Histology of selected tissues of the leopard seal and implications for functional adaptations to an aquatic lifestyle

Abstract

The microscopic anatomy of the cardio-respiratory system, digestive system, kidney, lymphatic system and integument was investigated in the leopard seal, Hydrurga leptonyx, by examining histological sections of tissues collected from leopard seals in Antarctica and New South Wales, Australia. The majority of the tissues had similar histological features to those described in terrestrial mammals and other pinniped species, particularly phocid seals. Differences noted included readily identifiable Purkinje cells within the endocardium, muscular rather than cartilaginous reinforcement of the smaller airways, a single capillary layer within the alveolar septa, limited and variable keratinization of the oesophageal epithelium, few lymphoid follicles within the lamina propria of the gastrointestinal tract, and an absence of a sporta perimedullaris musculosa described in the kidney of cetaceans and some pinniped species. Adaptations of the lung, spleen and integument, similar to those described in other pinnipeds, including reinforcement of the pulmonary terminal airways, prominent pulmonary interlobular septa, ample smooth muscle in the capsule and trabeculae of the spleen, increased thickness of the epidermis, well-developed dermal sebaceous glands, and a thick blubber layer, appear to confer upon the leopard seal advantages related to its aquatic lifestyle.

Introduction

Establishing the normal microscopic anatomy of a species is an important aid for the routine histopathological examination of tissues. Without such knowledge it is difficult to determine if features noted at the time of necropsy are related to individual variation, disease or are representative of the normal morphological structure of the tissue. Knowledge of species-specific microscopic anatomy can also provide information relating to the physiological and biological adaptations of the species.

The histology of the tissues of the leopard seal, Hydryrga leptonyx, is poorly described in the literature with few studies examining tissues of the leopard seal in sufficient detail or with sufficient sample size to draw any meaningful conclusions regarding their histology and its functional significance. Published reports of the histology of leopard seal tissues are limited to comments regarding the lungs of two seals (Denison & Kooyman, 1973), the histology of the aorta in one seal (Drabek, 1975), the absence of a sporta perimedullaris musculosa in the kidney of this species (Cave & Aumonier, 1964), plentiful lipids in the stratum corneum of the skin (Ling, 1974) and the organization of the hair in one seal (Scheffer, 1964). The scarcity of information detailing the histology of leopard seal tissues is due, in part, to the limited opportunities available for collecting tissue samples from this species.

Although little has been published on the histology of the leopard seal, the histology of several tissues has been described in other Antarctic pinnipeds, including the digestive system, lungs, spleen, kidney, aorta, thyroid gland and the integument, the integument in particularly in regard to the process of the moult (Hepburn, 1915; Cave & Aumonier, 1964; Scheffer, 1964; Ling & Thomas, 1967; Ling, 1968, 1970; Boshier & Hill, 1974; Eastman & Coalson, 1974; Drabek, 1975; Vardy & Bryden, 1981; Welsch & Drescher, 1982; Schumacher & Welsch, 1987; Little, 1991; Schumacher et al. 1995).

As the leopard seal is a marine mammal, adaptations of the microscopic anatomy may exist, conferring advantages upon this species for its aquatic lifestyle. To date, differences in the histology of pinnipeds and their terrestrial counterparts have been limited to notable differences in the structure of the lungs, kidney, spleen and integument, as well as less pronounced differences in other organs. However, for most tissues the histology is described as typically mammalian. Despite this, it may not be meaningful to extrapolate findings from one species to another, and differences have been noted in the histology of phocid and otariid species and were related to differences in their biology and lifestyle. For this reason, it is paramount that species-specific histological descriptions are generated.

The aim of the present study is to describe the histological features of selected leopard seal tissues and compare these findings with published reports in this and other pinniped species. Particular emphasis is directed towards describing histological features that have implications for functional significance related to the aquatic lifestyle of the leopard seal.

Materials and methods

Tissue samples were collected from four leopard seals along the fast ice edge and on ice floes in Prydz Bay near Davis Station (68°36′S, 78°02′E), eastern Antarctica, that died unexpectedly during or subsequent to immobilization with either a combination of 0.18–0.27 mg kg⁻¹ midazolam (Roche Products Pty. Ltd, NSW, Australia; reconstituted to 15 mg mL⁻¹ by Royal Hobart Hospital, Tasmania, Australia) and 1.0–1.5 mg kg⁻¹ pethidine (Sigma Pharmaceuticals, Clayton, Victoria, Australia; reconstituted to 150 mg mL⁻¹ by Royal Hobart Hospital) or a 1 : 1 ratio of 0.5–1.5 mg kg⁻¹ tiletamine/zolazepam (Telazol 100 mg mL⁻¹, Fort Dodge, NSW, Australia, or Zolatil 100, 100 mg mL⁻¹, Virbac Australia Pty. Ltd, NSW, Australia). Atropine (AstraZeneca Pty. Ltd, NSW, Australia; reconstituted to 16 mg mL⁻¹ by Royal Hobart Hospital) at a dose rate of 0.015 mg kg⁻¹ was administered with the immobilizing drugs. Tissue samples were also collected from five leopard seals that hauled out along the coast of NSW, Australia, during 2000–2003 and/or kept in captivity at Taronga Zoo, Sydney. Two of these seals died as a result of anaesthetic complications, two seals died from gastrointestinal disease, and one seal was euthanized owing to poor body condition and debilitation. Samples of cardiac muscle, trachea, lung, oesophagus, stomach, small intestine (jejunum and ileum), large intestine (colon), liver, pancreas, kidney, spleen, lymph node (tracheobronchial, axillary, mesenteric or jejunal), blubber and skin were collected during necropsy using a clean stainless steel knife or scalpel blade and tissues were placed immediately into clean plastic containers containing 10% neutral buffered formalin (concentrated buffered formalin, Fronine Pty Ltd, Riverstone, NSW, Australia). Formalin-fixed tissues were prepared by an automated tissue processor (Tissue-Tek® VIP Miles Scientific Miles, Ames, Technicon Bayer Diagnostics Australia). The tissues were embedded in paraffin wax and 5-µm sections were stained with haematoxylin and eosin (H&E) and used as the basis for tissue examination. Special stains were also employed for the examination of selected tissues, including Gomoris’ trichrome, which demonstrates collagen (blue) and muscle (red) for examination of the kidney; Resorcin-fuchsin-van Gieson's, which demonstrates elastic fibres (charcoal), collagen (red) and muscle (yellow) for examination of the lungs; Prussian Blue reaction (Perl's) for examination of the distribution of haemosiderin (blue) in the liver, spleen and lymph node; Periodic-Acid Schiff technique (PAS), which demonstrates glycogen (pink) for examination of cardiac muscle; and Toluidine blue for examination of mast cells in the spleen, lymph node and gastrointestinal tract. Owing to autolysis of some tissues, the limitations of sample collection, and the presence of haemorrhage and congestion in some of the tissues, not all histological features for each tissue could be described for every seal. Standard histological descriptive nomenclature and terminology was employed for the description of histology in the leopard seal tissues, and the description of the histology of domestic species employed for comparative purposes was taken from Dellman & Eurell (1998) and Bacha & Wood (1990).

Results

Cardio-respiratory system

Heart

The left and right ventricle of the heart of the leopard seal (n = 8) comprised three layers, the inner endocardium, myocardium and an outer epicardium. Three layers were observed within the endocardium. The innermost layer was an endothelium that overlaid an inner subendothelial layer consisting of connective tissue with collagen, elastic fibres, smooth muscle cells, nerves, small blood vessels and a prominent elastic layer. The outer subendothelial layer consisted of loosely arranged collagen, elastic fibres, blood vessels, lymphatic vessels, nerves, occasional adipose cells and variable numbers of modified cardiac myocytes, the Purkinje cells. In all sections in which the endocardial layer was present, the Purkinje cells were prominent, easily delineated from the underlying myocardium, were larger than the cardiac myocytes and had considerably lighter staining cytoplasm containing ample glycogen on application of PAS (Fig. 1A).

