JOURNAL OF MORPHOLOGY 207:173-184 (199])

Orbital Gland Structure and Secretions in the Atlantic Bottlenose Dolphin (Tursiops truncatus)

RAYMOND J. TARPLEY AND SAM H. RIDGWAY Naval Ocean Syst ems Center, San Diego, California 92152

ABSTRACT Orbital gland structure of the Atlantic bottlenose dolphin, Tur­siops truncatus, was examined at the macroscopic, light microscopic, and electron microscopic levels. The gland completely encircles the ocular globe in a belt-like fashion near the conjunctival fornix but is considerably more devel­oped medially. Duct openings are scattered throughout the fornix and over the surface of the palpebral conjunctiva. Microscopically, the gland has a tubuloal­veolar arrangement; alveolar cells contain numerous secretory vesicles which can be interpreted as two structural types by light and electron microscopy. Histochemical staining demonstrates that both types contain glycosaminogly­cans. Lipid analysis of the glandular secretion (dolphin tears) shows them to be non-oily and to contain only negligible amounts of cholesterol, triglycerides, phospholipids, and free fatty acids . The secretion is clear, slippery, and viscoelas­tic and well-adapted to protecting the eye and to reducing frictional forces between the eye surface and surrounding seawater.

Whereas serous-secreting lacrimal glands are said to be lacking in the ocular orbit of Cetacea (whales , dolphins, and porpoises), a well-developed gland has been noted in the orbital region by several investigators and has been presumed homologous with the Harderian gland (Slijper, '62; Walls, '67). Slijper ('62) suggested that this gland in Ceta­cea secretes an " oily substance which regu­larly bathes the cornea, thus protecting it and the eyelids against the harmful effects of seawater." Harder's gland was fi r st described in deer in 1694 (as reviewed in Sakai, '81 ) and has since been reported to occur in a wide variety of vertebrates including certain amphibians, reptiles, birds, and mammals (Prince, '56; Fahmy et al., '71; Burns, ' 78; Weaker, '81; Sakai and van Lennep, '84). However, there is considerable confusion re­garding the precise identity of the gland and its absence in several of the domestic mam­mals has been noted (Banks, 1981). The most detailed account of eye-associa ted glands in dolphins was presented by Waller and Harri­son ('78) who dichotomized the glandular mass into Harderian and conjunctival compo­nents.

Although its precise position has been said to vary with species, Harder's gland is gener­ally described as lying within the orbit in close association with the globe of the eye (Prince '56). Function of the gland has not

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been clearly established, but it has histori­cally been assumed to assist lubrication of the eye, particularly the movement of the third eyelid, or nictitating membrane, over the globe (Weaker et aI., '83). In fact, the gland has been claimed by some authors to be present only in those animals that have a nictitating membrane (a structure lacking in dolphins). There has also been suggestive evidence linking Harder's gland to reproduc­tive and circadian cycles in some mammaliall species (Wetterberg et al., '70; Payne et al ., ' 79 ; Weaker et al ., '83 ; Pevet et al., '84).

Tear secretion in the bottlenose dolphill has recently been investigated with reference to its physical and chemical composition (Uskova et al. , '75; Young, '84) as well as to its utility in minimizing the frictional chal­lenges to the surface of the eye in the aquatic medium (Haun et al ., '84). The potential significance of this secretion in reducing hy­drodynamic drag (Uskova et aI., '75) con­trasts somewhat with the traditional interpre­tation of the secretory product as an oily substance of the cetacean Harderian gland (Slijper, '62) since a fatty product would be less effective in reducing such friction. In the current inves tigation we attempt to addres:3

Address reprin t requests to Raymond J. Tarpley, Depar tmen to r Veterinary Anatomy, College of Veter inary Medicine, Texas A&M University. College Statio n . TX 77843.

174 R.J. TARPLEY AND S.H. RIDGWAY

this confusion by considering the structural features of this gland as they relate to the chemical composition of its secretory product and its proposed function within the marine environment.

