The structure of the conus arteriosus of the sturgeon (Acipenser naccarii) heart: II. The myocardium, the subepicardium, and the conus-aorta transition
Article first published online: 25 OCT 2002
Copyright © 2002 Wiley-Liss, Inc.
The Anatomical Record
Volume 268, Issue 4, pages 388–398, December 2002
How to Cite
Icardo, J. M., Colvee, E., Cerra, M. C. and Tota, B. (2002), The structure of the conus arteriosus of the sturgeon (Acipenser naccarii) heart: II. The myocardium, the subepicardium, and the conus-aorta transition. Anat. Rec., 268: 388–398. doi: 10.1002/ar.10170
- Issue published online: 2 NOV 2002
- Article first published online: 25 OCT 2002
- Manuscript Accepted: 17 AUG 2002
- Manuscript Received: 4 JUN 2002
- Ministerio de Ciencia y Tecnología. Grant Numbers: PB98-1428-CO2-02, BMC2000-0118-CO2-01
- Programma Nazionale di Ricerche in Antartide
- Acipenser naccarii;
- conus arteriosus;
- lymphoid tissue
Sturgeons constitute a family of living “fossil” fish whose heart is related to that of other ancient fish and the elasmobranches. We have undertaken a systematic study of the structure of the sturgeon heart aimed at unraveling the relationship between the heart structure and the adaptive evolutionary changes. In a related paper, data were presented on the conus valves and the subendocardium. Here, the structure of the conus myocardium, the subepicardial tissue, and the conus-aorta transition were studied by conventional light, transmission, and scanning electron microscopy. In addition, actin localization by fluorescent phalloidin was used. The conus myocardium is organized into bundles whose spatial organization changes along the conus length. The variable orientation of the myocardial cell bundles may be effective in emptying the conus lumen during contraction and in preventing reflux of blood. Myocardial cell bundles are separated by loose connective tissue that contains collagen and elastin fibers, vessels, and extremely flat cells separating the cell bundles and enclosing blood vessels and collagen fibers. The ultrastructure of the myocardial cells was found to be very similar to that of other fish groups, suggesting that it is largely conservative. The subepicardium is characterized by the presence of nodular structures that contain lympho-hemopoietic (thymus-like) tissue in the young sturgeons and a large number of lymphocytes after the sturgeons reach sexual maturity. This tissue is likely implicated in the establishment and maintenance of the immune responses. The intrapericardial ventral aorta shows a middle layer of circumferentially oriented cells and internal and external layers with cells oriented longitudinally. Elastin fibers completely surround each smooth muscle cell, and the spaces between the different layers are occupied by randomly arranged collagen bundles. The intrapericardial segment of the ventral aorta is a true transitional segment whose structural characteristics are different from those of both the conus subendocardium and the rest of the ventral aorta. Anat Rec 268:388–398, 2002. © 2002 Wiley-Liss, Inc.
Sturgeons constitute a family of living “fossil” fish that have inhabited the coastal and river waters since at least the low Jurassic era (Bemis et al., 1997). The heart of sturgeons is related to that of other ancient fish and the elasmobranches in that it has a long conus arteriosus endowed with a muscular coat and several rows of conus valves. Sturgeons are also related to the teleosts in that they have initiated the process of skeletal ossification. Despite their phylogenetic position, little is known of the cardiovascular changes that must have accompanied evolutionary adaptation (see Burggren et al., 1997) in sturgeons. Similarly, the structural characteristics of the sturgeon heart are almost unknown. Knowledge of these characteristics would provide valuable insight into the adaptive modifications that have taken place in the sturgeon and would make it possible to relate those changes to both the phyletic position and the design of the cardiovascular system. As part of a broader study on the structure of the sturgeon heart, we have recently presented a systematic analysis on the structure of the conus arteriosus (Icardo et al., 2002). Data on the valve leaflets, the endocardium, and the subendocardium were included in that study. The present paper reports on the structure of the conus myocardium, the subepicardium, and the conus-aorta transition.
MATERIAL AND METHODS
A total of 15 specimens of Acipenser naccarii (A. naccarii Bonaparte 1836) were collected at two different fish farms in Italy and Spain and were divided into three groups according to age and body weight: group 1 (G-1), 20 months, 0.5 kg, eight animals; group 2 (G-2), 4 years, 4 kg, five animals; group 3 (G-3), 6 years, 8 kg, two animals. All the sturgeons were collected between June and September. The fish were killed with a blow to the head, the ventral body wall was opened, and the hearts were extracted. The hearts were flushed first with saline to clear the blood and then with 3% glutaraldehyde and immersed in the same fixative for 4–6 hr. The conus was separated from the rest of the heart and treated according to the following procedures.
