSCs are the most abundant cell type of the neuroepithe-
lium and are characterized by having oval nuclei in the

123
 Author's personal copy
Polar Biol

Fig. 3 a–d Histological

sections of the Greenland shark
olfactory rosette, hematoxylin-
eosin staining. a Olfactory
lamellae are lined by
pseudostratified olfactory
epithelium. The elongated
nuclei in the upper layer belong
to the ciliated supporting cells.
The round nuclei in the middle
layer belong to olfactory
receptor cells. b Ionocytes are
well visible as large clear cells
with an apical cup (arrow).
Nuclei in the basal layer of the
epithelium belong to the basal
cells. c Mitoses occurring in the
upper layer of the epithelium
(arrow). d In the central raphe
the fila olfactoria are gathered in
large bundles, enclosed by large
cells (arrows) and larger
scattered cells (arrowheads). e–
f Masson’s trichrome. e The
border between the epithelium
and connective tissue is well
evident because of the blue
staining of collagen. BV blood
vessel, CT connective tissue.
f Basal nuclei can be elongated
(arrow) or roundish
(arrowhead). BCs basal cells,
BV blood vessel, CT connective
tissue, E epithelium, FO fila
olfactoria, ICs ionocytes, MCs
mucous cells, ORCs olfactory
receptor cells

most superficial zone. The ICs are large cells extending
from the basal lamina to the epithelium surface; they are
easily recognizable because of the pale cytoplasm and
apical cup morphology. MCs are typical mucous cells, and
their cytoplasm is filled with mucous droplets. The somata
of the olfactory receptor neurons, containing slightly large,
rounded nuclei, are found between the supporting cell and
basal cell layers. Such receptor cells are bipolar, with the
dendritic portion reaching the surface surrounded by
adjacent supporting cells. We found no crypt neurons.
In addition to mitoses occurring in basal cells, dividing
cells are also detected in the upper layer of the epithelium,
indicating at least a different, second population of pro-
liferating cells (Fig. 3c). The fila olfactoria are visible in
the lamellar lamina propria as thin bundles, while in the
central raphe they are gathered in thick bundles (Fig. 3d).
These bundles are surrounded by a cell layer, which
appears continuous, at least at the light microscope reso-
lution. The cells forming this layer are large, with big oval
nuclei instead of typically elongated ones as found in other
chondrichthyan fishes such as small-spotted catshark,
Scyliorhinus canicula, and rabbit fish, Chimaera monstrosa
(Ferrando unpublished data). In the Greenland shark, some
cells of this layer are even larger, double the size of the
others, and their function is unknown (Fig. 3d).
Immunohistochemistry
The immunofluorescence for anti-Gaai3 is detectable in the
olfactory mucosa: it is present in the sensory neurons,
especially in the dendritic part, and in the fila olfactoria in
the lamina propria (Fig. 4a, b). The large bundles of fibers
in the central raphe are also intensely immunofluorescent
(Fig. 4c). The pattern of anti-Gaao immunoreactivity is
similar to that observed for Gaai3, but immunoreactive
neurons appear less numerous and fiber bundles less
intensely fluorescent (Fig. 4d–f). No immunoreactivity is
detected for the anti-Gaaolf (Fig. 4g–i). The omission of
the primary antisera precluded any fluorescence (Fig. 4l–
n).

123
 Author's personal copy
Fig. 4 Histological section of the Greenland shark olfactory rosette,
immunofluorescence (green) and DAPI staining for nuclei (blue).
a Anti-Gaai3 in the olfactory mucosa. In the epithelium the olfactory
receptors neurons show immunofluorescence along the membrane and
especially in the dendritic zone (arrows). In the lamina propria, the
fila olfactoria are immunoreactive. b Same photo of a with DAPI
nuclear staining overlapped. c Anti-Gaai3 and DAPI in the central
raphe; the fila olfactoria are intensely immunoreactive. d Anti-Gaao
in the olfactory mucosa. In the epithelium, the olfactory receptor
neurons show immunofluorescence in the dendritic zone (arrows). In
the lamina propria, blood cells within the blood vessels show
Discussion
Structural organization of the olfactory rosette
and possible implications for ecology and lifestyle
The structure of the olfactory rosettes of the Greenland
shark is overall similar to that of other elasmobranchs (e.g.,
123
Polar Biol
autofluorescence. e Same photo of d with DAPI nuclear staining
overlapped. f Anti-Gaao and DAPI in the central raphe; the fila
olfactoria are immunoreactive. g Anti-Gaaolf in the olfactory
mucosa. No immunofluorescence is detectable. h Same photo of
g with DAPI nuclear staining overlapped. i Anti-Gaaolf and DAPI in
the central raphe; the fila olfactoria are not immunoreactive.
