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R E S E A R CH A R T I C L E
Whale tear glands in the bowhead and the beluga whales:Source and function
Susan J. Rehorek1 | Rapahela Stimmelmayr2,3 | John C. George2,4 |
Robert Suydam2,4 | Denise M. McBurney4 | JGM Thewissen4
1Department of Biology, Slippery Rock
University, Slippery Rock, Pennsylvania
2Department of Wildlife Management, North
Slope Borough, Barrow, Alaska
3Institute of Artic Biology, University of
Alaska, Fairbanks, Alaska
4Department of Anatomy and Neurobiology,
Northeast Ohio Medical University,
Rootstown, Ohio
Correspondence
Susan J. Rehorek, Department of Biology,
Slippery Rock University, Slippery Rock, PA
16057-1326.
Email: susan.rehorek@sru.edu
Funding information
North Slope Borough; Coastal Impact
Assistance Program, Fish and Wildlife Service,
U.S. Department of the Interior
Abstract
Orbital glands are found in many tetrapod vertebrates, and are usually separate struc-
tures, consisting of individual glands lying in the eyelids and both canthi of the orbit.
In cetaceans, however, the orbital glandular units are less distinct and have been
described by numerous authors as a single, periorbital mass. There are few histo-
chemical and immunhistochemical studies to date of these structures. In this study,
we examined the orbital glandular region of both the bowhead whale (Balaena
mysticetus: Mysticeti) and the beluga whale (Delphinapterus leucas: Odontoceti) using
histological, histochemical, and immunohistochemical techniques. Histologically, in
the bowhead, three glandular areas were noted (circumorbital, including Harderian
and lacrimal poles), palpebral (midway in the lower eyelid), and rim (near the edge of
the eyelid). In the beluga, there was only a large, continuous mass within the eyelid
itself. Histochemical investigation suggests neither sexual dimorphism nor age-
related differences, but both whales had two cell types freely intermingling with each
other in all glandular masses. Large cells (cell type 1) were distended by four histo-
chemically distinct intracellular secretory granules. Smaller cells (cell type 2) were not
distended (fewer granules) and had two to three histochemically distinct intracellular
secretory granules. The beluga orbital glands had additional lipid granules in cell type
1. Counterintuitively, both lipocalin and transferrin were localized to cell type 2 only.
This intermingling of cell types is unusual for vertebrates in whom individual orbital
glands usually have one cell type with one to two different secretory granules pre-
sent. The heterogeneity of the orbital fluid produced by cetacean orbital glands
implies a complex function, or series of functions, for these orbital glands and their
role in producing the tear fluid.
K E YWORD S
glands, lipocalin, orbital whales
1 | INTRODUCTION
Orbital glands, the source of tear fluid, are present in most tetrapod
vertebrates. They occur within three orbital regions: anterior
(Harderian gland), posterior (lacrimal gland), and within the eyelids
(palpebral and rim glands) (Rehorek, Hillenius, Lovano, & Thewissen,
2018). Up to three orbital glands have been described within Cetacea.
However, anatomical characterization of these structures is difficult,
as identification and characterization of these glands differs between
authors. This creates an impression of morphological diversity that
may not be accurate, and makes evolutionary comparison to terres-
trial mammals daunting (see Rehorek et al., 2018). For example, in
Received: 28 October 2019 Revised: 19 December 2019 Accepted: 20 December 2019
DOI: 10.1002/jmor.21099
Journal of Morphology. 2020;1–10. wileyonlinelibrary.com/journal/jmor © 2020 Wiley Periodicals, Inc. 1
several cetaceans, the anterior and posterior (when present) regions
are connected by a series of small intervening (conjunctival) glands,
resulting in the formation of a singular orbital gland. The palpebral
glands, however, are usually described as a singular glandular mass,
and are not separated into multiple smaller glands as described in ter-
restrial mammals (see Rehorek et al., 2018 for review). Histochemi-
cally, these glands in cetaceans are described as homogeneous and
mucoserous (Funasaka, Yoshioka, & Fujise, 2010; Tarpley & Ridgway,
1991; Waller & Harrison, 1978). Lipid granules have been described in
the female Harderian gland of the dolphin (Tursiops truncatus:
Bodyak & Stepanova, 1994) and is one of the few mammalian
instances of sexual dimorphism in the Harderian gland outside of
rodents (Payne, 1994). It is these orbital glands that are the source of
tear fluid, the fluid that washes over the eyeball in land mammals.