Although the dolphin eye is positioned lat­erally, the orientation of the globe and its allied structures will be discussed in this article as though frontally placed (that is , in relation to the optical axis) to facilitate com­parative referencing. Through gross dissec­tion of the orbit we define the position of the gland in relation to the globe, eyelids, and extrinsic ocular muscles (there is no nictitat­ing membrane). We describe the composition of glandular tissue by light and electron mi­croscopy and histochemistry. The chemical nature of dolphin tear secretion is presented as it relates to lipid fractions.

MATERIALS AND METHODS

The orbital gland was examined structur­ally and histochemically in 13 Atlantic bottle­nose dolphins, Tursiops truncatus, following fixation in 10% phosphate or acetate buffered formalin, Helly's solution, or Karnovsky's fixative. Fresh orbits were examined by mac­roscopic dissection, while chemically pre­served materials were studied by macro­scopic dissection, light microscopy, and electron microscopy using structural and his­tochemical interpretations.

Sections for light microscopy were taken from several regions within the gland and processed for histology using paraffin or plas­tic embedding methods. A variety of histolog­ical stains were used, including hematoxylin and eosin (H&E) for general structure, Ver­hoeff van Gieson's (VVG) for differentiating collagen and reticular fibers, and Masson's Trichrome (MT) to distinguish collagen and muscle tissue. Alcian blue/periodic acid-Schiff (AB/P AS) and periodic acid-Schiff (PAS) stains were used to detect glycosaminogly­cans in paraffin and plastic embedded sec­tions , respectively.

For electron microscopy, sections from sev­eral regions of the gland were immersed in Karnovsky's fixative (Karnovsky, '65) (Elec­tron Microscopy Sciences kit) within 1 hour post-mortem. After 24 hours the fixative was removed and replaced with 0.1 M cacodylate buffer (pH 7.3). After several washes in buffer, the tissues were post-fixed in 1% os­mium tetroxide in 0.1 M cacodylate buffer for 2 hours. Following several buffer and dis­tilled water washes, the tissues were en bloc stained with saturated aqueous acetate over-

night. Tissues were dehydrated in a graded ethanol series and embedded in Epon-Aral­dite. Epon-embedded tissues were sectioned at 0.5 f-Lm, stained with toluidine blue, and examined with brightfield light microscopy. Ultra-thin sections were stained with satu­rated aqueous uranyl acetate and Sato's lead (Sato, '76) and examined by electron micros­copy.

For correlative light and electron micros­copy, tissues were processed as above by the Image Analysis Laboratory of Texas A&M University, but without osmium tetroxide and uranyl acetate, and embedded in L.R. White (London Resin Co.). Two 0.5 f-Lm sec­tions were cut for light microscopy, one stained with PAS-light green and the other with toluidine blue. For electron microscopy, eight serial thin sections were taken from the same block following the 0.5 f-Lm sections and were placed on formvar-coated single hole grids. These were stained with saturated aqueous uranyl acetate and Sato 's lead . Cor­responding areas of each type of section were photographed and compared using a Zeiss PM-3 photomicroscope and a Zeiss 10-C transmission electron microscope.

Selected tissues were prepared for conven­tionallight microscopy using JB-4 methacry­late resin (Polysciences, Inc.) and 1.0 f-Lm sections were obtained with glass knives. These sections were stained with either H&E or PAS.

Secretions ofthe dolphin orbi tal gland were collected for analysis. Three dolphins were trained to hold in a relatively stationary man­ner with their eyes above water. After several days of training and numerous rewards for holding in this position, the dolphins would allow their trainers to collect the secretions in 2 ml syringes with the open tip placed at a canthus of the eye. A globule of secretion that formed at the canthus could be drawn into the syringe only with considerable suction. We frequently had to use a small plastic rod to break the strand of secretion leading from the canthus of the eye to the syringe. Vol­umes collected ranged from 0.25 to 1.50 m!. Portions of the collected secretions were ex­amined grossly and with a dissecting micro­scope. The remainder was frozen pending chemical analysis .