Fig. 1.

[A] Heart. Ventricle wall demonstrating endocardium (En), glycogen (pink) within Purkinje cells (PC), myocardium (My) and ventricular cavity (VC). PAS, scale bar = 100 µm. [B] Lung demonstrating charcoal elastic fibres (EF) in the pulmonary pleura. Resourcin-Fuchsin van Gieson's, scale bar = 20 µm. [C] Lung demonstrating interlobular septa (ILS), bronchi (BR), cartilage plates (C), alveolar duct (AD), alveolar sac (AS) and alveoli (A). H&E, scale bar = 200 µm. [D] Lung. Bronchus demonstrating the lumen (L), epithelium (E), cilia (arrow), smooth muscle (SM), cartilage plates (C), submucosal glands (G). H&E, scale bar = 50 µm. [E] Lung demonstrating smooth muscle (M) in the terminal airways and at the opening of the alveoli (A) and the single capillary layer (SCL) within the alveolar septa. H&E, scale bar = 100 µm. [F] Lung demonstrating the single capillary layer (arrows) within the alveolar septa. H&E, scale bar = 20 µm.

The myocardium, consisting of bundles of cardiac myocytes, was the thickest layer of the heart. Connective tissue, a dense capillary network and small amounts of adipose tissue were seen between the bundles of cardiac myocytes within this layer. The epicardium consisted of a mesothelium that overlaid a layer of connective tissue containing abundant elastic fibres that formed a meshwork around the blood vessels and nerves within the connective tissue. Variable amounts of adipose tissue were also present within this layer. Extending into the myocardium between the bundles of myocardial cells were connective tissue trabeculae and blood vessels from the epicardium.

Trachea

The trachea of the leopard seal (n = 6) had the typical mammalian structure. The mucosa was thrown into small folds in all of the sections examined, presumably due to tracheal collapse. The respiratory epithelium lining the lumen was ciliated, pseudostratified columnar epithelium with numerous goblet cells. The lamina propria was a loose connective tissue that contained numerous blood vessels located just beneath the epithelium and a prominent layer of longitudinally arranged elastic fibres. The connective tissue of the submucosa was denser than that of the lamina propria, and consisted of interweaving thick collagen bundles, plentiful blood and lymphatic vessels, nerves, elastic fibres, adipose tissue and plentiful mixed seromucous tubuloacinar glands in which serous glandular cells predominated.

Flattened C-shaped incomplete hyaline cartilage rings were a prominent feature of the tracheal wall, and the smooth muscle of the dorsally located trachealis muscle appeared to attach externally to the cartilage rings, but was not present in all of the sections examined. In one section there appeared to be isolated bundles of smooth muscle within the submucosa but it could not be determined if these were associated with the trachealis muscle. An adventitia of loose connective tissue consisting of collagen fibres, elastic fibres, blood vessels, nerves and variable amounts of adipose tissue was seen in all sections and a nerve plexus was seen in one out of six sections.

Lungs

Multiple sections of the lung of the leopard seal (n = 9) were examined. In all of these sections, some degree of congestion and pulmonary interlobular oedema was evident.

The lung was surrounded by thick visceral pleura with an outer mesothelial lining of squamous to low cuboidal cells overlying loose connective tissue containing smooth muscle, prominent elastic fibres, blood vessels, lymphatic vessels and nerves (Fig. 1B). The fibrous interlobular septa were prominent, partly due to oedema, and extended from the pleura to divide the pulmonary parenchyma completely into lobules (Fig. 1C).

In the majority of the airways the epithelium had a folded appearance, thought to be a consequence of airway collapse. The largest airways (Fig. 1D) were lined by ciliated, pseudostratified columnar epithelium with numerous goblet cells that overlaid a lamina propria of loose connective tissue containing prominent elastic fibres. A layer of smooth muscle that appeared to be complete in the majority of the large airways lay external to the lamina propria. The submucosa was a layer of loose connective tissue containing blood vessels, elastic fibres and varying numbers of seromucous (predominantly serous) glands. A number of these glands had a wide lumen and flattened lining epithelium, and in several of these glands the lumen contained concretions of secretory material. A layer of incomplete cartilage plates was observed in the large airways, between which were located submucosal glands. A surrounding layer of connective tissue or adventitia constituted the outermost layer of the large airways, and glands were also observed in this tissue. Lymphoid aggregates were not a common feature in the lamina propria and submucosa of the airways.

As airway diameter decreased, the layers of the airways became less well developed, the height of the epithelium decreased from pseudostratified columnar to low columnar to cuboidal, non-ciliated epithelium within the bronchioles. In addition, the number of secretory cells decreased, and submucosal glands were not observed in the smaller airways, becoming absent prior to the disappearance of cartilage in the airways, the lamina propria decreased in thickness, and there was a decrease in the amount of cartilage seen. Bronchioles lacked goblet cells and submucosal glands. Although cartilage was not generally seen in the bronchiolar walls, individual cartilage plates were occasionally seen in larger bronchioles lined by cuboidal epithelium, particularly those at the bronchi-bronchiolar junctions. A relative increase in the amount of smooth muscle within the airway wall was observed with decreasing airway size. A complete smooth muscle layer was observed in the smaller airways. Myoelastic sphincters were not a feature of the bronchioles in the leopard seal.

Beneath the cuboidal epithelium of respiratory bronchioles, a thin connective tissue layer and an oblique layer of smooth muscle were observed. The cuboidal epithelium of the respiratory bronchioles was interrupted by alveoli. Respiratory bronchioles branched to form alveolar ducts, and the latter branched into alveolar sacs whose walls were formed of alveoli. Variably sized bundles of smooth muscle, collagen and elastic fibres were located at the opening of the alveolus, giving the lip of the alveolus a rounded appearance (Fig. 1E). Within the alveolar septa, the majority of the alveoli had a single capillary layer (Fig. 1F), but in some areas there was an impression of a double capillary layer within the alveoli wall. Collagen fibres, elastic fibres and smooth muscle cells were observed in addition to the epithelial cells lining the alveolar septa.

Digestive system

In all of the tissues examined the tubular organs consisted of four layers. From the lumen to the external surface these were the tunica mucosa, tela submucosa, tunica muscularis and a tunica serosa (tunica adventitia in the oesophagus).

Oesophagus

The stratified squamous epithelium of the tunica mucosa of the oesophagus was markedly folded and slightly keratinized (1–2 layers) in all of the sections examined (n = 5). The lamina propria consisted of a network of collagen fibres containing blood vessels and the connective tissue of the lamina propria was denser than that of the submucosa. The lamina muscularis mucosa consisted of isolated bundles of smooth muscle in the submucosa above and between groups of submucosal glands in four of the five sections examined. The submucosa of loose connective tissue contained numerous large, longitudinally orientated blood vessels and plentiful mixed seromucous glands with mucous predominating, and numerous large ducts lined by stratified squamous epithelium. Although it was not possible to discern the full thickness and the number of layers of the tunica muscularis in all of the sections examined, an inner circular and outer longitudinal layer were common. Skeletal muscle formed the tunica muscularis in all of the sections (confirmed by identification of cross-striations under polarized light). In four out of five sections, small isolated smooth muscle bundles were seen adjacent to the submucosa in the inner layer of the tunica muscularis, and in one section occasional smooth muscle bundles were also seen in the outer muscle layer. The outermost layer of the oesophagus was an adventitia of loose connective tissue containing numerous blood vessels, lymphatic vessels and nerves.

