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.