Lipid analysis was conducted by the Lipid Research Laboratory of the University of Cal­ifornia at San Diego. Triglyceride and choles­terol assays were performed on the VP Super System Analyzer (Abbott Laboratories) with

ORBITAL GLAND STRUCTURE AND SECRETIONS IN DOLPHIN 175

Abbott enzymatic reagent and Boehringer Mannheim enzymatic cholesterol reagent . Phospholipids and neutral lipids were ex­tracted with methanol and chloroform. The chloroform phase was dried in a stream of nitrogen and the lipids were dissolved in 50 f.ll of chloroform. Two silica gel plates were activated in an oven at lloec for 60 minutes. Two TLC tanks were prepared using 1) hex­ane:diethyl ether:acetic acid (90:20:1.5 VN) for neutral lipids and 2) chloroform:methanol: water (65:25:4 VN) for phospholipids. Or­bital gland secretion was applied to the plate along with authentic phospholipid (phos­phatidylcholine and lysophosphatidylcho­line) and neutral lipid (free fatty acid, triglyc­eride, and cholesterol ester) standards. Plates were run in the respective solvent systems and were air dried. Samples were visualized by exposure to iodine vapors.

RESULTS

Macroscopic dissection

The orbital gland of the Atlantic bottlenose dolphin is an irregular multilobulated mass

o

Fig. 1. Illustration depicting anterior· oblique view (see arrow in inset for angle of view) of le ft eye encom­passed by the orbital gland (0). Note the prominent

which completely encircles the ocular globe in belt-like fashion on the corneal side of the globe's transverse equator (Fig. 1). It extends somewhat into the base of the eyelids (Fig. 2). In the fresh state it has a pinkish hue throughout. Some regions of the gland are more extensively developed than others; the ventromedial (rostral) aspect is consistently enlarged and projects deeper into the orbit ("ro" in Fig. 1). The gland thins mid-dor· sally, mid-ventrally, and, especially, mid · laterally. However, despite differences in de· gree of development, all gland regions are continuous, forming a unified mass.

No true capsule surrounds the gland since it is closely, although loosely, adhered to the extrinsic ocular musculature with sometime~; little intervening collagen. There are four rectus muscles (dorsal, ventral, lateral, ami medial). The bellies of the dorsal and ventraJ rectus muscles are broad and flat while those of the medial and lateral rectus muscles are more confined and cylindrical. Each rectm: muscle has two insertions in relation to the orbital gland-one more narrow insertion

medial (rostral) enlargement (ro) of the gland. A portion of the retractor bulbi muscles (rb) can be seen since this is an oblique view.

176 R.J. TARPLEY AND S.H. RIDGWAY

Fig. 2. Photograph of near-mid-sagittal section through ocular globe revealing orbital gland (g) in cross section both dorsally and ventrally. The limits of the gland are defined by the arrowheads. Note the envelop­ment (arrows) of the posterior aspect of the ventral gland portion by the ventral rectus muscle (vr). The ventral retractor bulbi muscle (rb) inserts in close proximity to the gland. Also indicated are the optic nerve (n), fibrous sheath (D surrounding the nerve, the sclera (8), lens (I) , cornea (co), and the conjunctival space (c) into which the orbital gland secretion empties. Scale bar = 1 cm.

upon the globe itself beneath the gland near the gland's posterior border and another more broad attachment to the periorbital soft tis­sues exterior to the gland. In this way the gland is embraced posteriorly by the inser­tive ends of the rectus muscles. Muscle fibers are shared anteriorly between rectus muscles where they meet at the exterior insertion . Such ajuncture between the medial and ven­tral rectus muscles creates a vertical groove on the medial surface of the ventromedial enlargement of the gland which continues over its dorsal aspect; as a result, a portion of the enlargement protrudes medially in the orbit beyond the rectus muscles while a sec­ond portion (the larger of the two) is tucked beneath the joined medial and ventral rectus muscles.