Stomach

The proper gastric (fundic) region of the stomach was examined in the leopard seal (n = 8) and the structure was found to be typically mammalian. In a number of sections the mucosal surface was autolytic and it was not possible to discern the full extent of the mucosa or the gastric pits. The epithelial lining was tall simple columnar with basally located oval or round nuclei. The gastric pits, lined by simple columnar epithelium, were invaginations of the lining epithelium and were continuous with the gastric glands proper. The loose connective tissue of the lamina propria contained long, straight, branched tubular gastric glands with chief and parietal cells. The chief cells were the most numerous of the cells seen, and the parietal cells were larger and their cytoplasm stained intensely eosinophilic (Fig. 2A). Within the lamina propria, scattered lymphoid cells were seen between the glands, prominent lymphoid follicles were absent, mast cells were observed particularly near the base of the gastric glands, and numerous small blood vessels were seen. The lamina muscularis mucosa was prominent and comprised multiple indistinct discontinuous layers of smooth muscle with large amounts of interspersed collagen. It was difficult to determine the orientation of the muscle layers in the lamina muscularis mucosa. Small numbers of smooth muscle cells from the lamina muscularis mucosa extended into the mucosa between the gastric glands.

Fig. 2.

[A] Stomach demonstrating gastric glands (G) and abundant parietal cells (arrows) within the gastric mucosa. H&E, scale bar = 50 µm. [B] Colon mucosa and submucosa (SMu) demonstrating the lumen (L), lymphoid infiltration (LI) of the lamina propria, crypts of Lieberkühn (C), lymphoid follicle (LF), and smooth muscle (SM) of the lamina muscularis mucosa. H&E, scale bar = 200 µm. [C] Liver demonstrating rows of hepatocytes separated by sinusoids and two central veins (arrows) with prominent collagen and smooth muscle in their walls. Gomoris’ trichrome, scale bar = 200 µm. [D] Liver. Distribution of haemosiderin within the cytoplasm of the hepatocytes and Kupffer cells (arrows) lining the hepatic sinusoids. Perl's, scale bar = 50 µm.

The submucosa of the stomach was a loose connective tissue consisting of collagen fibres with nerve plexuses, blood and lymphatic vessels, scattered lymphocytes, plasma cells and other leukocytes. No adipose tissue was seen within the submucosa. The tunica muscularis was only present in four of the eight sections examined and consisted of three layers of smooth muscle the orientation of which was difficult to discern. The innermost layers were generally circular and the outermost layer longitudinal in orientation. A myenteric nerve plexus was seen between the muscle layers. A serosal lining was only seen in three of the eight sections examined and consisted of mesothelium overlying loose connective tissue.

Small intestine

Multiple sections (1–5) of the small intestine (jejunum or ileum) of each seal (n = 7) were examined. Variation within and between seals was noted with regard to the cellular infiltrate of the lamina propria, the thickness of the submucosa, villi length, the number of lymphoid follicles, the prominence of the lamina muscularis mucosa, the number of goblet cells in the mucosa and the thickness of the tunica muscularis.

Simple columnar intestinal epithelial cells with a striated border and plentiful goblet cells lined the tunica mucosa. The villi were long and irregular in outline, and simple tubular glands (crypts of Lieberkühn) opened between the bases of the villi and extended as far as the lamina muscularis mucosa. Mitotic figures were seen among the cells lining the crypts of Lieberkühn, while Paneth cells were not apparent. Mucosal folds were not prominent. The vascular lamina propria formed the core of the villi and was diffusely infiltrated with variable numbers of lymphocytes, plasma cells and eosinophils; small numbers of mast cells were also observed, particularly near the base of the crypts of Lieberkühn. The prominence of the lamina muscularis mucosa varied from a thin area of indistinct multiple layers to more extensive bundles of smooth muscle in poorly defined layers. Smooth muscle cells from this layer extended into the lamina propria between the crypts of Lieberkühn. No clear delineation between the lamina muscularis mucosa and the submucosa could be discerned. Within the lamina propria in four out of seven seals, single to multiple lymphoid follicles, some with germinal centres, were noted. The lamina propria was more cellular in the regions of the lymphoid follicles and the follicles extended into the lamina muscularis mucosa, making the latter difficult to discern in the region of the follicles.

The submucosa was generally thick, with large amounts of collagen, large blood vessels, nerves and a prominent Meissner's plexus. Brunner's glands were not seen within the submucosa. The tunica muscularis consisted of an inner circular layer and an outer longitudinal layer of smooth muscle between which Auerbach's plexus was prominent, as were large blood vessels and nerves. The inner circular layer was thicker than the outer longitudinal layer, often 3–4 times thicker. The outermost layer of the small intestine, the serosa, consisted of a thin, loose connective tissue covered by mesothelium.

Large intestine

The mucosa of the large intestine of the leopard seal (n = 5) was non-villous. Prominent mucosal folds were seen in four out of five sections examined. The lumen was lined by a simple columnar intestinal epithelium with a striated border, and numerous long, straight tubular glands (crypts of Lieberkühn) were present. Plentiful goblet cells were observed particularly in the crypts, and were more numerous in the large intestine than in the small intestine. The lamina propria was relatively cellular with plentiful lymphocytes and plasma cells, and smaller numbers of polymorphonuclear leukocytes. The lamina muscularis mucosa was generally prominent and consisted of several indistinct discontinuous layers with no obvious organization, and the smooth muscle of this layer was admixed with the dense connective tissue of the submucosa. Lymphoid follicles extended from the lamina propria to the submucosa in three out of five seals examined (Fig. 2B), and where lymphoid follicles were seen, a heavier infiltration of the lamina propria with lymphoid cells was observed, and this was particularly prominent in two out of five seals making it difficult to discern the lamina muscularis mucosa in the region of the lymphoid follicles.

The submucosa was a loose connective tissue with collagen, large blood vessels, nerves and a prominent Meissner's plexus. The tunica muscularis was thick in most of the sections examined. The inner circular layer of smooth muscle of the tunica muscularis was thicker than the outer longitudinal layer, and between the two layers a prominent Auerbach's plexus and blood vessels and nerves were seen. Variation in the thickness of the outer longitudinal layer was not observed. The connective tissue of the serosa was more prominent in the large intestine than in the small intestine, and connective tissue septa from the serosa extended into the outer longitudinal layer of the tunica muscularis, forming indentations within this layer.

Liver

All of the liver sections examined (n = 9) had varying degrees of congestion. The following description applies to the normal architecture of the liver and this did not differ to any great extent from the general mammalian form.

The liver was covered by a serosa (visceral peritoneum) that overlayed a connective tissue capsule of variable thickness. Smooth muscle was either absent or seen in small amounts in the capsule, and small numbers of elastic fibres were seen. Interlobular connective tissue was not obvious. In most sections, variable amounts of collagen and smooth muscle were seen around the central veins (Fig. 2C). The portal areas were of the typical mammalian form, consisting of one or more branches of the hepatic artery, portal vein and bile ductule within a connective tissue framework. A smooth muscle sphincter was not observed around the portal vessels; however, the amount of connective tissue within the portal areas was variable. The bile ductule within the portal areas was lined by a simple cuboidal epithelium in five out of nine sections, and by either a simple cuboidal or a simple columnar epithelium in the remaining four sections. Larger bile ducts were lined by a simple columnar epithelium, and some of these cells had vacuoles in their apical regions presumably due to the presence of mucus.