l Omission of the primary antiserum: in the olfactory mucosa; non-
specific fluorescence is not detectable. m Same photo of l with DAPI
nuclear staining overlapped. n Omission of the primary antiserum: in
the central raphe nonspecific fluorescence is not detectable. DAPI
nuclear staining. E epithelium, BV blood vessel, FO fila olfactoria
Takami et al. 1994; Schluessel et al. 2008; Ferrando et al.
2010a) with olfactory lamellae closely packed along a
linear-curvilinear central raphe (Fig. 1b). Linear lamellar
organization occurs widely across neoselachian taxa
(sharks, skates and rays) and differs from the radial
olfactory lamellar array occurring in a few chondrichthyan
and several bony fishes (Cox 2008; Howard et al. 2013).
 Author's personal copy
Polar Biol
As to the fine organization of lamellae, and specifically
how sensory channels are formed by the free edges of the
lamellae, the Greenland shark rosette shows a ‘‘type II’’
lamellar array (sensu Cox 2013), that is, with free opposite
edges of lamellae nearly meeting. This lamellar organiza-
tion is typically found in sedentary shark species (e.g., the
zebra bullhead shark, Heterodontus zebra) and in species
that swim actively but slowly (such as the spinetail mobula,
Mobula japonica) (Cox 2013). The occurrence of type II
lamellar organization in the Greenland shark is therefore
not surprising, being in agreement with the expected slow
swimming speeds and prevalent benthic habit of this spe-
cies (MacNeil et al. 2012; Watanabe et al. 2012).
However, when considering other features possibly
related to lifestyle, such as the number of lamellae,
lamellar surface area and olfactory bulb mass (Kajiura
et al. 2005; Schluessel et al. 2008; Theiss et al. 2009), the
picture becomes complicated.
Each olfactory rosette comprises 22 lamellae and a total
of 44 lamellae per animal. Given that no significant vari-
ation seems to occur between genders and during ontogeny
(Theiss et al. 2009), the number of lamellae may be con-
sidered a species-specific trait. Compared to other elas-
mobranchs, the number of lamellae is relatively low.
Analyses across 16 shark and 5 ray species found that the
total number of lamellae ranged between 58 (in the Eastern
shovelnose ray, Aptychotrema rostrata) and 231 (in the
scalloped hammerhead, Sphyrna lewini), with benthic
species having significantly lower lamellar counts than
bentho-pelagic species (Schluessel et al. 2008). Similar
information on squaliform sharks (the order to which
Greenland sharks belong, Lynghammar et al. 2013) is
scarce, so comparison between taxa should be done cau-
tiously. However, the bentho-pelagic squaliform velvet
belly Etmopterus spinax (one female specimen, 37 cm total
length) (Ferrando, unpublished data) showed a similarly
low total number of lamellae (N = 50). We cannot exclude
that squaliformes may display a lower number of lamellae
compared to other elasmobranchs, but the link between the
lamellar count and ecology of squaliform species is still
uncertain and needs to be investigated further.
The total lamellar surface area of a single rosette of the
Greenland shark, on the other hand, was estimated at
117 cm2, which is high compared to the species studied by
Schluessel et al. (2008). Such a remarkable extent of the
olfactory epithelium might imply a relevant olfactory
potential for the Greenland shark that could also be related
to its ecology. According to Schluessel et al. (2008),
regardless of the body size, benthic species have a lower
sensorial epithelium surface area than bentho-pelagic spe-
cies. If the sensorial epithelium area is a good proxy for the
olfactory potential, the rationale for such a morpho-func-
tional difference would be that benthic animals readily
utilize sensory systems (vision, lateral line reception,
electro-reception) that receive short-range information,
while animals in open waters mostly rely on sensory sys-
tems that detect long distance cues (such as olfaction,
acoustics and magneto-reception). Hence, the relatively
extensive surface area of the olfactory epithelium places
the Greenland shark among the bentho-pelagic sharks
(Fig. 5). This finding is interesting and corresponds to data
from the literature regarding general observations (Coad
and Reist 2004) and migration behavior (Campana et al.
