Immune System Organs of Bony Fishes

C Soulliere and B Dixon, University of Waterloo, Waterloo, ON, Canada

r 2017 Elsevier Inc. All rights reserved.

Glossary feature from the earliest stages of development through to

Ontogenesis The development of an organism or maturity.
anatomical feature such as an organ or behavioral Parenchyma The distinct and essential organ tissue.

Lymphoid Organs of Bony Fishes

Bony fish lack both bone marrow and lymph nodes and therefore rely on other major lymphoid organs including the head kidney,
thymus, spleen and mucosa-associated lymphoid tissues (MALT) for defense against viruses, parasites, bacterial pathogens and
fungal infections (Kumar et al., 2016; Sahoo, 2006). The development of immunocompetent organs and the lymphoid cells that
these organs contain has been studied in several species including the channel catfish, common carp, rainbow trout, salmon, sea
bream and cobia (Klosterhoff et al., 2015; Sahoo, 2006). Studies suggest that there is a high level of variation in the age of
initiation of lymphoid organ ontogenesis, however generally in freshwater fish, such as salmon, trout and carp, lymphoid
organogenesis occurs in the following order: thymus, kidney and spleen (Klosterhoff et al., 2015; Sahoo, 2006). In marine fishes
including sea bream and cobia the general trend is that kidney is the first lymphoid organ to develop followed by spleen and then
thymus (Klosterhoff et al., 2015). Regardless of the observed differences in ontogenesis of lymphoid organs, like mammals, the
bony fishes have both primary lymphoid organs; the thymus and the head kidney, that are responsible for the production and
maturation of immune cells as well as secondary lymphoid organs; the spleen, head kidney and MALT (Sahoo, 2006). Addi-
tionally, the trunk kidney, liver and melano-macrophage centers (MMCs) play a role in the defense of bony fishes against
pathogens (Sahoo, 2006).

Primary Lymphoid Organs of Bony Fishes

Head Kidney
The kidney of fish is located outside the peritoneal cavity and is positioned behind the peritoneum along the dorsal body cavity
(Fig. 1; Abdel-Aziz et al., 2010; Sahoo, 2006). The organ is generally divided into the anterior pronephros or head kidney and the
posterior mesonephric renal kidney or opisthonephros, also referred to as the trunk kidney (Fig. 1; Kumar et al., 2016; Sahoo,
2006). As a fish matures the trunk kidney maintains excretory function and is primarily composed of excretory tissue with a small
amount of lymphoid tissue scattered throughout, while the head kidney loses excretory function as maturation occurs and adult
fish head kidney is primarily composed of hemopoietic tissue (Abdel-Aziz et al., 2010; Sahoo, 2006). Lymphohemopoietic cells
are scattered randomly throughout the stroma of the head kidney, which is comprised of heterogeneous fibroblastic reticular cells
and sinusoidal blood vessels (Sahoo, 2006; Zapata et al., 1996; Zapata, 1979). In the absence of bone marrow, the head kidney,

Head Kidney
Trunk Kidney

Head of Fish Tail of Fish

Fig. 1 Photograph depicting the kidney of a rainbow trout (black rectangle) along the dorsal body cavity outside the peritoneum. The head kidney
is on the right of the dotted line while the trunk kidney is on the left.

Reference Module in Life Sciences doi:10.1016/B978-0-12-809633-8.12179-X 1

2 Immune System Organs of Bony Fishes

which has been described as similar to mammalian bone marrow based on cellular composition, is a primary hemopoietic organ
of fish that functions as the lymphocyte precursor tissue responsible for the generation as well as maturation of B cells, and the
generation of T-cell progenitor cells that migrate to the thymus throughout life (Abdel-Aziz et al., 2010; Kumar et al., 2016;
Langenau et al., 2004; Zapata, 1979).