The hepatocytes were arranged in rows or laminae of single-layered cells, separated by sinusoids lined by endothelial cells and Kupffer cells (hepatic macrophages). Hepatocytes had a centrally located nucleus containing prominent nucleoli, the cytoplasm appeared granular and vacuoles (lipid droplets) were not obviously discerned in the majority of sections. Fine, granular, golden brown cytoplasmic pigment (demonstrated to be haemosiderin) (Fig. 2D) was obvious in the hepatocytes in four out of nine sections, and in the Kupffer cells in three out of nine seals.

Pancreas

The structure of the pancreas of the leopard seal (n = 8) followed the typical mammalian form. Both exocrine and endocrine parts could be identified. The pancreas was surrounded by a thin capsule of connective tissue. Septa from the capsule extended into the parenchyma of the organ, dividing it into lobules; within the interlobular connective tissue, nerve fibres and ganglia, blood vessels, and interlobular ducts lined by simple cuboidal to low columnar epithelium, or simple columnar epithelium in the larger ducts, were seen. Adipose tissue was observed in the interlobular connective tissue in only two out of eight sections. Within the exocrine component of the pancreas, the tubuloacinar secretory unit was composed of glandular epithelial cells arranged as acini surrounding a small lumen. The cells were pyramidal in shape, had a basal spherical nucleus surrounded by basophilic cytoplasm, and the apical cytoplasm of these cells was filled with eosinophilic granular material (zymogen). Intralobular ducts were lined by low cuboidal epithelium. The endocrine islets of Langerhans were variously distributed throughout the exocrine portion as lighter clusters of epithelial cells easily discerned within the exocrine tissue, and were not separated from the exocrine tissue by connective tissue. The islets were round or ovoid in shape and of varying overall size (Fig. 3A) containing from ten to over 100 cells arranged in irregular anastomosing cords.

Fig. 3.

[A] Pancreas demonstrating the variable size of the islets of Langerhans (I) and the interlobular connective tissue (arrows). H&E, scale bar = 200 µm. [B] Kidney demonstrating the interrenicular connective tissue between the cortex (C) of two adjacent reniculi. Gomoris’ trichrome, scale bar = 500 µm. [C] Kidney. Cortico-medullary junction demonstrating the cortex (C), large muscular arteries (A), connective tissue (CT), medulla (M), outer zone (OZ) and inner zone (IZ) of the medulla, and vasa rectae (arrows). Gomoris’ trichrome, scale bar = 500 µm. [D] Kidney demonstrating cortex (C), medulla (M), arteries (A) and connective tissue (CT) at the cortico-medullary junction and base of calyx (Cx), and a medullary pyramid (MP). Gomoris’ trichrome, scale bar = 500 µm.

Urinary system

Kidney

Several sections (2–4) of the kidney of each seal were examined (n = 8). The kidney was reniculate in form and each reniculus had a cortex, medulla and calyx, i.e. each reniculus was organized as a unipyramidal kidney. The boundary between each reniculus was indistinct, marked by incomplete connective tissue septa, most pronounced at the capsule and the calyx, and by large veins and arteries (Fig. 3B). The capsule was prominent and consisted primarily of collagen with scattered large vessels and nerve fibres. In some sections the capsule appeared to consist of two layers, an outer layer of collagen and blood vessels, and an inner layer of collagen closely adherent to the parenchyma. However, this division could have been a processing artefact. Fat was seen in the outer layer of the capsule in two out of seven seals. Beneath the capsule numerous large veins were prominent.

The renal cortex consisted of renal corpuscles and tubular structures that were typically mammalian, and it was possible to discern cortical labyrinth areas and medullary rays. The medullary pyramid was obvious with readily distinguishable outer and inner zones, and evident within the outer zone were groups of small blood vessels (vasa rectae). The interstitium was sparse in the cortex but more plentiful in the medulla. The cortico-medullary junction was distinct and was traversed by medullary rays. At the cortico-medullary junction, large muscular arcuate arteries were seen and small amounts of connective tissue and smooth muscle were observed (Fig. 3C), the smooth muscle projecting into the cortico-medullary junction particularly at the calyx (Fig. 3D).

A calyx lined by a transitional epithelium of 4–10 layers enclosed the medullary pyramid and the urinary space was readily discerned. The thickness of the transitional epithelium gradually reduced to 1–2 layers of cuboidal epithelium where the calyx was reflected over the medullary papilla. Beneath the epithelium at the base of the calyx a large amount of connective tissue containing nerves, blood vessels and small amounts of smooth muscle was seen. Where the calyx epithelium was reflected over the medullary papilla, a small amount of connective tissue was seen underlying it. Intrarenal branches of the ureter were lined by transitional epithelium and were surrounded by a thick layer of connective tissue with small amounts of smooth muscle intermingled with the innermost areas of collagen.

Lymphatic system

Lymph node

Two or three lymph nodes from each leopard seal (n = 8) were available for examination. The lymph nodes examined had a thick capsule consisting of collagen, smooth muscle cells, elastic fibres, lymphatic vessels, arteries and veins. Irregular septa from the capsule constituting a well-developed trabeculae framework divided the parenchyma of the lymph node. The trabeculae were broad and prominent, consisting of collagen, smooth muscle and elastic fibres, and the larger trabeculae contained lymphatic and blood vessels. A subcapsular sinus was evident.

The lymph node had an outer cortex and inner medulla although the cortico-medullary junction was not always well delineated. The degree of development of the lymphatic tissue varied depending on the degree of activity of the lymph node, thus explaining the observed variation in the number of follicles and their size, the thickness of the mantle zone of the follicles, and the cellularity of the paracortex and medulla. Generally, the cortex consisted of secondary follicles with germinal centres (Fig. 4A). A darker staining mantle zone that consisted of well-packed small lymphocytes surrounded the follicles. The follicles were separated by the paracortex or deep cortex, which consisted of lymphocytes arranged in sheets extending between the follicles and into the medulla. Macrophages and moderate amounts of cellular debris were seen within the germinal centre of a number of the follicles. Some lymph nodes lacked obvious follicles and were poorly populated with lymphocytes.

Fig. 4.

[A] Lymph node demonstrating the prominent capsule (C), trabeculae (T) and lymphoid follicles (F) within the cortex. H&E, scale bar = 200 µm. [B] Lymph node medulla demonstrating medullary cords containing plentiful eosinophils (arrows) and plasma cells (PC), and other cells separated by sinusoids and trabeculae (T). H&E, scale bar = 20 µm. [C] Spleen demonstrating smooth muscle (red) and collagen (blue) within the capsule (C) and trabeculae (T), white pulp (WP) and red pulp (RP). Gomoris’ trichrome, scale bar = 200 µm. [D] Spleen demonstrating a number of follicles (F) with germinal centres within the white pulp and the surrounding red pulp (RP). H&E, scale bar = 200 µm.

The medulla consisted of lymphatic tissue extending from the paracortex as medullary cords containing plasma cells, macrophages, lymphocytes and prominent eosinophils. Eosinophils were often observed in small groups within the reticular framework (Fig. 4B). A network of branching and anastomosing sinusoids and trabeculae separated the medullary cords. Mast cells were seen in small to large numbers in the medulla, capsule, subcapsular sinus and trabeculae, and in a number of sections, macrophages within the cortex and medulla contained brown cytoplasmic pigment, shown to be haemosiderin.