Biochemically, tear fluid consists of three components in terres-
trial mammals. These occur in distinct layers across the eye: an outer
lipid layer, a middle aqueous (proteinaceous) layer, and an innermost
mucus layer (King-Smith, Fink, Hill, Koelling, & Tiffany, 2004; Mishima,
1965; Tiffany, 1994). There is variation in these layers among aquatic
mammals. The outermost lipid layer is either missing (pinnipeds; Davis
et al., 2013; Uskova, Momot, & Surkina, 1976) or small (cetaceans:
Young & Dawson, 1992). There have been several biochemical studies
examining the precise compounds in the aqueous layer of the mam-
malian tear fluid, two of the most commonly reported proteinaceous
substances are tear-specific lipocalin and transferrin (see Table 1 for
summary). Several other proteinaceous substances were observed,
including lysozymes (Bright & Tighe, 1993; Gionfriddo et al., 2000;
Shamsi et al., 2011; Thörig, Van Agtmaal, Glasius, Tan, & Van
Haeringen, 1985; Young & Dawson, 1992), lactoferrin (Bright & Tighe,
1993; Brown, Brightman, Fenwick, & Rider, 1996; Gionfriddo et al.,
2000; Shamsi et al., 2011; Young & Dawson, 1992), lipophilin
(Azzarolo et al., 2004; Shamsi et al., 2011), and serum albumin
(Young & Dawson, 1992; Bright & Tighe, 1993; Gionfriddo
et al., 2000).
Tear lipocalin is a multifunctional protein. The functions ascribed
to this protein include: scavenging lipophilic substances (Redl, 2000),
stabilizing lipids in tear film (Redl, 2000; Tiffany, 2008; Glasgow &
Gasymov, 2011), and as a carrier for hormones, enzymes and endo-
nucelases (Glasgow & Gasymov, 2011). Both tear specific lipocalin
and transferrin are also metal chelating proteins, with a high affinity
for iron. Thus, they may work in a synergist manner with lysozymes as
broad bactericidal agents (Azzarolo et al., 2004; Gionfriddo et al.,
2000; Young & Dawson, 1992).
To date, there are few published studies conclusively linking tear
proteins immunohistochemically to their source cells in the orbital
glands (Franklin, Kenyon, & Tomasi, 1973; Gillette & Allansmith, 1980;
Inoue et al., 1993; Nguyen, Beuerman, Halbert, Qiangwei, & Sun,
1999; Pinard, Weiss, Brightman, Fenwick, & Davidson, 2003;
Stoeckelhuber et al., 2006; Vigneswaran, Wilk, Heese, Hornstein, &
Naumann, 1990). Most research suggests that the secretions originate
from a nearby gland (e.g., Dilly, 1994; Brown et al., 1996; Gionfriddo
et al., 2000; Shamsi et al., 2011) with few studies providing supporting
biochemical analysis of the glands (e.g., Azzarolo et al., 2004; Tiwari,
Ali, & Vemuganti, 2014).
The purpose of this study was twofold: (a) to characterize the
gross anatomy of the components of the ocular glandular complex,
consistent with the orbital gland anatomy as described in terrestrial
species, of two cetaceans, the beluga whale (Delphinapterus leucas:
Odontoceti, Monodontidae) and the bowhead whale (Balaena
mysticetus: Mysticeti, Balaenidae). (b) To subsequently perform a
detailed microanatomical, histochemical, and immunohistochemical
description of the orbital glands to elucidate their function.
2 | MATERIALS AND METHODS
2.1 | Source of animals
These observations are based on seven bowhead whales, Balaena
mysticetus Linnaeus, 1758, captured 2016–2018. In the fall
(September/October), the bowhead whales migrate from the Beaufort
and Chukchi Sea to the Bering Sea as the ice closes off the Artic
(George et al., 2016). A small number of migrating bowhead whales
are harvested by native Alaskans (Inupiat) during this seasonal migra-
tion in Utqiagvik (Barrow, Alaska). This harvest is regulated by the
AEWC (Alaska Eskimo Whaling Commission), permitted under the
U.S. Endangered Species Act and approved by the International Whaling
Commission. Samples of whales are collected by the Department of
Wildlife Management, North Slope Borough, under NOAA-NMFS per-
mits, and these specimens form the basis of our study (Table 2).