For conventional light microscopy and transmission electron microscopy (TEM), fragments of the conus were postfixed in 1% osmium tetroxide, dehydrated in graded acetone and propylene oxide, and embedded in Araldite (Fluka, Chemie GmbH, Buchs, Switzerland). Semithin sections were cut with a LKB III ultratome, stained with 1% toluidine blue, and observed with a Zeiss III photomicroscope. Ultrathin sections were cut with a Leica Ultracut UCT ultratome, stained with uranyl acetate and lead citrate, and examined with a Zeiss ME 10C.
For scanning electron microscopy (SEM), fragments of the conus (or the whole conus) were dehydrated in graded acetone, dried by the critical-point method with CO2 as the transitional fluid, coated with gold, and observed under a Philips SEM 501 microscope.
Four more specimens (0.5 and 8 kg, two of each) were fixed in 4% paraformaldehyde for 7 hr, washed in phosphate-buffered saline (PBS), and stained with fluorescein isothiocyanate (FITC)-conjugated phalloidin (Sigma, St. Louis, MO) for actin, as previously described (Germroth et al., 1995). Briefly, the conus was embedded in 10% acrylamide and sectioned at 200 μm with an FTB Vibracut vibratome along the longitudinal and transverse axes. Then the sections were washed in PBS and stained in gel overnight with a 1:40 solution of fluorescent phalloidin (Sigma). The sections were washed in PBS, mounted with Vectashield H-1000 (Vector Laboratories, Inc., Burlingame, CA), and observed with a laser confocal BioRad MRC 1024 microscope.
The myocardial layer is located between the subendocardium and the subepicardial layer. Myocardial cells are grouped into bundles whose spatial organization varies considerably along the conus length (Fig. 1). Near the ventricle, most myocardial bundles are oriented circumferentially. Above this level, myocardial cell bundles may be arranged into an inner circumferential and an outer longitudinal layer, into several layers with different orientations, or into a single circumferential layer (Fig. 1). Myocardial cells generally present a central nucleus with a large nucleolus and a peripheral ring of myofibrils (Fig. 2). Mitochondria are usually arranged in a central core (Fig. 2), but individual mitochondria are also found peripherally or interrupting the course of the myofibrils. Cytoplasmic areas occupied by randomly arranged filaments may also be observed. Myocardial cells present a peripheral vesicular system (Fig. 3), are rich in glycogen, and are joined to each other through intercalated discs (Fig. 4). Glycogen accumulates in the intermyofilament sarcoplasm (Fig. 4) or appears as dense granules between the myofilaments. Lipid droplets can also be observed. The peripheral vesicular system consists of single vesicles arranged around the cell periphery (Fig. 3). Some of these vesicles may open to the extracellular space. The intercalated discs are situated at the tapered end of the myocytes, present smooth surface areas, and mainly consist of long fascia adherens (Fig. 4). The dense layer that surrounds this area is irregular and may be very thick. We could not detect a longitudinal component in the intercalated disc, but longitudinal cell surfaces are frequently joined by desmosomes, desmosome-like plaques of variable length, and a small number of gap junctions. The lateral ends of the Z-band often coincide with areas of surface attachment. Each myocyte is surrounded by a continuous basement membrane (Fig. 2). Multivesicular bodies, similar to those described previously (Icardo et al., 2002), were also observed.
Myocardial cell bundles are separated by loose connective tissue septa containing numerous vascular profiles, interstitial cells, collagen bundles of variable size and orientation, and some elastin fibers. Myocardial vessels have a well-developed basement membrane, are formed by flat endothelial cells rich in vesicles of micropinocytosis, and present associated pericytes (Fig. 5). Pericytes are also rich in micropinocytotic vesicles. Interstitial cells are fibroblast-like and show very thin and long cell extensions that course through the extracellular space (Fig. 2), establishing separation between myocardial bundles and vessels, and enclosing groups of collagen fibers. SEM reveals that these cells are extremely flattened (Fig. 6), show cytoplasmic dehiscences, and appear to form a syncitium (Fig. 7) that encloses collagen fibers and separates myocardial bundles.