Spleen

The spleen of the leopard seal (n = 9) was covered by a mesothelial lining (serosa) overlying a thick capsule that contained arteries, large thin-walled vessels, presumably veins and lymphatic vessels depending on the presence of erythrocytes within the lumen, and nerves. Prominent trabeculae (Fig. 4C) extended from the capsule into the splenic parenchyma and both the capsule and the trabeculae were composed of abundant smooth muscle cells, collagen and elastic fibres. The splenic parenchyma was divided into red and white pulp (Fig. 4D). Lymphoid follicles and, less commonly, periarterial lymphatic sheaths (PALS) were present in the white pulp. The majority of the lymphoid follicles had germinal centres, some of which contained hyaline deposits and occasional variably brown-pigmented macrophages. At the marginal zone of the white pulp, plentiful macrophages were seen, some with brown cytoplasmic pigment that was shown to be haemosiderin (Fig. 5A). Occasional eosinophils were also identified in the marginal zone. Within the red pulp, erythrocytes, variably pigmented macrophages, lymphocytes, plasma cells, other leukocytes, reticular cells and a meshwork of reticular fibres were seen and although no obvious islands of haematopoiesis were evident, scattered erythroid and myeloid precursors were observed. Scattered megakaryocytes and precursors (Fig. 5B), sometimes in clusters of 2–4 cells, were seen within the red pulp often located close to veins near the trabeculae and the capsule. In some sections containing fewer erythrocytes, clusters of large mononuclear cells with abundant cytoplasm were more prominent and were thought to be macrophages or support cells. Sheathed capillaries (periarterial macrophage sheaths) were readily observed in the red pulp in a number of sections. Sinuses were not apparent in the sections examined. Mast cells were observed in small numbers in the red pulp, capsule and trabeculae.

Fig. 5.

[A] Spleen demonstrating the distribution of haemosiderin (blue) within the macrophages, particularly in the marginal zone of the red and white pulp and within the red pulp (RP), follicles (F) in the white pulp, trabeculae (T) and periarterial sheaths (arrows). Perl's, scale bar = 200 µm. [B] Spleen demonstrating the red pulp consisting of splenic cords and sinusoids, megakaryocytes (M) and haemosiderin (brown) within the cytoplasm of macrophages (arrows). H&E, scale bar = 20 µm. [C] Skin. Epidermis and papillary dermis (PD) of the skin showing the stratum corneum (SC), stratum spinosum (SS) and stratum germinativum (SG) of the epidermis and a hair follicle (H) extending to the surface of the skin. The pigmentation of the epidermis, particularly the stratum germinativum, is prominent. H&E, scale bar = 100 µm. [D] Dermis of the skin demonstrating cross-sections of compound hair follicles composed of a guard hair follicle (H), 1–2 secondary follicles (arrows) and sebaceous gland (S). Sweat glands are not readily discerned. H&E, scale bar = 100 µm.

Integument

Skin

The skin of the leopard seal (n = 4) was composed of an outer epidermis, dermis and an inner hypodermis (blubber layer, see below) and was a highly vascular tissue. The epidermis consisted of a relatively thick keratinized stratified squamous epithelium with some folds and dermal papillae. The outermost layer of the epidermis, the stratum corneum (horny layer), was a thick layer consisting of continuous keratinized lamellae. The stratum lucidum was absent. Although present, the stratum granulosum was generally not a well-defined layer and appeared to be discontinuous. Both the stratum granulosum and the keratohyalin granules within it were not prominent, which may be partly due to masking of this layer by the plentiful melanin granules observed in the epidermis. The stratum spinosum consisted of several layers of irregular polygonal cells that became more squamous towards the epidermal surface. The stratum germinativum, the deepest epidermal layer, consisted of a single layer of cuboidal to low columnar cells with large, ovoid nuclei. In all of the sections examined the epithelium, particularly the stratum germinativum and stratum spinosum, was heavily pigmented, with fine brown melanin granules (Fig. 5C).

In the underlying dermis, a thin superficial papillary layer and a substantially thicker deep reticular layer were indistinctly separated. The papillary layer consisted of loose connective tissue with greater cellularity compared with the reticular layer, and contained small blood vessels; in one section, melanocytes were seen. The reticular layer consisted of dense irregular connective tissue with variable amounts of adipose tissue and muscle fibres in the deeper portions, and abundant blood vessels. The collagen bundles within the reticular layer were thicker than those in the papillary layer. Hair follicles were embedded in the dermis and were seen at varying stages of activity. In three out of four sections, the majority of the hair follicles were simple, consisting of a large primary hair follicle producing a non-medullated guard hair, and associated well-developed sebaceous gland and small, simple tubular apocrine sweat gland. Groupings of cross-sections of the apocrine glands, presumably due to their coiled nature, were generally associated with the lower portion of each hair follicle. The secretory portion of the sweat glands had a large lumen lined by flattened cuboidal to low columnar epithelial cells. Compound hair follicles consisting of a primary hair follicle and associated structures as well as 1–2 secondary follicles of smaller diameter were commonly seen in only one in four seals (Fig. 5D). Arrector pili muscles were not associated with hair follicles in the leopard seal. Melanin pigment was present in the external root sheath of the hair follicles, particularly in the superficial portions, and occasionally, brown pigment was seen within the cells of the sebaceous glands. Sensory corpuscles were not observed in the sections examined.

Blubber

The blubber of the leopard seal (n = 4) consisted of uniformly sized adipose cells divided into indistinct lobules by septa of loose connective tissue. Sparse collagen and reticular fibres surrounded each adipose cell and fibrocytes were seen between individual adipocytes. Adipose cells appeared empty as a result of the loss of their single large lipid droplet in processing, and the nucleus of these cells was peripherally displaced. Dense bundles of collagen were seen ramifying throughout the blubber, and blood vessels were plentiful.

Discussion

Cardio-respiratory system

Apart from the prominent Purkinje cells in the leopard seal endocardium, the ventricular wall of the leopard seal was similar to that described in other pinniped species (Pfeiffer & Viers, 1995; Stewardson et al. 1999). The presence of Purkinje cells in the endocardium of the leopard seal may reflect species variation, or their absence in previous studies may be due to the lack of an endocardial layer in the sections examined. The Purkinje cells in the leopard seal were similar to those described in cetaceans, which are large and clearly demarcated from the underlying myocardium and were postulated to provide a selective advantage for the conduction of the cardiac impulse (Simpson & Gardner, 1972). It is assumed that this is also the case in the leopard seal.

The trachea of the leopard seal was similar to that described in the Weddell seal, Leptonychotes weddellii (Boyd, 1975), Cape fur seal, Arctocephalus pusillus (Stewardson et al. 1999), and ringed seal, Phoca hispida (Smodlaka et al. 2006). In the Weddell seal, in addition to the predominantly mucous-secreting seromucous tubuloacinar glands, large modified muco-serous glands lined by simple squamous epithelium termed diverticulae were observed in the trachea and pulmonary airways and were seen with increasing frequency in the tracheo-bronchial tree as far as the terminal bronchioles (Boyd, 1975). In the leopard seal, these structures identified in the trachea and large airways were thought to be the aforementioned seromucous glands exhibiting different stages of secretory activity. Glands were not observed in the more distal bronchi and terminal bronchioles in the leopard seal in contrast to the above description in the Weddell seal (Boyd, 1975).

The histological features of the lungs of the leopard seal were similar to those described in other pinniped species (Bélanger, 1940; Simpson & Gardner, 1972; Denison & Kooyman, 1973; Boshier & Hill, 1974; Boyd, 1975; Welsch & Drescher, 1982; Britt & Howard, 1983; Lowenstine & Osborn, 1990; Stewardson et al. 1999). However, when compared with the lungs of their terrestrial counterparts, pinniped lungs, including those of the leopard seal, possessed: a thick visceral pleura (Bélanger, 1940; Welsch & Drescher, 1982; Britt & Howard, 1983; Stewardson et al. 1999; Smodlaka et al. 2006); prominent connective tissue septa dividing the parenchyma into small lobules (Hepburn, 1915; Bélanger, 1940; Simpson & Gardner, 1972; Boshier & Hill, 1974; Boyd, 1975; Welsch & Drescher, 1982; Stewardson et al. 1999); more extensive connective tissue (Welsch & Drescher, 1982); plentiful, large glands and goblet cells in the mucosa of the large airways (Simpson & Gardner, 1972; Stewardson et al. 1999); and reinforcement of the terminal airways with muscle, such as around the openings of the alveoli and in the alveolar walls, and/or cartilage (Denison & Kooyman, 1973; Kooyman, 1973).