Two oblique muscles (dorsal and ventral) arise independently deep in the bony orbit and insert onto the globe. The dorsal oblique extends forward in the dorsomedial quadrant of the orbit and angles sharply laterally to travel beneath the medial insertion of the dorsal rectus . It continues to course laterally, finally inserting directly to the dorsolateral surface of the globe just beneath the poste­rior margin of the orbital gland. Conversely, the ventral oblique travels forward in the ventromedial quadrant of the orbit and an­gles sharply in the medial direction to travel between the exterior and interior bellies of

the ventral rectus muscle. It courses medi­ally with a slight posterior deflection to fol­low the posterior margin of the orbital gland, finally inserting onto the ventromedial as­pect of the globe just beneath the posterior edge of the gland.

Four retractor bulbi muscles, positioned as dorsolateral, dorsomedial, ventrolateral, and ventromedial bundles, together form a cone encompassing the posterior aspect of the globe and tapering to the rear of the orbit. The two dorsal bundles share crossed fibers along their anterior third before inserting independently upon the globe near its transverse equator beneath the belly of the dorsal oblique mus­cle. A similar arrangement exists for the two ventral bundles in relation to the ventral globe and ventral oblique muscle. There are no shared fibers between the dorsal and ven­tral groups of retractor bulbi muscles. There is very little positional overlap between the orbital gland and the retractor bulbi with the exception of the gland's ventromedial enlarge­ment (Fig. 1) ; the latter extends posteriorly to overlay the anterior portion of the medial retractor bulbi of the ventral pair.

Numerous excretory ducts drain the or­bital gland through pores encircling the eye within the conjunctival fornix as well as scat­tered randomly over the surface of the palpe­bral conjunctiva (Fig. 3). Two to three of the pores, consistently positioned in the medial aspect of the fornix, are larger than the oth­ers and drain the ventromedial lobe of the gland.

Light microscopy

Gland architecture is compound tubuloal­veolar with individual alveoli formed by a single layer of cuboidal to columnar cells (Fig. 4). Nuclei are generally oval and posi­tioned toward the basal lamina of the cell. The cytoplasm often has a foamy appearance in paraffin section due to the presence of vesicles adjacent to the alveolar lumen .

Alveoli empty into intralobular ducts lined by a stratified cuboidal to columnar epithe­lium (Fig. 5). These lead to interlobular ducts positioned within the connective tissue be­tween lobes. The lining of the interlobular ducts is further stratified and is often charac­terized by pronounced infoldings suggesting an irregular, expandable sac-like structure rather than a simple cylindrical duct (Fig. 5). Terminal interlobular ducts are continuous with the conjunctival epithelium (Fig. 6).

The close association between the orbital gland and surrounding musculature is evi-

ORBITAL GLAND STRUCTURE AND SECRETIONS IN DOLPHIN 177

Fig. 3. Photographs of Tursiops eyes illustrating or­bital gland duet openings into the conjunctival space. A: In a formalin-fixed specimen of the right eye, the dorsal (DE) and ventral (VE) eyelids have been transected and partially reflected (arrows) from the cornea (C) to demon­strate several of the excretory ducts (arrowheads) of the orbital gland emptying into the conjunctival fornix at the junction of the palpebral (PC) and bulbar (BC) conjuncti­vae. These orifices completely encircle the eye along the fornix. The larger of these openings (two of which are marked by the central two arrowheads) serve the ventro­medial enlargement of the orbital gland. Numerous smaller openings scattered over the surface of the palpe-

bral conjunctiva are not visible in this photograph bUl; can be seen in B. B: In a fresh specimen of the left eye, the lower eyelid (E) has been transected and pulled ventrally to expose several of the orbital gland duct openings (some indicated by arrowheads) over the surface of the palpe· bral conjunctiva (PC); digital pressure is being applied behind the orbital gland to enhance visibility of the openings. The cornea (C) and sclera with overlying bulbar conjunctiva (BC) of the ocular globe can be seen . The globe has been rolled upward so that it is being viewed from a ventral oblique angle. (Other examples 0:' the orbital gland duct openings in the palpebral conjunc tiva are shown microscopically in Fig. 6) .