Thymus
The thymus of bony fish, like that of other vertebrates, is a paired organ (Bowden et al., 2005; Chilmonczyk, 1992; Sahoo, 2006).
In fish the thymus is typically located close to the opercular cavity in the dorsolateral region of each gill chamber with one lobe, the
outer thymus, positioned superficially and the second lobe positioned more deeply in the gill chamber epithelium (Fig. 2; Bowden
et al., 2005; Chilmonczyk, 1992; Sahoo, 2006). Further, the thymus is positioned on a layer of connective tissue and is covered by
a modified stratified epithelium (Sahoo, 2006). Thus thymocytes are separated from the external environment by only a single
layer of epithelial cells (Sahoo, 2006). The histological arrangement of the thymus into adjacent zones varies with species, and in
some cases age, consequently, a bony fish thymus may have two, three or four zones, however, in the majority of cases the zone
boundaries are poorly defined (Bowden et al., 2005; Chilmonczyk, 1992; Sahoo, 2006). The cellular components of the bony fish
thymus include non-lymphoid cells of epithelial origin that exist alongside the thymocyte populations where the epithelial-
reticular cells of the stroma create a supportive three-dimensional framework for the thymocytes and other free mesenchymal cells
such as macrophages and mast cells (Chilmonczyk, 1992; Sahoo, 2006). The presence of additional cell types has also been
reported in the thymus of fishes including monocytes, melano-machrophages and plasma cells as well as, mucus secreting cells
which have been observed infiltrating the thymus parenchyma (Chilmonczyk, 1992; Sahoo, 2006). The production and
maturation of T cells appears to be highly conserved, and in zebrafish, it has been demonstrated by Langenau et al. (2004) that T-
cell progenitor cells originate in the head kidney and migrate to the thymus where they undergo maturation to become T cells.
While mature T-cell populations can be found in peripheral sites including the intestine, esophagus, ovary, and the base of the
olfactory epithelium, the thymus is the only reported site of T-cell maturation in zebrafish (Langenau et al., 2004). Furthermore,
except those listed above, most peripheral organs lack mature T-cell populations, however, studies have suggested that thymo-
cytes migrate from the thymus to the spleen and kidney with the majority of these cells settling in the spleen (Chilmonczyk,
1992; Irwin and Kaattari, 1986; Langenau et al., 2004; Sahoo, 2006; Tatner, 1985). Although it appears that age-related, cyclic,
hormonal and seasonal thymic changes occur in fish, the pattern of these changes and degree of involution varies from species to
species, and in some cases, it is unclear if the thymus undergoes involution at all (Bowden et al., 2005; Chilmonczyk, 1992;
Sahoo, 2006).

Operculum

Thymus

Gill Arches

Fig. 2 Photograph depicting the opercular cavity and gill chamber of a rainbow trout demonstrating the position of the thymus relative to the gill
arches and operculum.
 Immune System Organs of Bony Fishes 3

Secondary Lymphoid Organs of Bony Fishes

MALT
The aquatic environment contains ubiquitous microorganisms and fish that inhabit this environment that are in constant contact
with potential pathogens (Gomez et al., 2013; Rombout et al., 2014). The MALT of bony fish are the first line of defense against
invading pathogens, and include the skin, gut, gills and nose, as well as, the urogenital tract (Gomez et al., 2013; Rombout et al.,
2014; Sahoo, 2006; Tacchi et al., 2014). The surface of these tissues is coated with mucus that is secreted from up to four different
types of secretory cells: malpighian cells, sacciform cells, club cells and/or goblet cells, and contains important innate and adaptive
immune defense mechanisms involved in both cellular and humoral immunity (Gomez et al., 2013; Sahoo, 2006; Salinas et al.,
2011; Tacchi et al., 2014). Historically, the gut-associated lymphoid tissue, which has two compartments: the lamina propria (LP)
and the intraepithelial (IEL) region, the gill-associated lymphoid tissue (GIALT), which includes the gills as well as the interbranchial
immune tissue (ILT), and the skin-associated lymphoid tissue were considered the three main MALT of bony fish (Salinas et al.,
2011). However, recently Tacchi et al. (2014) demonstrated that the nasopharynx-associated lymphoid tissue of the bony fish
olfactory organ plays a critical role in mucosal immunity and, like the gut, lymphoid cells of this organ are scattered in the IEL and
LP. Several components and characteristics of bony fish mucosal immunity are similar to that of mammals, including the presence of
secreted IgM, a polymeric Immunoglobulin Receptor (pIgR) and production of a downstream secretory component, however, in fish,
Peyer’s patches, mesenteric lymph nodes, Microfold cells (M cells) and the IgM-joining J chain are absent, and fish also lack tonsils
and adenoids (Gomez et al., 2013; Rombout et al., 2014; Sahoo, 2006; Salinas et al., 2011; Tacchi et al., 2014). Further, the primary
mammalian mucosal-associated immunoglobulin, IgA, is not found in fish, however, IgT is present in the mucus of teleost fish and
IgZ, an orthologue of IgT, is present in zebrafish and carp (Danilova et al., 2005; Hansen et al., 2005; Rombout et al., 2014; Ryo et al.,
2010; Salinas et al., 2011; Zhang et al., 2010). Evidence suggests that IgT is a specialized immunoglobulin of fish mucosal immunity
(Salinas et al., 2011; Zhang et al., 2010). Although there is some variation among species, commonly, lymphoid cells are scattered
along the alimentary canal of fish (Salinas et al., 2011). Notably, within the LP, numerous cell types including but not limited to
macrophages, granulocytes, lymphocytes and plasma cells have been reported while the main immune cells observed in the IEL are
T cells mixed with a few B cells (Salinas et al., 2011). In GIALT immune cell types that have been reported include magcrophages,
neutrophils, eosinophilic granulocytes and antibody secreting cells, however, following an in vitro experiment where gill cells were
exposed to lipopolysaccharide and phytohemagglutinin leading to a mitogenic response few B cells and many T cells were observed
(Salinas et al., 2011). Further, evidence suggests that the predominant immune cell type of the ILT is the T-cell (Salinas et al., 2011).
The skin and associated mucus composition of fish varies between species, however, macrophages, granulocytes and lymphocytes are
commonly observed and mucus production is highest in the area that surrounds the gill cover compared to any other skin site
(Salinas et al., 2011). Remarkably, the olfactory IEL rather than the LP is rich with B cells and populations of mature T cells also
localize to the olfactory epithelium (Langenau et al., 2004; Tacchi et al., 2014). Overall, MALT are a complex and important immune
defense system and many aspects of how they function to protect the bony fish has yet to be revealed (Salinas et al., 2011).