The beluga, Delphinapterus leucas (Pallas, 1776), used in this study
were captured over the summer of 2017 (Table 2) in northern Alaska.
The belugas from the population that spend the summer season in the
eastern Chukchi Sea (O'Corry-Crowe et al., 2018) are harvested by
subsistence hunters from the village of Point Lay on the Chukchi Sea
TABLE 1 Summary of biochemical studies of tear fluidproteinaceous components (specifically only lipocalin and transferrin)in mammals
Secretion Attributed glandSourceanimal Citation
Lipocalin
(a.k.a. tear
specific
prealbumin)
Lacrimal gland Human Bright & Tighe,
1993; Redl, 2000
Glands of Krause
and Wolfring
Camel Shamsi et al., 2011
Unstated Sheep Shamsi et al., 2011
Unstated Rabbit Azzarolo et al.,
2004
Dolphin Young & Dawson,
1992
Transferrin Unstated Rabbit Azzarolo et al.,
2004
Unstated Llama Gionfriddo et al.,
2000
Unstated Cattle Gionfriddo et al.,
2000
2 REHOREK ET AL.
and samples are collected under a NOAA-NMFS permit. This harvest
is regulated by the Alaska Beluga Whale Committee, and permitted
under the U.S. Marine Mammal Protection Act.
2.2 | Histology, histochemistry, andimmunohistochemistry
Histological, histochemical, and immunohistochemical studies were
carried out on glandular regions, with swatches (1 × 1 × 1 cm)
harvested from the anterior (Harderian), posterior (lacrimal), palpebral,
and rim regions (see Table 2 for technique distribution).
Tissue swatches were preserved in 10% phosphate-buffered for-
malin and processed routinely for paraffin histology and sectioned at
7 μm. Several of these slides per specimen were then stained with
hematoxylin and eosin (HE), or Mason's trichrome (MT), or periodic
acid–Schiff (PAS; to visualize carbohydrates), or PAS with amylase (as a
negative control for glycogen), or Alcian blue (AB; pH 1.0 and pH 2.5,
to stain for acidic mucosubstances) (Drury &Wallington, 1980).
A subset of specimens was placed for a week in a 10% sucrose
solution (to prevent tissue from crystallizing) prior to sectioning on a
cryostat (Leica CM 185) at 16 μm increments. Frozen sections were
only stained with oil red O (ORO; to visualize hydrophobic lipids and
some hydrophilic lipids: Kiernan, 2015) using a method adapted from
Lillie (1954).
We determined the location of tear specific lipocalin-1 (H-8)
(Santa Cruz: sc-374,620) and transferrin (D-9) (Santa Cruz: sc-
375,871) using unstained paraffin sections of the orbital glands of
both species of whale. After sections were rehydrated, the slides
underwent heat-induced antigen retrieval using 0.01 mol/L sodium
citrate pH 6.0 to reverse the cross-linking between proteins in the tis-
sue and formalin. This was carried out for 10 min at 85–90�C, with a
further 20 min incubation (at room temperature). To prevent enzyme
activity naturally present in tissue from reacting with the DAB (dia-
minobenzidine), an additional step of 1% hydrogen peroxide was
applied to the slides for 5 min. To detect the proteins of interest, we
used an Immunoperoxidase Secondary Detection System Kit (Millipore:
DAB 150) and followed the accompanying protocol. Antibodies were
diluted in a concentration of 1:50 PBS. The sections were then coun-
terstained with 0.01% thionine (in 25% ethyl alcohol) for up to 1 min.
Slides were then dehydrated, cleared, and mounted in electron micros-
copy sciences mounting medium.
Positive staining was determined by the presence of DAB within
the tissue. To ensure positive staining was not exogenous, we used
two control methods. The first was a positive control for tissues
where lipocalin (snake Harderian gland: Steglich, Brown, Mitchell,
Millen, & Rehorek, 2019), and transferrin (cow anterior orbital gland:
Gionfriddo et al., 2000) are readily present. The second was a negative
control that performed the kit protocol without adding the primary
antibody to the control sections.