The outer surface of the conus is covered by the epicardial epithelium and presents a nodular appearance (see Fig. 1 from Icardo et al., 2002). The subepicardial space contains fat tissue, vessels, fibroblasts, and collagen. However, the most striking feature is the presence of large, ovoid-to-round nodular bodies containing a large vascular space lined by a continuous endothelium (Fig. 8). The wall of these nodes is formed by dense connective tissue, and the nodes are bounded by similar structures and loose connective tissue or adipose tissue. The inner vascular cavity is partially occupied by a mass of tissue that appears suspended from a wide base located on the epicardial side. This mass of tissue is cauliflower-like and consists of a central area and an irregularly lobed peripheral area (Fig. 8). The central area appears subdivided into two parts with different tissue architecture and cell type distribution (Fig. 9). Close to the epicardium, light microscopy indicates the presence of granulocytes, macrophages, lymphocytes, a small number of mitotic figures, and numerous arterioles and vascular spaces (Fig. 10). Large cells with a clear nucleus and pale cytoplasm are distributed throughout this area. Interstitial cells forming a barrier around the vessels can also be seen (Fig. 10). TEM reveals the presence of numerous young forms of the white blood cell series. Thus, we find neutrophilic myelocytes showing different degrees of nuclear lobation (Fig. 11), eosinophilic myelocytes containing specific granules with rhombohedral crystals embedded in a very dense matrix (Fig. 12), and basophilic myelocytes showing round or oval granules filled with a dense matrix (Fig. 13). In the latter, the formation of large granules by the fusion of smaller ones is a common finding. Medium-sized and small lymphocytes are also found. Some granulocytes appear to be in an early phase of differentiation and cannot be identified as belonging to any of the granulocyte types described above. Numerous interstitial cells and some amyelinic nerve fibers are also found. Erythrocytes are present throughout the tissue and in the vascular spaces, but we were not able to distinguish erythroblasts, thromboblasts, or young platelets. All the different cell types were usually mixed, but they can also be grouped into discrete colonies (Figs. 10 and 11).
Away from the epicardium, and surrounding the area just described, there is a compact tissue consisting of closely packed lymphocytes that alternate with clear, interstitial cells (Fig. 14). Lymphocytes show rounded nuclei, peripheral chromatin, and a reduced cytoplasm (Fig. 15). Mitotic lymphocytes may appear in the center of small lymphocyte groups (Fig. 16). The boundary between these two subdivisions shows some cell mixing and the presence of small lymphocytes entering vascular spaces lined by thin cell prolongations of interstitial cells (Fig. 17). Interstitial cells surround lymphocyte groups and show intermediate filament bundles (Fig. 18). These cells may also present complex interdigitations, desmosomes, and hemidesmosomes.
The most peripheral area of these tissue nodes is fragmented into cords or small lobes consisting of a cortical band rich in interstitial cells and a dense extracellular matrix core that contains a small number of cells (Fig. 19). Some of the cords cross the large vascular space and reach the myocardial side. Most of the cells found in these lobes are fibroblast-like cells that show extremely contort nuclei and numerous and tortuous cell prolongations enclosing the extracellular matrix. This matrix is formed by collagen fibers and very dense amorphous material (Fig. 20). Medium-sized and small lymphocytes may appear nested in this matrix. Numerous blood cells, including lymphocyte aggregates, can be observed in the large vascular cavity (Fig. 8). Most remarkably, the appearance of these structures undergoes profound changes with age. The distinct organization described above is lost in sexually mature sturgeons. The nodular tissue is reduced to the presence of highly vascularized cords connected by long stalks to the epicardial and myocardial sides (Fig. 21). The lympho-hemopoietic tissue has disappeared and the remaining tissue is infiltrated by a large number of lymphocytes. In yet older animals (14 kg, not included here), the subepicardium is largely occupied with white adipose tissue and most of the nodes have disappeared. However, numerous lymphocytes remain associated with the connective trabecular network that supports the subepicardial vessels.