Notable differences in the histology of the lungs in otariid and phocid seals have also been observed, particularly in regard to the small airways. In otariids, the small airways are reinforced by cartilage plates extending to the mouth of the alveolar sacs (Denison et al. 1971; Simpson & Gardner, 1972; Denison & Kooyman, 1973; Stewardson et al. 1999). However, cartilage has not been observed to this level in the lungs of phocid seals (Simpson & Gardner, 1972; Denison & Kooyman, 1973; Boshier & Hill, 1974; Welsch & Drescher, 1982) including the leopard seal in the present study, although individual cartilage segments were reported in the respiratory bronchiole and occasionally at the opening of the alveolar duct in the ringed seal (Smodlaka et al. 2006) and as far as the terminal bronchioles in the Weddell seal (Boyd, 1975). The smaller airways of phocid seals, however, contain more muscle than that seen in otariids (Denison et al. 1971). Like that of the leopard seal, the alveolar wall of the Weddell seal and crabeater seal, Lobodon carcinophaga, was composed of a connective tissue framework containing smooth muscle cells and elastic fibres, with denser smooth muscle and collagen forming the muscular opening of each alveolus (Boshier & Hill, 1974; Welsch & Drescher, 1982). A complete smooth muscle layer was also seen in the smaller airways of the leopard seal, as described in the lungs of the Cape fur seal (Stewardson et al. 1999). Myoelastic sphincters were not seen in the terminal sections of the bronchioles in the lungs of the leopard seal, as described in a number of small cetaceans and in the harbour seal, Phoca vitulina (Bélanger, 1940), and have not been reported in other phocid species including Ross seals, Ommatophoca rossii, and leopard seals (Kooyman, 1967 personal observations in Kooyman & Andersen, 1969) and the Weddell seal (Boyd, 1975). The glands in the large airways of the leopard seal appeared to be predominately serous secreting glands, similar to the finding in other Antarctic phocids (Boshier & Hill, 1974; Welsch & Drescher, 1982), whereas mucous glands have been reported in several otariid species (Simpson & Gardner, 1972; Stewardson et al. 1999).

Several histological features of the lungs of pinnipeds have functional significance related to their marine environment. The presence of a thick pleura and prominent interlobular septa is thought to impart rigidity to the lung, providing it with increased resistance to compression during a dive as well as facilitating rapid reopening of the alveoli upon resurfacing (Welsch & Drescher, 1982). The reinforcement of the terminal airways with muscle and/or cartilage has several proposed functions, including: prevention of airway collapse or compression of the lungs when diving (Boyd, 1975; Britt & Howard, 1983); enabling rapid ventilation at the surface between dives (Denison & Kooyman, 1973; Kooyman, 1973; Boshier, 1974); and preventing decompression sickness by facilitating complete alveolar emptying during a dive, isolating compressed gas in the lungs from the pulmonary capillary blood, and thus limiting the influx of nitrogen (Denison et al. 1971; Boshier & Hill, 1974; Welsch & Drescher, 1982). Also noted in one study was a greater amount of smooth muscle in the wall of the respiratory bronchioles in Weddell seals compared with crabeater seals, thought to be related to the greater diving depths attained by the Weddell seal (Welsch & Drescher, 1982).

The difference in the muscle/cartilaginous support observed in phocid and otariid seals has been explained by their different diving strategies. Phocids dive deeply and for long periods, staying at the surface for some time after a dive. By contrast, otariids dive less deeply and for shorter periods, but surface for single rapid ventilations before resubmerging (Denison & Kooyman, 1973). It is thought that the heavy reinforcement of the smaller airways in otariids functions not to limit nitrogen absorption during the course of a dive, but to allow for high flows at low lung volumes during expiration at the surface, and the muscular support of the smaller airways in phocids was shown to be sufficient to allow displacement of alveolar gas during a dive, thereby preventing decompression sickness (Denison & Kooyman, 1973).

The alveolar wall of the leopard seal contained smooth muscle cells and elastic fibres, as reported in other pinniped species (Boshier & Hill, 1974; Boyd, 1975; Welsch & Drescher, 1982; Stewardson et al. 1999). A single capillary layer was observed, similar to other phocids (Bélanger, 1940; Simpson & Gardner, 1972; Denison & Kooyman, 1973; Boshier & Hill, 1974; Boyd, 1975; Welsch & Drescher, 1982; Britt & Howard, 1983), contrasting with the double row of capillaries described in cetacea and some otariid species (Simpson & Gardner, 1972). However, in some alveolar septa, a double capillary layer appeared to be present. This was thought to be a sectioning artefact, pulmonary congestion, which was also reported to be responsible for the impression of a double capillary layer in the alveolar wall of the Weddell seal (Boyd, 1975), or folding/plication of the alveolar wall, as also reported in Weddell seals functioning to limit reabsorption of nitrogen (Boshier, 1974; Boshier & Hill, 1974). However, in another study of Weddell and crabeater seals it was suggested that the observed folding or plication could be an artefact (Welsch & Drescher, 1982).

Lymphatic aggregations in the lamina propria and submucosa of the airways were not prominent in the lungs of the leopard seals examined but have been reported in pinnipeds (Simpson & Gardner, 1972; Stewardson et al. 1999). Pulmonary parasites were not commonly observed and may be the reason for the absence of prominent lymphatic aggregations in the airways of these seals. However, scattered lymphocytes and plasma cells were observed in the lamina propria and submucosa of the airways, as described in the Cape fur seal (Stewardson et al. 1999).

Digestive system

Adaptations of the histology of the digestive system to an aquatic lifestyle in the leopard seal were not obvious, a similar finding to that reported in Weddell and crabeater seals (Eastman & Coalson, 1974; Schumacher et al. 1995).

The oesophagus of the leopard seal was similar to that described in the Weddell seal (Eastman & Coalson, 1974). However, differences included slight keratinization of the stratified squamous epithelium, the absence of a well-developed lamina muscularis mucosae, described as a single thick longitudinal layer in the Weddell seal, and the predominance of skeletal muscle in the tunica muscularis of the leopard seal. It is assumed that the anterior oesophagus was sampled in the present study due to the presence of an adventitia as opposed to a serosa, and the predominance of skeletal muscle in the tunica muscularis. Most probably, differences in the site of sampling of the oesophagus will explain the difference in the type of muscle seen in the inner circular layer of the tunica muscularis of the two species (predominantly smooth muscle in the Weddell seal), although species differences cannot be excluded as the cause. The mucous secretion of the plentiful submucosal glands observed in the oesophagus of the Weddell seal (Eastman & Coalson, 1974) and the leopard seal in the present study is thought to augment salivary secretion.

The stomach of the leopard seal was similar to that described in several pinniped species (Simpson & Gardner, 1972; Eastman & Coalson, 1974; Schumacher et al. 1995; Stewardson et al. 1999). Primary lymphoid follicles reported in the Cape fur seal (Stewardson et al. 1999) and Weddell seal (Eastman & Coalson, 1974) were not evident in the lamina propria of the leopard seal stomach and although this finding may be a species difference, it could be due to the site of sample collection. Lymphoid follicles were particularly noted in the pyloric region of the stomach in the Cape fur seal (Stewardson et al. 1999), whereas in the present study, the proper gastric (fundic) region was sampled. The layers of the tunica muscularis were not readily distinguishable in the leopard seal, although they appeared to be similar to the description of these layers in the Cape fur seal (Stewardson et al. 1999) and the Weddell seal (Eastman & Coalson, 1974). Abundant parietal cells were present in the gastric glands of the leopard seal, which is similar to the finding in the Cape fur seal (Stewardson et al. 1999) and California sea lion, Zalophus californianus (Simpson & Gardner, 1972).