2013). Given the unique ability of the olfactory system to
track a constantly moving world, with selection for
decoding and mapping patterns of odorants, land-marking
and linking locations in olfactory space for navigation
(Jacobs 2012), a good olfactory capability is of great
importance for the Greenland shark, capable of swimming
under the sea-ice cover and performing vertical excursions
(Skomal and Benz 2004). This also nicely meets the nav-
igational demand of the Greenland shark during in-shore/
off-shore movement excursions related to the different
habitat preferences across life stages as well as during
long-distance migrations and pelagic swimming (Campana
et al. 2013).
A comprehensive review of the relative importance of
the various sensory systems assisting the Greenland sharks
during complex activities is beyond the scope of the pre-
sent work, but the potentially high olfactory capability
deduced from the epithelial surface area suggests that this
slow-swimming bentho-pelagic predator greatly relies on
olfaction for long distance cues, but also for foraging, until
in close proximity, similar to what is described for the
night-active and slow-swimming nurse shark Gingly-
mostoma cirratum (Gardiner et al. 2014). Moreover, the
Greenland shark seems to spend most of its life in deep and
Fig. 5 Total surface area of the olfactory epithelium of the Greenland
shark as a function of body length compared to that of other bentho-
pelagic and benthic elasmobranch species (modified from Schluessel
et al. 2008)
123
 Author's personal copy
low-light environments (e.g., Yano et al. 2004, 2007; Fisk
et al. 2012; Nielsen et al. 2014; Campana et al. 2013)
where vision is reduced and olfactory cues would nicely
assist this shark in detecting both living prey and carcasses.
The olfactory epithelium
The general organization of the olfactory mucosa in the
Greenland shark and cell types found in the epithelium are
similar to those described for other elasmobranchs.
Ionocytes, previously described in other species (Fer-
rando et al. 2006a; Ferrando 2008), are here confirmed as a
cell type characteristic of elasmobranchs, possibly involved
in the ionic regulation of the olfactory mucus.
No crypt neurons were observed, although this particular
neuronal cell is present in other elasmobranches (Ferrando
et al. 2006b, 2007) as well as many bony fishes (e.g.,
Hansen and Finger 2000). However, we cannot exclude
their occurrence because the crypt neurons in other fish
species are described as randomly distributed and scattered
within the olfactory epithelium (Hansen and Finger 2000;
Ferrando et al. 2006b; Schmachtenberg 2006; Bettini et al.
2009), and in the present work we could not perform a
detailed analysis of the epithelium of the entire rosette.
Moreover, various papers report that the presence of crypt
neurons may vary ontogenetically and seasonally (Ham-
dani et al. 2008; Camacho et al. 2010; Bettini et al. 2012).
Noteworthily, higher densities of crypt neurons within the
olfactory epithelium were detected in prenatal, larval and
mature stages, with a lower presence in immature fish
(Bettini et al. 2012; Bazàes et al. 2013). Accordingly,
further studies on different life stages of the Greenland
shark are required to assess the presence of this type of
neuronal cell. In general, it would be important to study the
olfactory neurogenesis in this species, which is supposed to
have an exceptionally long life span (MacNeil et al. 2012).
Mitoses in the apical part of the epithelium, never
observed in the olfactory epithelium of bony fishes in
normal conditions, confirms a previous observation in the
small-spotted catshark (Ferrando et al. 2010a). Neverthe-
less, for this species, the apical mitoses present axes
orthogonal to the basal lamina. For the Greenland shark, on
the other hand, the axes were parallel to the basal lamina,
suggesting a possible symmetric cell division. The func-
tional meaning of apical mitoses actually in contact with
the external environment is unknown.
At least one type of the large cell observed along the
olfactory bundles could be ensheathing cells (Barraud et al.
2010), a unique class of glial cells recently considered as a
model in neural regeneration studies in mammals (e.g.,
Yang et al. 2015).