Conus-Ventral Aorta Transition
When viewed from outside, this area shows a regular, circular boundary in its tissue structure (see Fig. 1 from Icardo et al., 2002). On the inner side, the conus endocardium is continuous with the aortic endothelium (Fig. 22). At the level of the myocardial ring, which forms the distal conus border, the conus subendocardial cells are continuous with the aortic smooth muscle cells (Fig. 23). Furthermore, elastin fibers may extend across the conus border linking aortic and conus cells. The collagenous component of the aorta extends downstream and passes into the inner border of the myocardial ring (Fig. 22). Despite this smooth transition, the changes in architectural arrangement and cell phenotype are quite marked. The aortic wall consists of a discontinuous internal elastic lamina subjacent to the endocardium (Fig. 23), a middle layer of smooth muscle cells, and an adventitial layer (Fig. 24). Interestingly, smooth muscle cells organize into an inner longitudinal layer, a middle circumferential layer, and an external longitudinal layer (Fig. 24). Muscle cell layers are separated by large extracellular spaces. The external longitudinal layer is poor in cells and very rich in large collagen bundles, the amount of collagen increasing toward the aortic adventitia (Fig. 22). On the other hand, subendocardial conus cells are myofibroblasts (Icardo et al., 2002), while aortic cells are typical smooth muscle cells (Fig. 25). Elastin fibers surround each smooth muscle cell and are oriented along the main cell axis. Large collagen bundles, very discrete amounts of elastin, and amorphous material occupy the space between the cell layers. Collagen fibers appear oriented in different directions and can be seen in close contact with the elastin fibers and with the surface of the muscle cells (Fig. 25). In contrast, the ventral aorta above the pericardial sac shows a continuous subendocardial elastic lamina and has mostly lost the inner longitudinal cell layer (Fig. 26). However, the outer longitudinal cell layer is still present. An intima-like layer located between the internal elastic lamina and the smooth muscle cell layer is clearly visible, becoming more prominent with age. Also, cell organization becomes more irregular with age and fibrous plaques appear between the endocardium and a disrupted internal elastic lamina (Fig. 26). Under the fibrous plaques there is an increase in cell and extracellular matrix density. This includes the presence of smooth muscle cells.
Our structural findings in this and in the previous paper are summarized in Figure 27. The sturgeon conus is organized into concentric layers. The external, subepicardial layer contains thymus-like nodes and variable amounts of adipose tissue. The middle layer consists of myocardium organized into bundles whose orientation varies along the conus length. Myocardial cell bundles are separated by loose connective tissue septa that are continuous with the collagenous component of both the subepicardium and the subendocardium. The inner layer is an elastic coat that contains collagen and elastin material. The distribution of these components varies between the proximal and distal segments of the conus. Finally, the inner conus surface shows three valve rows, two proximal and one distal, separated by a space devoid of valvular structures.
The conus myocardium is compact and well vascularized. Myocardial vessels appear to arise from the cranial coronary artery, which runs along the ventral surface of the conus. In elasmobranches, the conus myocardium has been reported to be formed by circularly arranged myocytes (Santer, 1985; Farrell and Jones, 1992). In the sturgeon, the architectural arrangement is far more complicated and varies along the conus length. Organization of the myocardium into several spatial planes may be more effective in emptying the conus lumen during conus contraction and in bringing the conus valves together to prevent reflux of blood. The conus myocardial cells share many structural features with the ventricular myocardial cells of sturgeons (Mykeblust and Kryvi, 1979), elasmobranches, and teleosts (Santer, 1985; Satchell, 1991; Farrell and Jones, 1992). It has a peripheral system of vacuoles (caveolae) and a poorly developed sarcoplasmic reticulum; and it lacks T-tubules. Despite the absence of T-tubules, calcium is readily diffused in fish because of the small diameter of myofibrils and the tendency of myofibrils to arrange in a single peripheral ring (see Satchell, 1991). The peripheral vacuoles appear to be connected to the cisternae of the endoplasmic reticulum in the sturgeon ventricle (Mykeblust and Kryvi, 1979), although we were not able to observe such a connection. The structure of the intercalated discs is far less complicated than those in higher vertebrates (Cobb, 1974), and gap junctions and desmosomes occur along the lateral cell surface. The absence of nerve fibers in the conus myocardium is also consistent with previous studies in elasmobranches (Yamauchi, 1980). The nerve and nerve endings described in the sturgeon ventricle (Mykeblust and Kryvi, 1979) appear to correspond to the multivesicular bodies reported here. The many similarities between the myocardial cells of different fish groups support the idea that the structure of the myocardium is largely conservative (Mykeblust and Kryvi, 1979; Santer, 1985).