3 | RESULTS
Species-specific and regional (anatomical, microanatomical, and histo-
chemical) variation was observed in this ocular glandular region. How-
ever, glandular regions showed no evidence of sexual dimorphism or
any difference between juvenile and adult whales.
3.1 | Anatomy and histology
Orbital glands were present in both species, but they were distributed
differently. In the bowhead whale, there were three regions of orbital
glandular tissue (Figure 1a,b). The first was the largest, with its glandu-
lar tissue distributed circumorbitally in a semi-continuous mass at the
intersection between the eyelid and the eyeball. Two large masses of
this region: anterior (Harderian pole) and posterior (lacrimal pole) mass
extended beyond the conjunctiva and into the adjacent subcutaneous
adipose tissue. Dissection was difficult at this stage, and thus no
TABLE 2 North Slope Borough, Department of Wildlife Management (NSB_DWM) specimens used in this study
Species Specimen number Year Season collected Sex Total body length (m) Age (years) Techniques
Bowhead 2016B16 2016 Fall Male 9.2 13 HC, IC
2016B14 2016 Fall Female 9.72 6 HC, IC
2017B1 2017 Spring Female 8.94 1.2 IC
2017B9 2017 Fall Male 9.42 7 HC
2017B11 2017 Spring Male 10.4 20 HC
2018B1 2018 Spring Female 8.94 1.2 IC
2018WW2 2018 Spring Male 4.19 0 (full term) IC
Beluga 2017LDL1 2017 Summer Female 3.40 >8 HC, IC
2017LDL2 2017 Summer Male 3.34 >8 HC, IC
2017LDL12 2017 Summer Male 2.56 <5 HC, IC
2017LDL26 2017 Summer Male 3.64 >8 HC, IC
Note: Estimated age determination via baleen length in bowhead, in the form of incremental baleen growth (as per Lubetkin, Zeh, Rosa, & George, 2008;
Lubetkin, Zeh, & George, 2013). Beluga age was estimated by converting length to age (Suydam, 2009).
REHOREK ET AL. 3
measurements were made. Smaller intervening glandular masses could
not be found gross-anatomically. The second region included glands
within the eyelid (palpebral). These glandular masses occur in a con-
densed area in the lower eyelid and can occasionally be palpated.
Large ducts were identified upon gross dissection. The third region
with glands is at the rim of the eyelid. Diffuse glands occur throughout
the rim, but could not be discerned grossly.
In contrast, the beluga whale had only one glandular mass. It fills, but
did not extend beyond, the conjunctival region of the eyelid (Figure 1c,d).
The extent of this glandular mass was difficult to determine gross-
anatomically and there did not seem to be any regional enlargements.
3.2 | Histochemistry
All orbital glands in both species, the bowhead and the beluga, pos-
sessed two secretory cell types. Firstly, there were the large cells (cell
type 1, lg in Figure 2a), which were distended with numerous secre-
tory granules. Secondly, there were the smaller cells (cell type 2)
which were not distended with secretory granules (sm in Figure 2a). In
the bowhead, there was additional regional histological variation. The
large, circumorbital glandular region, especially in the expanded
Harderian and lacrimal regions, contained many Pacinian corpuscles
(pc in Figure 2b) in the stroma. The palpebral glands consisted mainly
of cell type 1, with large ducts lined by nonsecretory simple cuboidal
cells (dc in Figure 2c). The rim glands were diffuse with both cell types
present in equal amounts and no Pacinian corpuscles (Figure 2d). In
the beluga, the two cells types are also more equally distributed
(Figure 2e) and similar specialized duct cells were observed (Figure 2f).
The histochemical results are summarized in Table 3. Using paraf-
fin histochemical techniques, the orbital glands of both whale species
showed the same histochemical profile. Four different secretory gran-
ules could be identified in cell type 1 of the whale orbital glands
(Figure 3a): (1) Glycolipid (PAS ++: pink granules), (2–3) glycoproteins
TABLE 3 All orbital glands have thesame two cells types (cell type 1 = largecell; cell type 2 = smaller cell)Chemical staineda
PAS PAS/amylase AB pH 1.0 PAS/AB pH 2.5 ORO
CH Glycogen Acidic MS Glycoprotein Lipid
Cell type 1
Bowhead
++ − ++ +++ −
Cell type 1
Beluga
++ − ++ +++ +
Cell type 2 ++ − +/− +/− −
Duct − − − − −
Note: Histochemical observations are typically shown in such a tabular manner.