178 R.J. TARPLEY AND S.H. RIDGWAY

Fig. 4. Light micrograph demonstrating lobular as­seMbly (LO) of orbital gland secretory units (arrow­heads). Hematoxylin and eosin stain. Scale bar = 300 f.Lm.

dent microscopically. In some regions the absence of a well-developed fibrous capsule is especially apparent where muscle fibers ex­tend for a short distance between glandular lobules (Fig. 7).

Electron microscopy

Two types of cytoplasmic vesicles are n u­merous within alveolar cells although no sin­gle cell contains both. Most prominent are large pale vesicles which crowd toward the alveolar lumen (Fig. 8). Other cells contain smaller, much denser vesicles which tend to be more widely distributed within the cyto­plasm and less often in contact with neighbor­ing vesicles (Fig. 9). Although no cell has both vesicle types, cells containing one or the other vesicle often share the same alveolus (Fi~. 10). Ot~er alveoli appear essentially dedIcated to eIther the pale or the dark vesi­cles, respectively.

Moderately developed microvilli line the alveolar lumen. Adjacent alveolar cells are

Fig. 5. Light micrograph of cavernous duct system (d) leading from orbital gland lobules (g). Hematoxylin and eosin stain. Scale bar = 300 )Lm.

joined by a complex interdigitation of cell borders, with numerous desmosomes. Mito­chondria and rough endoplasmic reticulum are abundant. Myoepithelial cells can occa­sionally be identified exterior to alveolar cells (Fig. 11).

Histochemistry

Histochemically, alveolar cells are positive for glycosaminoglycans throughout the or­bital gland. Reactions to AB/PAS staining in paraffin section and PAS staining in plastic section suggest the presence of two varieties (Fig. 12). Alveoli are often essentially seE!re­gated by chemical type, with the larger otthe two vesicles predominating and positioned near the alveolar lumen; however it is not uncommon to find both types within the same alveolus. Correlative light and electron micro­scopy using serial sections from a single plas­tic-embedded block demonstrate that the two vesicle types evident in electron microscope preparations correspond to the two types seen in toluidine blue-stained thick plastic sec­tions and in PAS preparations (Fig. 13a--c).

Analysis of secretions

. The glandular secretion is readily apparent In the normal dolphin; it covers the entire cornea, conjunctiva, and exposed portions of the globe (Fig. 14). It is clear, and has a slippery yet viscous and somewhat elastic consistency to the touch. Whenever drawn into the syringe for collection, it often slides out the syringe orifice. If placed in distilled water, globules of secretion sink slowly but maintain their form for some time. Globules broken up or agitated in water produce no oil or sheen on the water surface as would be expected if the secretion contained signifi­cant amounts of oil. However, a foamy sub­stance does appear on the surface following agitation. Triglyceride and cholesterol assays with the Super System Analyzer and the enzymatic cholesterol reagent show amounts within the lower limits of detection of the t~sts. More sensitive chromatographic analy­SIS proves the secretion to contain both phos­pholipid and neutral lipid (cholesterol ester, free fatty acid, and triglyceride) with the predominant lipid being triglyceride; how­ever, total lipid is less than 1% of the total specimen.

DISCUSSION

The eye-associated gland in cetaceans has historically been interpreted as the gland of Harder (Slijper, '62; Walls '67). However,

ORBITAL GLAND STRUCTURE AND SECRETIONS IN DOLPHIN 179

~~ C d

Fig. 6. Light micrograph showing junction of orbital gland ducts (d) into the conjunctival space (c) . Several orbital gland lobules (g) are visible. Hematoxylin and eosin stain. Scale bar = 50 fJ.m.

there is much confusion in the literature with regard to the presence, positioning, structure, and secretory product of this gland in the various vertebrate species (Sakai, '81), creating difficulties in establishing such ho­mologies with certainty based upon these criteria.