In vertebrates, the receptor neurons express G protein
coupled receptors (GPCRs) in a one-neuron–one-receptor
123
Polar Biol
fashion; these GPCRs can belong to the family of the
olfactory receptors (ORs), the trace amine-associated
receptors (TAARs), vomeronasal receptors 1 (V1Rs) or
vomeronasal receptors 2 (V2Rs) (Mombaerts 2004;
Liberles 2009). Each receptor family is known to be
coupled to a defined G protein alpha subunit; specifi-
cally, ORs and TAARs are coupled to the subunit Gaolf,
V1Rs are coupled to the subunit Gai, and V2Rs are
coupled to Gao (Berghard and Buck 1996; Jia and
Halpern 1996; Hansen et al. 2004; Hansen and Zielinski
2005; Liberles and Buck 2006; Hashiguchi and Nishida
2007). Generally, ORs and TAARs are expressed in
ciliated receptor neurons, while V1Rs and V2Rs are
expressed in microvillous receptor neurons (Hamdani
and Døving 2007).
In the Greenland shark, we did not detect immunofluo-
rescence for Gaolf. This agrees with previous reports on
the absence of sensory cilia in chondrichthyans (Holl 1973;
Theisen et al. 1986; Takami et al. 1994; Ferrando et al.
2009; 2010b; Quintana-Urzainqui et al. 2014) and with the
presence of only two ORs in the genome of the Elephant
shark, the only chondrichthyan genome sequenced to date
(Venkatesh et al. 2014). Instead, the detection of
immunoreactivity for Gao and Gai in virtually all the
receptor neurons supports the presence, also in the
Greenland shark, of microvilli as the main olfactory den-
dritic specialization.
This peculiar condition of chondrichthyans, unique
across vertebrates, has been discussed elsewhere (Ferrando
and Gallus 2013) and could represent a crucial point in the
evolution of chondrichthyans as the top predators in marine
ecosystems.
Concluding remarks
Here the structure of the olfactory rosette of the Greenland
shark is described for the first time. Overall, the lamellar
organization (linear-curvilinear along a central raphe) and
general organization of the olfactory epithelium (presence
of ionocytes and absence of sensory cilia, among others)
conform to the characteristics of elasmobranch olfactory
rosette.
In accordance with the known ecological habits of the
Greenland shark, its olfactory rosette has a lamellar array
(type II sensu Cox 2013), typical of slow-swimming,
prevalently benthic species. On the other hand, based on
the olfactory capability deduced from the total surface area
of the olfactory epithelium, this species resembles bentho-
pelagic rather than strictly benthic sharks. The extent of the
olfactory epithelium in this species suggests a well-devel-
oped olfactory sense, which plays a key role in foraging as
well as long-distance migrations.
 Author's personal copy
Polar Biol
Acknowledgments The Italian National Program of Research in
Antarctica (PNRA) funded this research (Project 2013/AZ.1.11). The
samples were collected during the TUNU-V Expedition in 2013
within the framework of the international TUNU-Programme lead by
the UiT The Arctic University of Norway. The authors are very
grateful to colleagues and crew on board the R/V Helmer Hanssen for
their kind collaboration.
References
Barraud P, Seferiadis AA, Tyson LD, Zwart MF, Szabo-Rogers HL,
Ruhrberg C, Liu KJ, Baker CVH (2010) Neural crest origin of
olfactory ensheathing glia. PNAS 107:21040–21045. doi:10.
1073/pnas.1012248107
Bazàes A, Olivares J, Schmachtenberg O (2013) Properties, projec-
tions, and tuning of teleost olfactory receptor neurons. J Chem
Ecol 39:451–464. doi:10.1007/s10886-013-0268-1
Benz GW, Borucinska JD, Lowry LF, Borucinska JD, Lowry LF,
Whiteley HE et al (2002) Ocular lesions associated with attach-
ment of the copepod Ommatokoita elongata (Lernaeopodidae:
Siphonostomatoida) to corneas of Pacific sleeper sharks Somnio-
sus pacificus captured off Alaska in Prince William Sound.
J Parasitol 88:474–481. doi:10.1645/0022-3395%282002%
29088%5B0474%3AOLAWAO%5D2.0.CO%3B2
Berghard A, Buck LB (1996) Sensory transduction in vomeronasal
neurons: evidence for G alpha o, G alpha i2, and adenylyl
cyclase II as major components of a pheromone signaling
cascade. J Neurosci 16:909–918
Berland B (1961) Copepod Ommatokoita elongata (Grant) in the Eyes
of the Greenland Shark—a Possible Cause of Mutual Depen-
dence. Nature 191:829–830. doi:10.1038/191829a0
Bettini S, Lazzari M, Ciani F, Franceschini V (2009) Immunohisto-
chemical and histochemical characteristics of the olfactory
system of the guppy, Poecilia reticulate (Teleostei, Poecilidae).