Spleen
The spleen, a small, encapsulated tissue, is a secondary lymphoid organ positioned near the liver and intestine and frequently
associates with pancreatic tissue (Van Muiswinkel et al., 1991; Zapata, 1982). Fibroblasts and collagenous fibers form the con-
nective tissue that encapsulates the parenchyma which contains blood vessels, red and white pulp and ellipsoids, which are
terminal capillaries that have a thick endothelial cell layer that is surrounded by a covering of reticular fibers and macrophages
(Espenes et al., 1995; Klosterhoff et al., 2015; Sahoo, 2006; Van Muiswinkel et al., 1991; Zapata, 1982). In bony fishes the red
and white pulp is not as clearly distinct as it is in mammals and in fishes the red pulp, which constitutes most of the splenic tissue
is made up of a cellular reticulum containing interspersed hemopoietic tissue and blood sinuses, while the white pulp, which
is poorly developed consists primarily of lymphoid cells diffusely dispersed within a reticular network (Sahoo, 2006;
Van Muiswinkel et al., 1991; Zapata, 1982). The types of immune cells that have been described in splenic lymphoid tissue
include, monocytes and macrophages, lymphocytes, plasma cells, granulocytes and dendritic-like cells (Press et al., 1994; Zapata,
1982). Notably, rosette forming lymphocytes, blast cells, plasma cells, macrophages/monocytes and granulocytes have been
reported in rainbow trout spleen, and several groups have also demonstrated that lymphocytes, including thymocytes from the
thymus, migrate preferentially to the spleen (Chiller et al., 1969; Chilmonczyk, 1992; Irwin and Kaattari, 1986; Tatner, 1985,
Tatner and Findlay, 1991; Zapata, 1982). Aggregation of lymphoid cells in the white pulp has been observed around splenic
arteries and ellipsoids, as well as around small MMCs (Sahoo, 2006; Van Muiswinkel et al., 1991; Zapata, 1982). MMCs have also
been observed in the red pulp of the bony fish spleen (Van Muiswinkel et al., 1991). It also appears that ellipsoids which are
involved in plasma filtration are responsible for entrapment of immune complexes and likely play an important role in the rapid
delivery of antigens to the lymphoid cells surrounding them (Espenes et al., 1995; Sahoo, 2006; Van Muiswinkel et al., 1991).

Kidney
The head kidney functions not only as a primary lymphoid organ, but also, as a secondary lymphoid organ rich with monocytes and
macrophages that are involved in phagocytosis of invading pathogens and secretion of cytokines, such as the pro-inflammatory
4 Immune System Organs of Bony Fishes

cytokines interleukin-1b and tumor necrosis factor, thus playing a key role in initiation of the immune response of fish (Kumar et al.,
2016; Press et al., 1994; Sahoo, 2006). Additionally, Chiller et al. (1969) have reported observation of rosette forming lymphocytes,
blast cells, plasma cells, macrophages/monocytes, and granulocytes in rainbow trout head kidney tissue. Furthermore, MMCs have
been reported in head kidney and it has been suggested that the trunk kidney, which also contains MMCs, as well as, some scattered
lymphoid tissues, may play a role in immune function (Agius and Roberts, 2003; Kumar et al., 2016; Sahoo, 2006; Zapata et al.,
1996).