The ventromedial (ventrorostral) enlarge­ment of the dolphin orbital gland is analo-

Fig. 7. Light micrograph demonstrating close associa­tion between the orbital gland (g) and surrounding ex­traocular musculature (m). Some muscle fibers can be seen between gland lobules (arrowheads). Hematoxylin and eosin stain. Scale bar = 100 fJ.m .

gous in location to that frequently described for the Harderian gland on the nasal side of the ocular globe in a variety of species (Sakai, '81). The distinctive, enlarged orifices drain­ing the ducts from the ventromedial enlarge­men t support homology of this portion of the dolphin gland with that of Harder since the medial canthus is a common site for the Harderian duct emptying in other species (Warwick, '76) . However, the remaining, belt­like, globe-encircling portion of the gland is oddly positioned in this respect. Accordingly, Waller and Harrison ('78) have reserved tr..e term Harder's gland for the ventromedial portion of the total gland mass only, whHe homologizing that portion of the gland sur­rounding the ocular globe with the glands of Krause. The latter opinion was based on their dissections of a Pontoporia (La Plata river dolphin) fetus in which they determined the gland to be derived from conjunctival epithe­lium. The "conjunctival" glands reported by Slijper ('62) may actually reference this encir­cling segment of the orbital gland since no other glands were found within the orbit or associated with the eyelids in our study.

In mammals, the Harderian gland is fre­quently linked functionally to the needs of the nictitating membrane ("third eyelid"),

180 R.J. TARPLEY AND S.H. RIDGWAY

Figures 8-11

ORBITAL GLAND STRUCTURE AND SECRETIONS IN DOLPHIN 181

Fig, 12, Light micrograph showing positive his­tochemical reaction for glycosaminoglycans (arrowheads) in the cytoplasm of alveolar cells throughout the gland, When viewed in color, some vesicles appear as a pale blue while others stain magenta, Note that cells lining the excretory duct (d) are negative for glycosaminoglycans, Alcian blue/periodic acid-Schiff stain , Scale bar = 80 "m,

providing a lubricant to reduce friction as the latter passes over the eye (Weaker et at, '83). It is well developed in rabbits and a variety of rodents (Davis, '29; Mykytowycz, '66; Bube­nik et al., '76). However, the gland has not been universally identified in all mammals possessing a nictitating membrane. Banks ('81) states, for example, that the Harderian gland is found only in the pig and ox among the domestic mammals.

While cetaceans lack a nictitating mem­brane, lubrication remains the likely primary function of orbital gland secretion in this mammal group. During attempts to treat eye injuries or foreign bodies in dolphins we have noticed that secretions can be released rap­idly and in considerable volume. For exam­ple, several milliliters of secretion can appear

Fig, 8, Transmission electron micrograph of a single alveolus containing pale cytoplasmic vesicles (arrow­heads) adjacent to alveolar lumen (I ). Microvilli are appar­ent within the lumen, extending from the apical portion of alveolar cells. Scale bar = 2 "m,

Fig, 9, Transmission electron micrograph demonstrat­ing dark cytoplasmic vesicles (arrowheads) in a single alveolus. The alveolar lumen (I) is marked. Scale bar = 2 "m.

Fig. 10. Transmission electron micrograph of alveo­lus comprising mixed cells serving a single lumen (I ), Some cells contain light vesicles (large arrowheads) while others contain dark vesicles (small arrowheads), Scale bar = 2 "m.