Anat Rec 292:1569–1576. doi:10.1002/ar.20944
Bettini S, Lazzari M, Franceschini V (2012) Quantitative analysis of
crypt cell population during postnatal development of the
olfactory organ of the guppy, Poecilia reticulata (Teleostei,
Poecilidae), from birth to sexual maturity. J Exp Biol
215:2711–2715. doi:10.1242/jeb.069039
Bigelow HB, Schroeder WB (1948) Sharks. In: Fishes of the Western
North Atlantic. Part one: Lancelets, Cyclostomes, Sharks.
Memoir Sears Foundation for Marine Research, Yale University,
pp 53–576
Borucinska JD, Benz GW, Whiteley HE (1998) Ocular lesions
associated with attachment of the parasitic copepod Omma-
tokoita elongata (Grant) to corneas of Greenland sharks,
Somniosus microcephalus (Bloch & Schneider). J Fish Dis
21:415–422. doi:10.1046/j.1365-2761.1998.00122.x
Camacho S, Ostos-Garrido DA, Carmona R (2010) Study of the
olfactory epithelium in the developing sturgeon. Characteriza-
tion of the crypt cells. Chem Senses 35:147–156. doi:10.1093/
chemse/bjp091
Campana SE, Fisk AT, Klimley AP (2013) Movements of Arctic and
northwest Atlantic Greenland sharks (Somniosus microcephalus)
monitored with archival satellite pop-up tags suggest long-range
migrations. Biol Deep-Water Chondrichthyans Deep Sea Res
Part II Topical Stud Oceanogr 115:109–115. doi:10.1016/j.dsr2.
2013.11.001
Christiansen JS (2012) The TUNU-programme: Euro-Arctic marine
fishes—diversity and adaptation. Adaptation and evolution in
marine environments, vol 1. Springer, Berlin, pp 35–50
Christiansen JS, Mecklenburg CW, Karamushko OV (2014) Arctic
marine fishes and their fisheries in light of global change. Glob
Change Biol 20(2):352–359. doi:10.1111/gcb.12395
Coad BW, Reist JD (2004) Annotated list of the Arctic marine fishes
of Canada. Fisheries and Oceans Canada, Winnipeg
Compagno LJV (1984) FAO species catalogue. Vol. 4. Sharks of the
world. An annotated and illustrated catalogue of shark species
known to date. Part 1—Hexanchiformes to Lamniformes. Food
& Agriculture Org
Corsolini S, Ancora S, Bianchi N, Mariotti G, Leonzio C, Chris-
tiansen JS (2014) Organotropism of persistent organic pollutants
and heavy metals in the Greenland shark Somniosus micro-
cephalus in NE Greenland. Mar Pollut Bull 87:381–387. doi:10.
1016/j.marpolbul.2014.07.021
Cox JPL (2008) Hydrodynamic aspects of fish olfaction. J R Soc
Interface 5:575–593. doi:10.1098/rsif.2007.1281
Cox JPL (2013) Ciliary function in the olfactory organs of sharks and
rays. Fish Fisheries 14(3):364–390. doi:10.1111/j.1467-2979.
2012.00476.x
Ebert DA, Stehmann MFW (2013) Sharks, batoids and chimaeras of
the North Atlantic. Food and Agriculture Org
Ferrando S (2008) Ionocytes in the olfactory epithelium of developing Raja
clavata. Italian J Zool 75:233–236. doi:10.1080/11250000801934120
Ferrando S, Gallus L (2013) Is the olfactory system of cartilaginous
fishes a vomeronasal system? Front Neuroanat 7:37. doi:10.
3389/fnana.2013.00037
Ferrando S, Bottaro M, Gallus L, Girosi L, Vacchi M, Tagliafierro G
(2006a) Na?/K? ATPase immunoreactivity in olfactory epithe-
lium of small-spotted catshark Scyliorhinus canicula (Linnaeus,
1758): possible presence of ion exchanging cells? J Fish Biol
69:278–282. doi:10.1111/j.1095-8649.01157.X
Ferrando S, Bottaro M, Gallus L, Girosi L, Vacchi M, Tagliafierro G
(2006b) Observations of crypt neuron-like cells in the olfactory
epithelium of a cartilaginous fish. Neurosci Lett 403:280–282.