The histological features of the small and large intestine of the leopard seal were similar to those reported in other pinniped species (Eastman & Coalson, 1974; Schumacher et al. 1995; Stewardson et al. 1999). Plicae circulares were observed in the small intestine of the Weddell seal (Eastman & Coalson, 1974) but were not prominent in the leopard seal. Brunner's glands were also absent in the sections of the small intestine of the leopard seal examined, most likely due to the site of sampling being confined primarily to the jejunum and ileum. Brunner's glands have a restricted distribution in the duodenum, and species variation in their distribution has also been noted (Simpson & Gardner, 1972). Variation in the thickness of the outer longitudinal layer of the tunica muscularis was not observed in the large intestine of the leopard seals in the present study. Similarly, in the colon of the Weddell seal, the thickness of the outer longitudinal layer of the tunica muscularis did not vary, presumably due to the absence of taeniae coli (Eastman & Coalson, 1974), but taenia were reported in the colon of the Weddell seal in a later study (Schumacher et al. 1995). Diffuse infiltration of the lamina propria in both the small and the large intestine of the leopard seal by lymphoid cells, polymorphonuclear leukocytes and eosinophils has been previously reported in pinnipeds (including Simpson & Gardner, 1972; Eastman & Coalson, 1974) and is most probably related to the heavy parasite burdens present in the leopard seals examined. The number of mononuclear, polymorphonuclear cells and eosinophils varied between sections, and greater numbers were associated with cestodes within the lumen. In some sections, marked infiltration of the lamina propria with mononuclear cells was seen.

The liver of the leopard seal was similar to that described in other pinnipeds (Eastman & Coalson, 1974; Stewardson et al. 1999). Despite the increase in the amount of connective tissue in some portal areas and an increase in the connective tissue and smooth muscle component of the vascular structures within them, a thick muscular wall of branches of the portal veins, reported to be a sphincter for blood pooling during deep dives in cetaceans (Simpson & Gardner, 1972; Lowenstine & Osborn, 1990), was not observed. A number of central veins did possess a thick collagen and smooth muscle coat and this has not been previously reported in pinnipeds. Haemosiderin was present in the hepatocytes in a number of the sections examined and the presence of pigment in hepatocytes has been previously reported in pinnipeds (Eastman & Coalson, 1974; Britt & Howard, 1983; Lowenstine & Osborn, 1990) and is postulated to represent a normal storage pool of iron (Britt & Howard, 1983).

Few studies have documented the histology of the pancreas of pinnipeds. An interesting feature of the pancreas of the leopard seal was the varying size of the islets of Langerhans and this feature has been reported in other marine mammals (Lowenstine & Osborn, 1990) including the Weddell seal (Eastman & Coalson, 1974). Its functional significance is unclear.

Kidney

The structure of the reniculate kidney of the leopard seal was similar to that described in other seals (Bester, 1975; Vardy & Bryden, 1981; Britt & Howard, 1983; Stewardson et al. 1999). The reniculate form is thought to accommodate an increase in the number of glomeruli in the kidney (Harrison & Tomlinson, 1963; Vardy & Bryden, 1981) and may have developed to increase the area of interface between the kidney medulla and pelvis in larger mammalian species (Vardy & Bryden, 1981). The boundaries between the renicule in the kidney of the leopard seal were incomplete and often indistinct, formed by connective tissue, veins and arteries. In the Weddell seal and Cape fur seal this boundary was formed by arteries and veins and not connective tissue septa (Vardy & Bryden, 1981; Stewardson et al. 1999); however, younger Weddell seals had greater amounts of connective tissue surrounding the interrenicular veins compared with adult seals (Vardy & Bryden, 1981). Although the interrenicular connective tissue in subadult leopard seals appeared to be reasonably prominent, it was difficult to determine if it was comparatively greater than that of adult seals due to the limited sections of adult kidney available for examination.

A complete layer of collagen and smooth muscle extending from the calyx wall and extending along the cortico-medullary junction (sporta perimedullaris musculosa) was absent, despite the increase in the amount of collagen fibres and to a lesser extent smooth muscle at the corticomedullary junction, a similar finding to a previous study in the leopard seal (Cave & Aumonier, 1964). The sporta perimedullaris musculosa has been documented in cetaceans (Cave & Aumonier, 1964; Simpson & Gardner, 1972) but has not been reported in pinnipeds (Cave & Aumonier, 1964; Simpson & Gardner, 1972; Bester, 1975; Stewardson et al. 1999), except in the Weddell seal where it is described as small and consisting of a few, thin, widely spaced strands of collagen and muscle (Vardy & Bryden, 1981). Secondary medullary pyramids, which increase the interface between the renal medulla and the pelvis, were absent in the leopard seal kidney sections examined in the present study, but have been reported in a Weddell seal pup but not adult Weddell seals, and were thought to be related to age (Vardy & Bryden, 1981). Both adult and subadult leopard seals were examined in the present study without this feature being noted.

Lymphatic system

The histology of the lymph node of the leopard seal was similar to previous reports of the lymph node in pinnipeds (Lowenstine & Osborn, 1990; Welsch et al. 1997) and terrestrial mammals. The activity state and appearance of the lymph nodes examined was variable and the key factors thought to be contributing to this variation include history of antigen exposure, the particular lymph node examined, the age of the seal from which the lymph node was taken, as well as cause of death. In lymph nodes with cortical follicles, the majority were secondary follicles similar in appearance to the follicles in harbour and grey seals, Halichoerus grypus (Welsch et al. 1997). In other pinniped species, lymph nodes were described as sparsely and diffusely populated with lymphocytes with inactive germinal centres, a finding attributed to washout due to autolysis (Lowenstine & Osborn, 1990), and the age, health and lack of immunogenic stimuli in the animals examined (Simpson & Gardner, 1972). Sparsely populated lymph nodes were also seen in the present study and this finding was attributed to acute lymphoid depletion due to endogenous steroid release (K. Rose, Veterinary and Quarantine Centre, Taronga Zoo Mosman, NSW, pers. comm.). As a consequence, in some of the lymph nodes examined in the leopard seal, the cortico-medullary junction was difficult to delineate clearly, a finding not reported in harbour and grey seals (Welsch et al. 1997).

Plasma cells were common in the medulla of the lymph nodes of the leopard seals examined, similar to findings for harbour and grey seals (Welsch et al. 1997). Mast cells were also numerous in some sections, but their location varied somewhat from that previously reported (Welsch et al. 1997). Eosinophils were plentiful, often in clusters similar to previous findings in other pinniped species (Simpson & Gardner, 1972; Welsch et al. 1997). The presence of plentiful eosinophils is a common finding in pinniped lymph nodes and is thought to reflect the ubiquitous presence of parasites in these species (Simpson & Gardner, 1972). The majority of the leopard seals examined had moderate to heavy gastrointestinal parasite burdens.