The presence of extremely flat interstitial cells is an interesting finding. These cells extend over long distances, spanning the myocardial bundles and separating muscle bundles, collagen fibers, and vessels. These cells resemble the stellate cells found in the interstitial space of several mammals and fish organs (Fujita et al., 1986; Martinotti et al., 1991). Interstitial cells here may have a similar function to that of stellate cells, separating territories on the one hand and providing a structural nexus for the conus myocardium on the other.
The most remarkable structural feature in the subepicardium is the presence of nodular structures containing lymphocytes and young forms of the white blood cell series in a very precise distribution. The outer part of the central tissue area of those nodes shares all the histological characteristics of the cortical area of the thymus of both fish (Zapata et al., 1996) and higher vertebrates (Fawcett, 1994). The most central part contains granulocytes and macrophages and resembles, at least at first sight, the thymus medulla. However, the presence of young forms of the granulocyte series indicates that this central area is a developing center for the white blood cell series. Thus, the subepicardium of the sturgeon A. naccarii appears to be a highly complex lympho-hemopoietic organ and is presumably involved in establishing immune responses in sturgeons. Whether the subepicardial tissue has two separate components, thymic and hemopoietic, or is a modified thymus with medullary hemopoietic capabilities is a matter of debate. We have not found any structure resembling the medullary Hassall's corpuscles, but the sturgeon thymus (Fange, 1982) and, indeed, all ectotherms lack these corpuscles. Sturgeons present a cervical thymus (Fange, 1982), and the presence of thymic tissue in two different locations is striking. However, this is not a singular case. Some marsupials present both, a cervical and a thoracic thymus (Yadav et al., 1972), which probably originate from different pharyngeal pouches. However, the point at which precursor lymphoid cells colonize the subepicardial tissue is unknown. The atrophy of the subepicardial tissue by the time the sturgeons reach sexual maturation is another similarity with the thymus. Atrophy of the thymic tissue and fat infiltration occurs in many fish groups (Fange, 1986), and in higher vertebrates with age.
The subepicardial tissue of the young A. naccarii heart was described in an early histological study as a complex lymphoid gland containing lymphocytes, macrophages, granulocytes, macrophages, and mitotic cells (Scatizzi, 1933). Thus, it was considered to be a site of lymphocyte, erythrocyte, and granulocyte production (Scatizzi, 1933; Zapata and Cooper, 1990). However, we have not found structural evidence that may suggest that the subepicardium is a site of erythrocyte or platelet production. The sturgeon subepicardium is described in other reports to contain lymph node-like structures (Fange, 1986). This apparent discrepancy is probably due to the fact that only large sturgeons were included in later studies. The histological findings in old sturgeons (Scatizzi, 1933; Fange, 1986) match those presented here in the oldest specimens examined, when the tissue is atrophic and appears infiltrated with lymphocytes. Our findings suggest that the sturgeon subepicardium is implicated in both the establishment and the maintenance of the immune responses in sturgeons.
Finally, the conus-aorta junction is another point of interest. The smooth transition between the conus subendocardium and the aorta indicates not only a close structural relationship, but also the existence of close interactions in the embryonic period. The arrest of the aortic tissue at the level of the conus myocardium suggests the existence of intrinsic myocardial signals that may regulate the characteristics of the subendocardium. The structure of the intrapericardial segment of the ventral aorta is very different from that of the elastic arteries in higher vertebrates (Fawcett, 1994). However, the types of cardiovascular modifications underlying the architectural differences between the sturgeon aorta and that of higher vertebrates (see Shadwick, 1999) are currently unknown. The intrapericardial segment of the ventral aorta shows well-developed inner and outer longitudinal layers of muscle cells, and the internal elastic lamina is discontinuous and located subendocardially. Also, elastin fibers surround the muscle cells instead of forming layers alternating with muscle cell layers. The ventral aorta above the pericardial sac shows a well-developed internal elastica and a thick layer of circumferentially oriented cells. The inner longitudinal cell layer is mostly residual, and a well-developed intima-like layer appears between the inner elastica and the smooth muscle cells. We indicated previously (Icardo et al., 2002) the lack of evidence for the existence of an intermediate segment between the conus myocardium and the aorta. However, the differences between the intrapericardial and extrapericardial segments of the ventral aorta found here suggest that the first is a true transitional (or intermediate) segment whose origin may be different from the rest of the ventral aorta. Whether this transitional segment may be equivalent to the truncus of higher vertebrates is still unclear. In the two aortic segments, the arrangement of the smooth muscle cells is irregular. The observations of elastin surrounding the muscle cells indicate that, at least at some points, elastin may form fenestrated lamina. The fibrous plaques appear in the oldest animals and are pathological changes linked to the onset of atherosclerosis. This may seem a surprising finding in sturgeons, but it also occurs, for instance, in the dogfish coronaries (Muñoz-Chapuli et al., 1991). This speaks in favor of the universality of patho-physiological mechanisms.