Fig, 11, Transmission electron micrograph of myoep­ithelial cells (arrowheads) surrounding basal portion of alveolar cells , Scale bar = 2 "m.

in a few seconds after ophthalmic ointment is applied, creating a flow which is usually suffi­cient to immediately wash away the medica.­tion. Three structural features of the dolphin orbital gland described in this study appear capable of supporting rapid and voluminous expulsion of secretory product. Contraction of myoepithelial cells (reported also in Waller and Harrison, '78) embracing alveolar cells likely aid expulsion of secretory product from the alveolar lumen. Such cells have beeC1 described in the Harderian glands of rat , mice, and rabbits (Chiquoine, '58). Similarly, at the macroscopic level, the insertional em­brace of rectus muscles over and beneath the gland's exterior may assist in emptying of the gland through voluntary muscular contrac­tion. Such skeletal muscle assistance may also be afforded through the action of the oblique muscles beneath the border of the gland along the globe'S transverse equator as well as retraction of the globe by the retrac­tor bulbi muscles (modifying pressures among tissues surrounding the gland). Finally, the cavernous reservoirs formed by interlobular ducts may serve to store secretory product until needed.

In addition to its value as a lubricant, the secretion may serve osmotic functions as well. Sodium and chloride levels in dolphin blood are approximately one-third and one-fourth, respectively, those in seawater (Ridgway et al., '70; Ridgway, '72), and Young ('84) re­ports similar levels for sodium in dolphin tears. Therefore we might expect these secre­tions to protect the exposed eye somewhat from the relatively hypersaline conditions of seawater.

Yet another utility of tear secretion may relate to an ability to guard against a variety of biological organisms which might reach the eye region in the medium of the sea. It can be suggested that the mucous nature of the secretion provides a physically protective barrier against various planktonic organ­isms. Further, Young ('84) noted the pres­ence of lysozyme in bottlenose dolphin tears , rendering this secretion, at least to some extent, bacteriolytic,

The liberal use of tear secretion as needed for such a variety of lubricative and protec­tive functions would not likely hinder vision in water since the refractive index of dolphin tear secretion has been reported as not signif­icantly different from seawater (Young, '84:1.

In most mammals the secretory product of the Harderian gland is said to be lipid­secreting, and Sakai ('81) uses this feature to

182 RJ. TARPLEY AND S.H. RIDGWAY

Fig. 13. Serial sections (a--c) through a single orbital gland alveolar unit (open arrow designates alveolar lu ­men) which has been embedded in L.R. "Vhite plastic for correlative microscopy. Both dark (white bordered arrow­head) and light (unbordered arrowhead ) intracytoplas­mic vesicles can be followed through light microscopic structural staining with toluidine blue (a) , light micro­scopic histochemical stain ing with periodk acid-Schiff (b), a nd transmission electron microscopic s ta ining with

uranyl acetate and Sato's lead (c). This procedure demon­strates that the dark and light vesicles seen with tolu­idine blue light microscopy correspond with dark and light vesicles visible with electron microscopy. The his­tochemical preparation in b demonstra tes that both vesicles are positive for glycosaminoglycans, although the reaction is much stronger for the dark vesicles in this preparation lacking A1cian blue staining for acid glycosami­noglycans. Scale bars = 5 f.lm (a), 5 f.lm (b), 2 f.lm (c).

ORBITAL GLAND STRUCTURE AND SECRETIONS IN DOLPHIN 183

Fig. 14 . Photograph of tear secretion (arrowheads) pulled by syringe (8) from the nearly closed eyelids of a live dolphin in left lateral recumbency to demonstrate the tear 's s tringy, adhering consistency. The shadow of the operator's hand assists visibility of the essentially trans­parent secretion. For orientation, the lower eyelid (E ), the commissure of the mouth (e) , and the dorsal (D) and rostral (R) aspects of the dolphin, as well as shadows cast by the tea r string (T8) and syr inge (88), are indicated .

define the gland in mammals. Similarly, both Kellogg ('38) and SIUper ('62) describe the ocular secretions of cetaceans as "oily." How­ever, our gross examination does not confirm this oily consistency, but rather demon­strates a secretion which is viscous in flow, tenacious to the cornea and sclera, yet slip­pery to the touch in agreement with the observations of Waller and Harrison (' 78). Mixing the secretion with distilled water leaves no visible oily surface sheen. Slijper's examination of the secretions could possibly have been limited to dissection of specimens on board a whaling ship or of beach-stranded specimens in which oil from the fatty skin and blubber could have contaminated the glandular secretions.