Abbreviations: AB, Alcian blue; CH, carbohydrate; MS, mucosubstance; ORO, oil red O; PAS, periodic
acid–Schiff.aAs defined by Drury & Wallington, 1980.
F IGURE 1 Schematics of thebowhead (a, b) and the beluga (c, d)whale orbital glands. These are lateral(a, c) and schematic mid-sagittal (b, d)views of the left eye. Three sets ofglands in the bowhead whale includethe circumorbital glands, at least onesolid glandular mass in the middle ofthe eyelid (palpebral gland) and a ringof diffuse rim glands. The belugawhale has only one glandular mass(palpebral glands) which resideswholly within the eyelid
4 REHOREK ET AL.
(PAS/AB2.5 +++: both purple and indigo granules) and (4) Sulphated
acid mucins (AB1.0 ++; light blue granules). Cell type 2 contained only
the glycolipid (PAS ++: pink granules), with occasionally some glyco-
protein (PAS/AB2.5 +/−: both purple and indigo granules).
Cryohistochemistry showed that there is a difference between
these whale species. No lipid was found in the orbital glands of any of
the bowhead orbital glands (Figure 3b). Some secretory granules in
cell type 1 of the beluga orbital gland stained with ORO (Figure 3c) of
both sexes.
3.3 | Immunohistochemistry
Similar results were observed in both species of cetaceans. The secre-
tory granules of cell type 2 reacted positively to the lipocalin anti-
bodies of all glands (Figure 3d) as did the duct lining cells associated
with the cell type 2 (Figure 3e). Cell type 2 secretory granules also
reacted positively to the transferrin antibodies in all glands (Figure 3f).
Neither the secretory granules of cell 1 nor the nonsecretory duct
lining cells of any glands in either species stained for lipocalin or trans-
ferrin antibodies.
4 | DISCUSSION
Cetaceans are replete with orbital glands, which seem to encompass
the entire orbital region. They differ from other mammals in glandular
identity (which affects nomenclature and inferences about phylogeny)
and glandular structure (which affects inferences about composition
and function).
4.1 | Gland identity
There is much disparity in the literature regarding cetacean orbital
gland nomenclature and distribution (see Rehorek et al., 2018 for
review). Some published reports refer to a singular large, circumorbital
gland as the orbital (Tarpley & Ridgway, 1991) or the Harderian
F IGURE 2 Histochemicallystained whale orbital glands. In bothspecies, there are two secretory celltypes, large and small. Three differentglandular regions were sampled in thebowhead, each with a slightlydifferent ratio of small to large cells.The three regions included thecircumorbital at the junction of the
eyelid and the eyeball, which includesthe lacrimal portion (a and b: NSB-DWM 2016B16), and palpebral in themiddle of the eyelid (c: NSB-DWM2016B14) and subconjunctival at therim of the eyelid (d: NSB-DWM2016B14) regions. Beluga whaleeyelid (e: NSB-DWM 2017LDL1)showed one large gland in the bodyof the eyelid. Ducts could be clearlyseen as non-secretory structures (f:NSB-DWM 2017LDL26) moreuniformity. Abbreviations: dc, ductcells; dl, lumen; lg, large cells (Type 1);pc, Pacinian corpuscles; sc, secretorycells; sm, small cells (Type 2). Stains:(a,c-f) PAS/AB 2.5, (b) Mason'strichrome
REHOREK ET AL. 5
(Bodyak & Stepanova, 1994; Funasaka et al., 2010; Ortiz et al., 2007)
gland. Other authors identify groups of two to three individual glands
that include the Harderian, lacrimal, and intervening conjunctival
glands (Pütter, 1903; Rodrigues et al., 2015; Waller & Harrison, 1978;
Weber, 1886). Yet, other studies mention glands in the eyelid (Pütter,
1903; Rehorek et al., 2018), referring to them as palpebral glands
(Stenella attenuata: Delphinidae: Rehorek et al., 2018) or “unnamed
eyelid glands” (Hyperoodon rostratus Ziphiidae, Delphinapterus leucas
Monodontidae, Phocoena communis Phocoenidae, Delphinus delphis
Delphinidae, Balaenoptera ostrata Balaenopteridae: Pütter, 1903).