doi:10.1016/j.neulet.2006.04.056
Ferrando S, Bottaro M, Pedemonte F, De Lorenzo S, Gallus L,
Tagliafierro G (2007) Appearance of crypt neurons in the
olfactory epithelium of the skate Raja clavata during develop-
ment. Anat Rec A Discov Mol Cell Evol Biol 290:1268–1272.
doi:10.1002/ar.20584
Ferrando S, Gambardella C, Ravera S, Bottero S, Ferrando T, Gallus L,
Manno V, Salati AP, Ramoino P, Tagliafierro G (2009)
Immunolocalization of G-protein alpha subunits in the olfactory
system of the cartilaginous fish Scyliorhinus canicula. Anat Rec
Adv Integr Anat Evol Biol 292:1771–1779. doi:10.1002/ar.21003
Ferrando S, Gallus L, Gambardella C, Ghigliotti L, Ravera S,
Vallarino M, Vacchi M, Tagliafierro G (2010a) Cell proliferation
and apoptosis in the olfactory epithelium of the shark Scyliorhi-
nus canicula. J Chem Neuroanat 40:293–300. doi:10.1016/j.
jchemneu.2010.08.004
Ferrando S, Gallus L, Gambardella C, Vacchi M, Tagliafierro G
(2010b) G protein alpha subunits in the olfactory epithelium of
the holocephalan fish Chimaera monstrosa. Neurosci Lett
472:65–67. doi:10.1016/j.neulet.2010.01.059
Fisk AT, Tittlemier SA, Pranschke JL, Norstrom RJ (2002) Using
anthropogenic contaminants and stable isotopes to assess the
feeding ecology of Greenland sharks. Ecology 83:2162–2172.
doi:10.2307/3072048
Fisk AT, Lydersen C, Kovacs KM (2012) Archival pop-off tracking
of Greenland sharks Somniosus microcephalus in the High
Arctic waters of Svalbard, Norway. Mar Ecol Progr Ser
468:255–265. doi:10.3354/meps09962
Gardiner JM, Atema J, Hueter RE, Motta PJ (2014) Multisensory
integration and behavioral plasticity in sharks from different
ecological niches. PLoS One 9(4):e93036. doi:10.1371/journal.
pone.0093036
Hamdani EH, Døving KB (2007) The functional organization of the
fish olfactory system. Prog Neurobiol 82:80–86. doi:10.1016/j.
pneurobio.2007.02.007
123
 Author's personal copy
Hamdani EH, Lastein S, Gregersen F, Døving KB (2008) Seasonal
variations in olfactory sensory neurons—fish sensitivity to sex
pheromones explained? Chem Senses 33:119–123. doi:10.1093/
chemse/bjm072
Hansen A, Finger TE (2000) Phyletic distribution of crypt-type
olfactory receptor neurons in fishes. Brain Behav Evol
55:100–110. doi:10.1159/000006645
Hansen A, Zielinski BS (2005) Diversity in the olfactory epithelium
of bony fishes: development, lamellar arrangement, sensory
neuron cell types and transduction components. J Neurocytol
34:183–208. doi:10.1007/s11068-005-8353-1
Hansen A, Anderson KT, Finger TE (2004) Differential distribution
of olfactory receptor neurons in goldfish: structural and molec-
ular correlates. J Comp Neurol 477:347–359. doi:10.1002/cne.
20202
Hashiguchi Y, Nishida M (2007) Evolution of Trace Amine-
Associated Receptor (TAAR) gene family in vertebrates:
lineage-specific expansion and degradation of vertebrate
chemosensory receptors expressed in the olfactory epithelium.
Mol Biol Evol 24:2099–2107. doi:10.1093/molbev/msm140
Holl A (1973) Feinstruktur des Riechpithels von Chimaera monstrosa
(Holocephali). Mar Biol 23:59–72. doi:10.1007/BF00394112
Howard LE, Holmes WM, Ferrando S, Maclaine JS, Kelsh RN,
Ramsey A, Abel RL, Cox JP (2013) Functional nasal morphol-
ogy of Chimaerid fishes. J Morphol 274:987–1009. doi:10.1002/
jmor.20156
Hussey NE, Kessel ST, Aarestrup K, Cooke SJ, Cowley PD, Fisk AT,
Harcourt RG, Holland KN, Iverson SJ, Kocik JF, Flemming
JEM, Whoriskey FG (2015) Aquatic animal telemetry: a
panoramic window into the underwater world. Science
348(6240):1255642. doi:10.1126/science.1255642
Idrobo CJ, Berkes F (2012) Pangnirtung inuit and the Greenland
shark: co-producing knowledge of a little discussed species.