However, in deference to the earlier de­scriptions of its oily composition and to con­firm our physical observations suggesting the absence of oiliness, we subjected dolphin or­bital gland secretions to lipid analysis, finding only negligible amounts of cholesterol, triglyc­erides, phospholipids, and free fatty acids . You ng ('84) also reported low levels of choles­terol in ocular secretions from the bottlenose dolphin . Physical and chemical analysis by Pickwell (in Haun et al., '84) showed the secretions to consist mainly of "a homoge­neous, colloidal material" of high molecular weight (greater than 1,000,000 daltons) which

"may be a mucopolysaccharide." Sudan Black B stains for lipids were negative in their study. Uskova et al. ('75), in a biochemical and histochemical investigation of tear secre­tion in the bottlenose dolphin, identified born acid and neutral mucopolysaccharides.

These findings are in agreement with our correlative light and electron microscopic re­sults, with histochemical bridging demon­strating that both light and dark vesicles observed in electron micrographs consisted of glycosaminoglycans (a current term for the mucopolysaccharide group). Further, our AB/PAS staining of paraffin-embedded, for­malin-fixed tissue sections revealed both acid and neutral glycosaminoglycan varieties .

The structural and functional departures from the mammalian norm based on this and previous studies have led us to refrain for the present from equating the entire cetacean orbital gland with the traditional gland of Harder until thorough embryological studies can support such a homology. The lengthy time scale of cetacean evolution, dating back some 55 million years (Gingerich et al ., '83; , has provided a sufficient interval for consider­able morphologic and functional change, mak­ing it difficult to presume homologies based on gland positioning, structure, or chemica.l composi tion of its secretory product.

In summary, the slippery, viscoelastic se­cretion of the dolphin orbital gland appears well suited to bathe and protect the eye within the marine environment while providing a clarity which does not hinder passage of light into the eye. Seawater streaming past the cornea as the animal swims rapidly exposes it to much more friction than the eye of a terrestrial animal running at a similar speed. Such environmental friction or hydrody­namic drag creates the need for a secretory product which is much more viscous and tenacious than are the serous tears of terres­trial mammals.

ACKNOWLEDGMENT8

We are grateful to K. Neck, M. Frey, B. Merka, and M. Costello for their assistance in tissue preparations and S . Parthasarathy and J. Juliano for lipid analyses of dolphin tears. We thank J. Geraci, J . Mead, S. Hersh, and J . Whaley for facilitating our rapid collection of tissues following the death of dolphins which died during an unusual mortality along th(~ eastern U.S. coast during late 1987 and early 1988. We appreciate the cooperation of E. Asper, B. Andrews, J . McBain, J . Antrim , and the Animal Care staff of Sea World, Inc.

184 R.J . TARPLEY AND S.H. RIDGWAY

for assisting our collection from dolphins which died from natural causes. We appreci­ate review of the manuscript by F. Wood. This work was supported through a postdoc­toral fellowship from the U.S. National Acad­emy of Sciences to R. Tarpley conducted at the Naval Ocean Systems Center, San Diego, CA.

LITERATURE CITED

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Bubenik, G.A., G.M. Brown, and L.J. Grota (1976) Immu­nohistochemical localization of melatonin in the rat Harderian gland. J. Histochem . Cytochem. 24: 1173-1177

Burns, R.B. (1978) Histology of Harder's gland of the rockhopper penguin , Eudyptes cres/alu s. Zool. J . Linn. Soc. 64:129-133.

Chiquoine, A.D. (1958) The identification and electron microscopy of myoepithelial cells in the Harderian gland. Anat. Rec. 132 '569-583.

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