Thus, when it comes to orbital glandular identity in cetaceans there is
much variation. This could be indicative of much variation in orbital
gland distribution within cetacean or it could merely reflect different
sampling techniques. Further comparative analysis is required.
In mammals, the many orbital glands are found in three regions:
anterior, posterior and within the eyelid (palpebral) itself. Two sets of
palpebral glands are found in mammals (see Rehorek et al., 2018 for
review). First there are the rim glands. These are modified sweat
glands (glands of Möll) or modified sebaceous glands (glands of Zeis
and Meibomian glands) (Gartner, 2017). Bowhead rim glands lack simi-
larities with either type as they do not have the features of a modified
sweat gland (coiled tubular gland) or those of modified sebaceous
gland (holocrine secretory mechanism). Instead, the bowhead rim
glands are similar in structure to all the other orbital glands.
The other set of glands reside within the body of the eyelid.
These are the palpebral glands. There are two types of palpebral
glands, which are distinguished from each other based on relative
location in the eyelid, the more lateral (peripheral) glands of Wolfring
and the more medial (ocular) glands of Krause (Rehorek et al., 2018).
These palpebral glands are histologically similar to the lacrimal gland
(Conrady, Joos, & Patel, 2016) and bear similarities to whale eyelid
glands. The bowhead would thus have both a series of Wolfring
glands near the rim and a series of deeper, more compact glands of
Krause. The single mass of eyelid glands in the beluga makes identifi-
cation of the palpebral glands more difficult, although it may be
resolved with embryological observations.
In balaenopterid baleen whales, the Harderian gland is large, the
lacrimal gland absent, and some small conjunctival glands do occur
F IGURE 3 Histochemical andimmunohistochemical sections ofwhale orbital glands. Highermagnification of secretory cells inbowhead palpebral gland, indicatingthe four secretory granules that couldbe identified with the stain (nos 1–4)(a: NSB-DWM 2016B14).Cryosections of bowhead palpebral
gland (b: NSB-DWM 2016B14)Beluga orbital gland (c: NSB-DWM2017LDL2) showing lipid (stainedorange/red) only found in the orbitalgland of both sexes of Beluga whale.These secretions were limited to thelarger cells. Immunohistochemicalstaining for lipocalin (brown) the bothbowhead lacrimal (d: NSB-DWM2016B16) and palpebral (e: NSB-DWM 2016b16) glands andtransferrin (brown) lacrimal gland (f:NSB-DWM 2016B16) was restrictedto the smaller cells and part of theductal lining in the palpebral gland.Abbreviations: dc, duct cells; dl,lumen; lg, large cells (Type 1); sc, smallcells (Type 2). Stains: (a) PAS/AB 2.5,(b, c) oil red O, (d, e) lipocalin and (f)transferrin
6 REHOREK ET AL.
(Weber, 1886; Pütter, 1903; Funasaka et al., 2010; Rodrigues et al.,
2015). In contrast, our bowhead evidence suggests that lacrimal
glands are present in balaenids. However, we caution against ascribing
great importance to such a difference for taxonomic purposes. It is
possible that cursory, gross-anatomical investigation is insufficient to
recognize smaller glandular masses in balaenopterids.
In contrast, some small dolphins (Odontoceti) appear to possess a
lacrimal gland (Bodyak & Stepanova, 1994; Ortiz et al., 2007; Pütter,
1903; Tarpley & Ridgway, 1991; Waller & Harrison, 1978; Weber,
1886) and palpebral glands as fetuses (Rehorek et al., 2018). The only
other description of the beluga is for a fetus, and indicates a singular
glandular mass with temporal (lacrimal) and nasal (Harderian) enlarge-
ments and small intervening (conjunctival) glands (Pütter, 1903). The
absence of the circumorbital gland in the adult beluga, as described in
this study, could be age-related or could be caused by difficulty of dis-
tinguishing glandular and adipose tissue. Nevertheless, it appears that
the amount of orbital glandular material in cetaceans, like the
bowhead and the beluga whales, may have been underestimated. The
cetacean orbital region is replete with glandular material.