We have shown in this and in our previous paper (Icardo et al., 2002) that the sturgeon conus is a very complex structure. It is a contractile part of the heart, capable of controlling the reflux of blood. In addition, the conus subepicardium appears to be a complex lympho-hemopoietic tissue and is most likely involved in the establishment and maintenance of immune responses. Similar systematic studies in other fish groups would lead to a better understanding of the relationship between the structure of the heart and the phyletic and adaptive modifications involved in cardiovascular evolutionary changes.
Some of the sturgeons used in this study were kindly donated by A. Domezain, Sierra Nevada Fishery at Riofrio, Granada, Spain. The authors thank Drs. M. Lafarga and M.T. Berciano, for useful histological and structural insights, and R. Garcia-Ceballos, M. Mier, and L. González, for expert technical assistance.
- 1997. An overview of Acipenseriformes. Environ Biol Fish 48: 25–71. , , .
- 1997. Vertebrate cardiovascular systems. In: DantzlerWH, editor. Handbook of physiology, Sect. 13, Comparative physiology, Vol. 1. New York: Oxford University Press. p 215–308. , , .
- 1974. Gap junctions in the heart of teleost fish. Cell Tissue Res 154: 131–134. .
- 1982. A comparative study of lymphomyeloid tissue in fish. Dev Comp Immunol 2: 23–33. .
- 1986. Lymphoid organs in sturgeons (Acipenseridae). Vet Immunol Immunopathol 12: 153–161. .
- 1992. The heart. In: HoarWS, RandallDJ, FarrellAP, editors. Fish physiology, Vol. XII, The cardiovascular system, Part A. San Diego: Academic Press. p 1–87. , .
- 1994. W. Bloom and D.W. Fawcett: a textbook of histology, 12th ed. New York: Chapman & Hall. p 432–444. .
- 1986. Fine-structural characteristics of the liver of the cod (Gadus morhua macrocephalus), with special reference to the concept of a hepatoskeletal system formed by Ito cells. Cell Tissue Res 244: 63–67. , , , .
- 1995. Confocal microscopy of thick sections from acrylamide gel embedded embryos. Microsc Res Tech 30: 513–520. , , .
- 2002. The structure of the conus arteriosus of the sturgeon (Acipenser naccarii) heart. I. The conus valves and the subendocardium. Anat Rec 267: 17–27. , , , .
- 1991. Scanning electron microscopy of stellate cells of mammalian adrenal cortex as revealed by NaOH maceration. J Anat 179: 33–38. , , , , .
- 1991. Coronary myointimal lesions in the dogfish shark Scyliorhinus canicula. J Comp Pathol 105: 387–395. , , .
- 1979. Ultrastructure of the heart of the sturgeon Acipenser stellatus (Chondrostei). Cell Tissue Res 202: 431–438. , .
- 1985. Morphology and innervation of the fish heart. Adv Anat Embryol 89: 1–102. .
- 1991. Physiology and form of fish circulation. Cambridge: Cambridge University Press. p 10–40. .
- 1933. L'organo linfomieloide dello sturione. Arch Zool Ital 18: 1–26. .
- 1999. Mechanical design in arteries. J Exp Biol 202: 3305–3313. .
- 1972. The thymus glands of marsupial Setonix brachyurus (quokka) and their role in immune processes. Structure and growth of the thymus glands. Aust J Exp Biol Med Sci 50: 347–356. , , .
- 1980. Fine structure of the fish heart. In: BourneG, editor. Heart and heart-like organs, Vol. 1. New York: Academic Press. p 119–148. .
- 1990. The immune system: comparative histophysiology. Chichester: John Wiley & Sons. p 33–52. , .
- 1996. Structure of the lymphoid organs in elasmobranches. J Exp Zool 275: 125–143. , , , , .