MMCs

Macrophages in the head kidney as well as other fish organs including the trunk kidney, spleen, thymus, gills, brain, gonads,
intestinal submucsa and periportal regions of the liver that are pigmented are referred to as melano-macrophages (Agius and
Roberts, 2003; Kumar et al., 2016; Sahoo, 2006). Melano-macrophages may be observed singly or in aggregates called MMCs, and
are found primarily in the reticulo-endothelial supporting matrix of the hemopoietic tissues of fish (Agius and Roberts, 2003;
Kumar et al., 2016; Sahoo, 2006). Four pigments have been observed in melano-macrophages: melanin, lipofuscin, ceroid and
haemosiderin, and generally multiple pigments are observed not only within MMCs but also within a single malano-macrophage
(Agius and Roberts, 2003; Kumar et al., 2016; Sahoo, 2006). There are numerous functions attributed to MMCs, including the
accumulation of resistant pathogens and parasitic spores, antigen processing and entrapment of immune complexes leading to
high levels of antibody production (Agius and Roberts, 2003; Sahoo, 2006).

Other Important Immune-Related Organs of Bony Fishes

Liver
While little is known regarding the role of the liver in the immune response of bony fish, evidence of MMCs surrounding the portal
vein suggests that it likely plays a nominal role in defense against invading pathogens (Sahoo, 2006; Press and Evensen, 1999;
Wolke, 1992). Based on morphological criteria, Kupffer cells, which are liver specific macrophages observed in mammals, have
been described in a small handful of fish species, however, the general consensus remains that these cell are rare or do not exist at
all in most bony fish (Dalmo et al., 1997; Sahoo, 2006). Furthermore, though it appears to function less efficiently in the liver than
in spleen and kidney, it has also been reported that a reticulo-endothelial system present in the liver of bony fish plays a
scavenging role important in immune defense (Dalmo et al., 1997; Sahoo, 2006).

Heart

There is no direct evidence linking immune function in fish with the heart, however, some researchers, have observed the uptake of
foreign particles by the endocardial cells of the heart, and have hypothesized that this is the beginning of a phagocytic process or
initiation of transport to specialized phagocytic cells, such as, macrophages (Dalmo et al., 1997; Sahoo, 2006).

Conclusions

The lymphoid organs of the bony fish immune system are critical to successful immune defense. The primary lymphoid tissues are
essential in establishing a heavy arsenal of immunocompetent cells while secondary lymphoid tissues are key players in mounting
an immune response. Although some variation among species is evident, and there are exceptions to the descriptions discussed
here, the above information provides information that relates to the majority of bony fishes. The observed variations are likely due
to the large number of species included under the bony fish umbrella, the differing evolutionary age of these species and the
diverse microenvironments they inhabit.

References

Abdel-Aziz, E.S.H., Abdu, S.B., Ali, T.E.S., Fouad, H.F., 2010. Haemopoiesis in the head kidney of tilapia, Oreochromis niloticus (Teleostei: Cichlidae): A morphological (optical
and ultrastructural) study. Fish Physiology and Biochemistry 36, 323–336.
Agius, C., Roberts, R.J., 2003. Melano-macrophage centres and their role in fish pathology. Journal of Fish Diseases 26, 499–509.
Bowden, T.J., Cook, P., Rombout, J.H.W.M., 2005. Development and function of the thymus in teleosts. Fish and Shellfish Immunology 19, 413–427.
Chiller, J.M., Hodgins, H.O., Chambers, V.C., Weiser, R.S., 1969. Antibody response in rainbow trout (Salmo gairdneri) I. Immunocompetent cells in the spleen and anterior
kidney. Journal of Immunology 102, 1193–1201.
Chilmonczyk, S., 1992. The thymus in fish: Development and possible function in the immune response. Annual Review of Fish Diseases 2, 181–200.
 Immune System Organs of Bony Fishes 5