4.2 | Gland structure
Histologically, the orbital glands of cetaceans are poorly described in
the literature. The presence of a distinct non-secretory duct system
has been previously described in Tursiops truncatus (Tarpley &
Ridgway, 1991) and three species of balaenopterid whales (common
minke whale [Balaenoptera acutotostrata], sei whale [Balaenoptera
borealis], and Bryde's whale [Balaenoptera edeni]: Funasaka et al.,
2010). This duct system is restricted to the larger glandular masses
(lacrimal, Harderian, and palpebral glands) and not the smaller, inter-
vening diffuse glandular mass. Thus, the presence of these ducts may
simply be a function of glandular size.
Funasaka et al. (2010) showed that balaenopterid orbital glands
possess secretory cells that are positive for both acidic and neutral
glycosaminoglycans, although other cell types were not specified.
Other descriptions identify multiple histochemically (Tarpley &
Ridgway, 1991) or ultrastructurally (Bodyak & Stepanova, 1994; Ortiz
et al., 2007; Waller & Harrison, 1978) characterized cell types, but do
not identify specific anatomical sites within the glandular mass. Our
study identified two histologically and histochemically distinct cell
types in the bowhead and the beluga (large cell type 1 and small cell
type 2). These appear to be largely separated in distinct lobules. These
cells (in our study) may correspond to the secretory cells of the male
dolphin Tursiops truncatus by Bodyak and Stepanova (1994). Type
1 cells of these authors are characterized by many ultrastructurally
different types of secretory granules and could be the large cell type
1 identified by us, as characterized by histochemically different types
of secretory granules. Cell type 2 of Bodyak and Stepanova (1994)
could be the smaller cell type 2, with only one type of secretory
granule.
Lipid granules have thus far only been described in female Type
1 Harderian gland secretory cells of Tursiops truncatus (Bodyak &
Stepanova, 1994). We observed lipid in the beluga, but there was no
evidence of sexual dimorphism. The absence of lipoidal secretions in
balaenopterid (Funasaka et al., 2010) and balaenid (this study) whales
suggests that lipoidal orbital gland secretions may be characteristic of
delphinoids or odontocetes. To firmly establish this, further compara-
tive studies are needed.
The histochemical nature of the two secretory cells types in the
bowhead and the beluga, large (cell type 1) and small (cell type 2),
could be taken to suggest that large cells are highly secretory and that
smaller cells are quiescent. Immunohistochemical observations indi-
cate otherwise, with the presence of tear-specific lipocalin and trans-
ferrin in the smaller cell type 2. These are both proteins (Azzarolo
et al., 2004; Young & Dawson, 1992), with no glycol or lipid compo-
nents (as defines a glycolipid: Drury & Wallington, 1980), and thus
cannot account for the PAS ++ stain seen in these cells. The neutral
glycoprotein (PAS/AB2.5 ++, responsible for the purple and indigo
granules) was not present in Type 2 cells, and thus could not account
for the lipocalin and transferrin. Furthermore, biochemical analysis of
the tear film of Tursiops truncatus (Young & Dawson, 1992) showed
lipocalin, but no transferrin. The precise role of these chemicals in tear
fluid is unknown, but it has been speculated that these are bactericidal
proteins, with a strong affinity for iron, thereby inhibiting bacterial
growth by competing for the iron (Gionfriddo et al., 2000). Addition-
ally, tear specific lipocalin and transferrin may work in combination
with lysozymes as a broad bactericidal agent (Young & Dawson,
1992). Thus, the presence of these proteins, in the smaller cells, indi-
cates that the cetacean orbital glands are continuously producing a
complex, bactericidal rich fluid.