The spleen of the leopard seal was similar to that described in pinnipeds (Schumacher & Welsch, 1987; Lowenstine & Osborn, 1990; Stewardson et al. 1999) and terrestrial mammals. A notable feature of the spleen common to the leopard seal and other pinniped species is the presence of a thick capsule and prominent trabeculae. The storage function and release of blood from the pinniped spleen has been well documented (including Qvist et al. 1986; Schumacher & Welsch, 1987; Ponganis et al. 1992; Castellini & Castellini, 1993; Cabanac et al. 1997, 1999) and is facilitated by the well-developed capsule and trabeculae system and their smooth muscle content (Schumacher & Welsch, 1987). These features were better developed in the Weddell seal than in the crabeater seal and Antarctic fur seal, Arctocephalus gazella (Schumacher & Welsch, 1987), presumably due to the greater diving ability of the Weddell seal. Contraction of the spleen has been reported to be the cause of the increase in haematocrit, and subsequent increase in oxygen availability during diving (Qvist et al. 1986; Zapol et al. 1989; Ponganis et al. 1992; Hurford et al. 1996) and other periods of apnoea in seals (Castellini & Castellini, 1993; Castellini et al. 1996).

The predominance of well-developed follicles in the white pulp, plentiful plasma cells (Schumacher & Welsch, 1987; Stewardson et al. 1999) and eosinophils in the red pulp (Schumacher & Welsch, 1987) are thought to reflect an active immune response and high parasite burdens, as previously reported in Antarctic seals (Schumacher & Welsch, 1987). This is assumed to be the case in the leopard seals in the present study; however, although present, eosinophils were not plentiful in the marginal zone of the spleen. As noted in the present study, brown pigment (presumably haemosiderin) was reported in the splenic macrophages in several studies of pinnipeds (Simpson & Gardner, 1972; Schumacher & Welsch, 1987; Stewardson et al. 1999), and was particularly prominent in the red pulp and marginal zone, indicating areas of erythrocyte destruction (Schumacher & Welsch, 1987). Extramedullary haematopoiesis was also reported to be common in young pinnipeds, especially elephant seals, Mirounga sp. (Lowenstine & Osborn, 1990), and islands of haematopoiesis were reported in the red pulp of Antarctic seals (Schumacher & Welsch, 1987), but were not seen in the leopard seal, nor in a study of the spleen of the Cape fur seal (Stewardson et al. 1999). Despite the lack of islands of haematopoiesis in the spleen of the leopard seal, the presence of erythroid precursors within the red pulp could indicate that extramedullary haematopoiesis is a function of the spleen of this species when required. Thrombopoiesis, a function of the red pulp of the spleen of the leopard seal, was also reported in Antarctic seals (Schumacher & Welsch, 1987) but not in Cape fur seals (Stewardson et al. 1999). Mast cells were observed in small numbers in the spleen of the leopard seal, and were reported to be quite numerous in the medulla, capsule and trabeculae in the spleen of Antarctic seals (Schumacher & Welsch, 1987). Although it is not possible without ultrastructural studies to determine definitively if the leopard seal spleen is sinusal or non-sinusal, other phocid seals including the hooded seal, Cystophora cristata (Cabanac et al. 1999), and Weddell and crabeater seals are reported to have non-sinusal spleens (Schumacher & Welsch, 1987) and presumably this is the case in the leopard seal.

Integumentary system

The histology of the skin of the leopard seal was similar in most respects to that described previously in pinnipeds (including Montagna & Harrison, 1957; Harrison & Tomlinson, 1963; Scheffer, 1964; Ling & Thomas, 1967; Ling, 1968, 1974; Simpson & Gardner, 1972). The increased thickness of the epidermis, the absence of arrector pili muscles and thus flattened hairs, and the secretion of the large and numerous sebaceous glands prevents waterlogging of the skin and hairs and reflect adaptations to an aquatic lifestyle (Montagna & Harrison, 1957).

Apart from in one leopard seal, the vast majority of the hair follicles were simple, not compound, and where compound follicles were present, the majority contained generally one, occasionally two under-hairs. Similarly, in a previous study, an adult leopard seal was reported to have compound follicles with only one under-hair (Scheffer, 1964). Phocid seals are reported to have compound hair follicles (Montagna & Harrison, 1957; Harrison & Tomlinson, 1963; Stutz, 1967; Ling, 1974) with few if any secondary or under-hairs (Ling, 1970), although greater numbers of under-hairs were reported in ice-dwelling seals (Ling, 1974). Air bubbles adhere to the hairs when the seal dives, particularly the under-hairs, and may provide an insulative quality to the skin in addition to keeping the skin dry (Montagna & Harrison, 1957; Ling, 1968). Simple follicles have been reported in adult southern elephant seals, Mirounga leonina (Ling, 1968), northern elephant seals, Mirounga angustirostris, and Hawaiian monk seals, Monachus schauinslandi (Scheffer, 1964). The absence of large numbers of under-hairs in the follicles may indicate that these species use other mechanisms for insulation such as modification of blood flow (Ling, 1968) and a thick blubber layer.

Although scattered clusters of keratohyalin granules were identified, the absence of a well-developed and prominent stratum granulosum as noted in the present study has also been reported in harbour seals and southern elephant seals (Montagna & Harrison, 1957; Ling, 1968) and is thought to result in a more pliable horny layer (Ling, 1968) so that the stratum corneum is a more permanent layer as opposed to a surface of scaly keratin that could become waterlogged and flake off (Montagna & Harrison, 1957). The stratum granulosum has been reported to be most evident at around the time of the moult in other pinniped species, but is well developed in the densely haired regions of otariids (Ling, 1974). The skin samples examined in the present study were collected from non-moulting leopard seals, so it is possible that the absence of a prominent stratum granulosum represents a normal variation in the prominence of this layer, depending on the stage of the skin cycle in this species.

Large amounts of lipid have been reported in the horny layer of the skin of the leopard seal and other seal species (Montagna & Harrison, 1957; Ling, 1968, 1974) but could not be demonstrated in the formalin-fixed tissues used in the present study. The sebum produced by the large sebaceous glands may also aid the maintenance of the stratum corneum, making it impervious and waterproof, and may prevent it from drying out and flaking off (Montagna & Harrison, 1957; Ling, 1968). Lipids within the horny layer may also have insulative properties (Montagna & Harrison, 1957; Ling, 1968). Abundant melanin pigment was seen in the epidermal cells of the leopard seal similar to other seal species, and is thought to function in protecting the epidermal cells from sunlight, as well as playing a role in heat absorption and insulation (Montagna & Harrison, 1957; Harrison & Tomlinson, 1963).

The blubber of the leopard seal was similar histologically to that described in seals (Ling, 1974) and cetaceans (Simpson & Gardner, 1972) and is reported to be an aid to buoyancy (Harrison & Tomlinson, 1963; Kooyman, 1973), insulation (Harrison & Tomlinson, 1963; Bryden, 1964; Scheffer, 1964; Ling, 1974; Laws, 1977), an energy store (Ling, 1974; Laws, 1977) and an aid in locomotion by shaping and streamlining the body (Ling, 1974). A thick blubber layer was observed in the leopard seals sampled in Antarctica and is a useful adaptation for an Antarctic phocid.

Conclusion

In the present study, we describe the normal microscopic anatomy of the leopard seal. The histology of the tissues examined is consistent with the limited descriptions in the literature of this species, and is also consistent with previous findings in other pinniped species, and more specifically, phocid seals. A number of features were observed that appeared to confer upon the leopard seal advantages for an aquatic lifestyle and these were mainly limited to the lungs, spleen and integument. The findings presented will constitute an important reference detailing the normal histology of the leopard seal for future workers and studies investigating the cause of mortality in leopard seals in Antarctica, on an individual and on a population level when disease outbreaks occur, and in individual leopard seals in captivity. Further investigations could be employed, including electron microscopy, and histochemical and immunocytochemical techniques to elucidate more detailed descriptions of cell structure and function in the leopard seal.