Hum Ecol 40:405–414. doi:10.1007/s10745-012-9490-7
Jacobs LF (2012) From chemotaxis to the cognitive map: the function of
olfaction. PNAS 109:10693–10700. doi:10.1073/pnas.1201880109
Jia C, Halpern M (1996) Subclasses of vomeronasal receptor neurons:
differential expression of G-proteins (Gi2a and Goa) and
segregated projections to the accessory olfactory bulb. Brain
Res 719:117–128. doi:10.1016/0006-8993(96)00110-2
Jungblut LD, Paz DA, López-Costa JJ, Pozzi AG (2009) Heteroge-
neous distribution of G protein alpha subunits in the main
olfactory and vomeronasal systems of Rhinella (Bufo) arenarum
tadpoles. Zool Sci 26:722–728. doi:10.2108/zsj.26.722
Kajiura SM, Forni JB, Summers AP (2005) Olfactory morphology of
carcharhinid and sphyrnid sharks: Does the cephalofoil confer a
sensory advantage? J Morphol 264:253–263. doi:10.1002/jmor.
10208
Kiraly SJ, Moore JA, Jasinski PH (2003) Deepwater and other sharks
of the U.S. Atlantic Ocean exclusive economic zone. Mar Fish
Rev 65(4):1–63
Kyne PM, Sherrill-Mix SA, Burgess GH (2006) Somniosus micro-
cephalus. The IUCN Red List of Threatened Species. Version
2015.2. www.iucnredlist.org. Downloaded 05 July 2015
Leclerc LM, Lydersen C, Haug T, Glover KA, Fisk AT, Kovacs KM
(2011) Greenland sharks (Somniosus microcephalus) scavenge
offal from minke (Balaenoptera acutorostrata) whaling opera-
tions in Svalbard (Norway). Polar Res 30:7342. doi:10.3402/
polar.v30i0.7342
Leclerc LM, Lydersen C, Haug T, Bachmann L, Fisk AT, Kovacs KM
(2012) A missing piece in the Arctic food web puzzle? Stomach
contents of Greenland sharks sampled in Svalbard, Norway.
Polar Biol 35:1197–1208. doi:10.1007/s00300-012-1166-7
Liberles SD (2009) Trace amine-associated receptors are olfactory
receptors in vertebrates. Ann NY Acad Sci 1170:168–172.
doi:10.1111/j.1749-6632.2009.04014.x
123
Polar Biol
Liberles SD, Buck LB (2006) A second class of chemosensory
receptors in the olfactory epithelium. Nature 442:645–650.
doi:10.1038/nature05066
Lynghammar A, Christiansen JS, Mecklenburg CW, Karamushko
OV, Møller PR, Gallucci VF (2013) Species richness and
distribution of chondrichthyan fishes in the Arctic Ocean and
adjacent seas. Biodiversity 14(1):57–66. doi:10.1080/14888386.
2012.706198
MacNeil MA, McMeans BC, Hussey NE, Vecsei P, Svavarsson J,
Kovacs KM, Lydersen C, Treble MA, Skomal GB, Ramsey M,
Fisk AT (2012) Biology of the Greenland shark Somniosus
microcephalus. J Fish Biol 80:991–1018. doi:10.1111/j.1095-
8649.2012.03257
McMeans BC, Arts MT, Lydersen C, Kovacs KM, Hop H, Petersen
SF, Fisk AT (2013) The role of Greenland sharks (Somniosus
microcephalus) in an Arctic ecosystem: assessed via stable iso-
topes and fatty acids. Mar Biol 160:1223–1238. doi:10.1007/
s00227-013-2174-z
Meng Q, Yin M (1981a) A study of the olfactory organ of the shark.
Trans Chin Ichthyol Soc 2:1–24
Meng Q, Yin M (1981b) A study on the olfactory organ of skates, rays
and chimaeras. J Fisheries China 5(3):209–228
Meredith TL, Kajiura SM (2010) Olfactory morphology and phys-
iology of elasmobranchs. J Exp Biol 213:3449–3456. doi:10.