Nevertheless, the secretions produced by cetacean orbital glands
individual cells are not homogeneous, and are instead a mixture of
components including serous (PAS/AB +++), mucous (AB ++), lipoidal
(ORO+, PAS ++), tear-specific lipocalin, and transferrin. These secre-
tions are produced by two intermingling cells throughout the entire
region. This is far more complex than the orbital gland secretions in
most vertebrates, in whom the individual glands are relatively homo-
geneous. Harderian glands usually produce one or two different
species-specific and histochemically distinct secretory substances,
including mucoserous (mostly reptiles and amphibians: Chieffi et al.,
1992; Rehorek, 1997), serolipoidal (many mammals and gecko lizards:
Payne, 1994; Rehorek, Firth, & Hutchison, 1997), mucolipodal (birds:
Burns, 1992), or just lipoidal (rodents: Payne, 1994). Lacrimal glands
are usually either mucous (some mammals and reptiles: Rehorek,
1997; Menaka & Puri, 2015) or seromucous (other mammals: Men-
aka & Puri, 2015; Shaker & Al-Obeady, 2016). Finally, meibomian
glands are the source of the lipid layer in the tear fluid (Millar &
Schuett, 2015). The histochemical nature of the other eyelid glands is
unknown, with only mucous (with possible serous and lipodal contri-
butions) secreting palpebral glands in alligators (Rehorek, Legenzoff,
Carmody, Smith, & Sedlmayr, 2005). The variable amount of lipid (pre-
sent only in odontocetes) in cetaceans is consistent with the role of
lipid as an evaporative barrier in terrestrial but not aquatic vertebrates
(Davis et al., 2013; Young & Dawson, 1992). A heterogeneous secre-
tory cell may be an adaptation to an aquatic lifestyle. Since the tear
REHOREK ET AL. 7
film is continuously washed away, a more efficient method of mixing
a continuously produced large amount of orbital secretions is needed.
The presence of many Pacinian corpuscles (i.e., lamellar corpuscles)
within the stroma of the circumorbital gland in the bowhead whale is
intriguing. Rare reports of ectopic Pacinian corpuscles in glandular tissue
(e.g., prostate, pancreas), and lymphatic tissues have been reported in
humans (Feito, Cobo, Santos-Briz, & Vega, 2017; García-Suárez et al.,
2010; Pai, 2017; Standop, Ulrich, Schneider, Andrén-Sandberg, & Pour,
2001). The physiological significance of these anatomical rarities is
unknown. As a component of the somatosensory system, Pacinian cor-
puscles (mechanoreceptors) detect stimuli (e.g., gross pressure changes,
high frequency vibrations) that ultimately give rise to sensations
(mechanotransduction principle: Quindlen, Lai, & Barocas, 2015). Pacinian
corpuscles can be used to perceive sound waves in water (Munger & Ide,
1987). Plantar Pacinian corpuscles have been proposed as the anatomic
mechanism for seismic communication in elephants (Bouley, Alarcon,
Hildebrandt, & O'Connell-Rodwell, 2007). It is possible that the bowhead
too, can detect vibrations with these Pacinian corpuscles.
5 | CONCLUSIONS
The cetacean orbital glandular region, which superficially resembles a
mass of glandular tissue, is a highly integrated system of
multifunctional secretory cells arranged as glandular masses or diffuse
tissue. This is consistent with the production of a continuous stream
of a complex, heterogeneous orbital fluid which covers the entire eye.
Though presumably protective in nature, the precise function of the
glands is difficult to ascertain. These cetacean orbital glands produce
many different secretions per cell, unlike that of their terrestrial coun-
terparts. Future studies of these glands needs to include more
detailed immunohistochemical examination of the glands and bio-
chemical analysis of the tear fluid.
ACKNOWLEDGMENTS
The authors wish to thank Martin Buckley for proof reading this man-
uscript. Collection of the bowhead and the beluga material was car-
ried out under an NOAA-NMFS permit (Nos 17350-01 and
17350-02) to J.C.G. This study was partially funded by substantial
grant from the Coastal Impact Assistance Program, Fish and Wildlife
Service, U.S. Department of the Interior, and the North Slope Bor-
ough). We thank the Alaska Eskimo Whaling Commission and the Bar-
row Whaling Captains Association, and the bowhead whaling crews
of Utqiagvik for their help and support. We also thank the Alaska
Beluga Whaling Committee, Point Lay subsistence hunters, the NSB
school district for their help and support. Bailey McKenna assisted in
making Figure 1.
DATA AVAILABILITY STATEMENT
Research data are not shared.
ORCID
Susan J. Rehorek https://orcid.org/0000-0003-2550-905X
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How to cite this article: Rehorek SJ, Stimmelmayr R,
George JC, Suydam R, McBurney DM, Thewissen J. Whale
tear glands in the bowhead and the beluga whales: Source and
function. Journal of Morphology. 2020;1–10. https://doi.org/
10.1002/jmor.21099
10 REHOREK ET AL.
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