Dalmo, R.A., Ingebrigtsen, K., Bøgwald, J., 1997. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). Journal of Fish
Diseases 20, 241–273.
Danilova, N., Bussmann, J., Jekosch, K., Steiner, L.A., 2005. The immunoglobulin heavy-chain locus in zebrafish: Identification and expression of a previously unknown
isotype, immunoglobulin Z. Nature Immunology 6, 295–302.
Espenes, A., Press, C.M., Dannevig, B.H., Landsverk, T., 1995. Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss). Cell and Tissue
Research 282, 41–48.
Gomez, D., Sunyer, J.O., Salinas, I., 2013. The mucosal immune system of fish: The evolution of tolerating commensals while fighting pathogens. Fish and Shellfish
Immunology 35, 1729–1739.
Hansen, J.D., Landis, E.D., Phillips, R.B., 2005. Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway
in teleost fish. Proceedings of the National Academy of Sciences of the United States of America 102, 6919–6924.
Irwin, M.J., Kaattari, S.L., 1986. Salmonid B lymphocytes demonstrate organ dependent functional heterogeneity. Veterinary Immunology and Immunopathology 12, 39–45.
Klosterhoff, M.C., Pereira Junior, J., Rodrigues, R.V., et al., 2015. Ontogenic development of kidney, thymus and spleen and phenotypic expression of CD3 and CD4 receptors
on the lymphocytes of cobia (Rachycentron canadum). Anais da Academia Brasileira de Ciências 87, 2111–2121.
Kumar, R., Joy, K.P., Singh, S.M., 2016. Morpho-histology of head kidney of female catfish Heteropneustes fossilis: Seasonal variations in melano-macrophage centers,
melanin contents and effects of lipopolysaccharide and dexamethasone on melanins. Fish Physiology and Biochemistry, 1–20. doi:10.1007/s10695-016-0218-2.
Langenau, D.M., Ferrando, A.A., Traver, D., et al., 2004. In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish. Proceedings of the National
Academy of Sciences of the United States of America 101, 7369–7374.
Press, C.M., Dannevig, B.H., Landsverk, T., 1994. Immune and enzyme histochemical phenotypes of lymphoid and nonlymphoid cells within the spleen and head kidney of
Atlantic salmon (Salmo salar L.). Fish and Shellfish Immunology 4, 79–93.
Press, C.M., Evensen, O., 1999. The morphology of the immune system in teleost fishes. Fish and Shellfish Immunology 9, 309–318.
Rombout, J.H., Yang, G., Kiron, V., 2014. Adaptive immune responses at mucosal surfaces of teleost fish. Fish and Shellfish Immunology 40, 634–643.
Ryo, S., Wijdeven, R.H., Tyagi, A., et al., 2010. Common carp have two subclasses of bonyfish specific antibody IgZ showing differential expression in response to infection.
Developmental and Comparative Immunology 34, 1183–1190.
Sahoo, P.K., 2006. Immunocompetent organs in teleosts. In: Swain, P., Sahoo, P.K., Ayyappan, S. (Eds.), Fish and Shellfish Immunology: An Introduction. Delhi: Narendra
Publishing House, pp. 1–11. unknown ed.
Salinas, I., Zhang, Y.A., Sunyer, J.O., 2011. Mucosal immunoglobulins and B cells of teleost fish. Developmental and Comparative Immunology 35, 1346–1365.
Tacchi, L., Musharrafieh, R., Larragoite, E.T., et al., 2014. Nasal immunity is an ancient arm of the mucosal immune system of vertebrates. Nature Communications 5, 1–11.
Tatner, M.F., 1985. The migration of labelled thymocytes to the peripheral lymphoid organs in the rainbow trout, Salmo gairdneri Richardson. Developmental and Comparative
Immunology 9, 85–91.
Tatner, M.F., Findlay, C., 1991. Lymphocyte migration and localisation patterns in rainbow trout Oncorhynchus mykiss, studied using the tracer sample method. Fish and
Shellfish Immunology 1, 107–117.
Van Muiswinkel, W.B., Lamers, C.H.J., Rombout, J.H.W.M., 1991. Structural and functional aspects of the spleen in bony fish. Research in Immunology 142, 362–366.
Wolke, R.E., 1992. Piscine macrophage aggregates: A review. Annual Review of Fish Diseases 2, 91–108.
Zapata, A., 1979. Ultrastructural study of the teleost fish kidney. Developmental and Comparative Immunology 3, 55–65.
Zapata, A., 1982. Lymphoid organs of teleost fish. III. Splenic lymphoid tissue of Rutilus and Gobio. Developmental and Comparative Immunology 6, 87–94.
Zapata, A.G., Chiba, A., Varas, A., 1996. Cells and tissues of the immune system of fish. In: Iwama, G., Nakanishi, T. (Eds.), The Fish Immune System: Organism, Pathogen
and Environment. San Diego, CA: Academic Press, pp. 19–20. unknown ed.
Zhang, Y.A., Salinas, I., Li, J., et al., 2010. IgT, a primitive immunoglobulin class specialized in mucosal immunity. Nature Immunology 11, 827–835.