1242/jeb.045849
Mombaerts P (2004) Genes and ligands for odorant, vomeronasal and
taste receptors. Nat Rev Neurosci 5:263–278. doi:10.1038/
nrn1365
Nielsen J, Hedeholm RB, Simon M, Steffensen JF (2014) Distribution
and feeding ecology of the Greenland shark (Somniosus
microcephalus) in Greenland waters. Polar Biol 37:37–46.
doi:10.1007/s00300-013-1408-3
Quintana-Urzainqui I, Rodrı́guez-Moldes I, Candal E (2014) Devel-
opmental, tract-tracing and immunohistochemical study of the
peripheral olfactory system in a basal vertebrate: insights on
Pax6 neurons migrating along the olfactory nerve. Brain Struct
Funct 219:85–104. doi:10.1007/s00429-012-0486-2
Rasband WS (1997–2014) ImageJ, U.S. National Institutes of Health,
Bethesda, Maryland, USA. http://imagej.nih.gov/ij/
Schluessel V, Bennet MB, Bleckmann H, Blomberg S, Collin SP
(2008) Morphometric and ultrastructural comparison of the
olfactory sensory system in elasmobranchs: the significance of
structure-function relationships based on phylogeny and ecology.
J Morphol 269:1365–1386. doi:10.1002/jmor.10661
Schmachtenberg O (2006) Histological and electrophysiological
properties of crypt cells from the olfactory epithelium of the
marine teleost Trachurus symmetricus. J Comp Neurol
495:113–121. doi:10.1002/cne.20847
Skomal GB, Benz GW (2004) Ultrasonic tracking of Greenland
sharks, Somniosus microcephalus under Arctic ice. Mar Biol
145:489–498. doi:10.1007/s00227-004-1332-8
Takami S, Luer CA, Graziadei PPC (1994) Microscopic structure of
the olfactory organ of the clearnose skate, Raja eglanteria. Anat
Embryol 190:211–230. doi:10.1007/BF00234300
Theisen B, Zeiske E, Breuker H (1986) Functional morphology of the
olfactory organs in the spiny dogfih (Squalus acanthias L.) and
the small-spotted catshark (Scyliorhinus canicula L.). Acta Zool
67:73–86. doi:10.1111/j.1463-6395.1986.tb00851.x
Theiss SM, Hart NS, Collin SP (2009) Morphological indicators of
olfactory capability in Wobbegong sharks (Orectolobidae, Elas-
mobranchii). Brain Behav Evol 73:91–101. doi:10.1159/
000209865
Venkatesh B, Lee AP, Ravi V, Maurya AK, Lian MM, Swann JB,
Ohta Y, Flajnik MF, Sutoh Y, Kasahara M et al (2014) Elephant
shark genome provides unique insights into gnathostome evo-
lution. Nature 505:174–179. doi:10.1038/nature12826
 Author's personal copy
Polar Biol
Wakabayashi Y, Ichikawa M (2008) Localization of G protein alpha
subunits and morphology of receptor neurons in olfactory and
vomeronasal epithelia in Reeve’s turtle, Geoclemys reevesii.
Zool Sci 25:178–187. doi:10.2108/zsj.25.178
Watanabe YY, Lydersen C, Fisk AT, Kovacs KM (2012) The slowest
fish: swim speed and tail-beat frequency of Greenland sharks.
J Exp Mar Biol Ecol 426–427:5–11. doi:10.1016/j.jembe.2012.
04.021
Yang H, He B-R, Hao D-J (2015) Biological roles of olfactory
ensheathing cell in facilitating neural regeneration: a systematic
review. Mol Neurobiol 51:168–179. doi:10.1007/s12035-014-
8664-2
Yano K, Stevens JD, Compagno LJV (2004) A review of the
systematics of the sleeper shark genus Somniosus with
redescriptions of Somniosus (Somniosus) antarcticus and Som-
niosus (Rhinoscymnus) longus (Squaliformes: Somniosidae).
Ichthyol Res 51:360–373. doi:10.1007/s10228-004-0244-4
Yano K, Stevens JD, Compagno LJV (2007) Distribution, reproduc-
tion and feeding of the Greenland shark Somniosus (Somniosus)
microcephalus, with notes on two other sleeper sharks, Somnio-
sus (Somniosus) pacificus and Somniosus (Somniosus) antarcti-
cus. J Fish Biol 70:374–390. doi:10.1111/j.1095-8649.2007.
01308.x
123