FOLIA PARASITOLOGICA 46: 1-9, 1999

Immune mechanisms in fish skin against monogeneans - a model

Kurt Buchmann
Department of Veterinary Microbiology, Section of Fish Diseases, Royal Veterinary and Agricultural University, 13 Blowsvej,
DK-1870 Frederiksberg C, Denmark

Key words: Monogenea, fish immunity, skin immune system, cytokines, complement, humoral factors, cellular
responses
Abstract. Host responses against skin inhabiting monogeneans are commonly observed but the responsible immune mechanisms
in the fish skin are insufficiently described. Based on recent knowledge of fish immunity and skin response mechanisms in
mammals a model for the skin immunity in fish to monogenean infections is proposed. Important cellular components of the
model are the epithelial cells, the mucous cells and leucocytes. The release of cytokines, e.g. IL-1, following mechanical or
chemical injury of the epithelial cells, initiates a series of events leading to decrease of the ectoparasite population. Cytokines
(e.g. IL-1, TNF, INF) are suggested to affect secretions from mucous cell and attract neutrophils and macrophages. Leukotrienes
are probably involved in the inflammatory reactions. The subsequent production of humoral substances (among others
complement factors and peptides) could be responsible for the antiparasitic response in the later stages of infection. Although
non-specific factors dominate the response, the involvement of specific antibodies and lymphocytes cannot be excluded.

It has been demonstrated through a number of

investigations that teleosts are capable of mounting
protective host responses against infections with
ectoparasitic monogeneans (Vladimirov 1971, Scott
1985, Buchmann and Bresciani 1998). Already seven
decades ago different marine fish species in the New
York Aquarium exhibited varying responses to
infections with Benedenia (Epibdella) melleni
(MacCallum) (Jahn and Kuhn 1932, Nigrelli and Breder
1934, Nigrelli 1937). Later, carp (Cyprinus carpio) was
shown to respond effectively against the gill parasitic
Dactylogyrus vastator Nybelin (Paperna 1964,
Vladimirov 1971). Likewise, the European eel Anguilla
anguilla was seen to develop partial immunity to the
gill monogeneans Pseudodactylogyrus anguillae (Yin et
Sproston) and P. bini (Kikuchi) (Buchmann 1988,
Slotved and Buchmann 1993). The viviparous
gyrodactylids are no exception to this rule as a range of
experiments have documented that sticklebacks (Lester
and Adams 1974), guppies (Scott 1985, Richards and
Chubb 1996) and salmonids (Cusack 1986, Cone and
Cusack 1988, Bakke et al. 1991, Bakke and MacKenzie
1993, Malmberg 1993, Buchmann 1997, Buchmann and
Uldal 1997, Buchmann and Bresciani 1998) activate
repelling mechanisms against these monogeneans in the
later stages of infection. Despite this evident response to
monogenean ectoparasites the involved mechanisms are
still insufficiently elucidated. However, recent progress
in fish immunology and studies on infections with
Gyrodactylus derjavini Mikailov on rainbow trout skin
have pointed to both cellular and humoral factors as the
main actors in the play. The present work treats the
existing knowledge on interactions between fish and

monogenean, draws parallels to ectoparasitic infections

of mammals and finally presents a hypothetical model
for the intricate immunity mechanisms in teleost skin.
THE RAINBOW TROUT EPIDERMIS
The rainbow trout skin is composed of the outer
epidermis beneath which the dermis (mainly consisting
of connective tissue) is located (Hibiya 1982, Yasutake
and Wales 1983). Various cell types are found in the
epidermis. The outermost layer is composed of flattened
epithelial cells tightly bound to each other. Their outer
cell membranes are folded into fine microridges
producing fingerprint like patterns on the cell surface
(Buchmann and Bresciani 1998). Beneath the epithelial
cells mucous (goblet) cells are distributed. They are
opening into small pores between the outer epidermal
cells. Mucous or goblet cells are numerous, exhibiting
cell densities of several hundreds per square mm
(Buchmann and Bresciani 1998). Due to their content of
acid mucopolysaccharides they will stain intensely blue
with Alcian blue and the carbohydrate moieties also
react with lectins (Fig. 1). The content of the goblet
cells are released onto the surface of the fish. Deeper in
the tissue are found chromatophores as melanophores
and xanthophores. Chromatophores have not been
connected to any function in skin immunity but they
occur abundantly on the corpus of rainbow trout and
less frequently on the fins. In the process of
inflammation different leucocytes as macrophages,
neutrophils, eosinophils and other granulocytes are
found distributed between the deeper cells in the
epidermis in carp (Cross and Matthews 1993). In

Address for correspondence: K. Buchmann, Department of Veterinary Microbiology, Section of Fish Diseases, Royal Veterinary and Agricultural
University, 13 Blowsvej, DK-1870 Frederiksberg C, Denmark. Phone: ++45 35282700; Fax: ++45 35282711; E-mail:
kurt.buchmann@vetmi.kvl.dk

rainbow trout neutrophils and macrophages occur

abundantly in inflammatory reactions of the superficial
tissue layers (Finn and Nielsen 1971). The mast cell
(Reite 1998) is also an important epidermal cell.

Fig. 1. Mucous cells in pectoral fin from rainbow trout. Scale

bar = 60 m. Rainbow trout fins were fixed in 4% neutral
formaldehyde for 1 month. Following rinsing in distilled
water mucous cells were visualized by detecting galactose
derivatives in the mucopolysaccharides by incubating the fins
(blocked for 1 h with 2.5% bovine serum albumin in
phosphate buffered saline [PBS]) with biotinylated
concanavalin A (Sigma C-2272) 10 g/ ml for 1 h, rinsing in
PBS with 0.05% Tween 20 310 min, incubating with avidine
and biotinylated alkaline phosphatase (DAKO K376).
Following a final wash the lectin binding was detected as red
colouring by incubation in Fast Red and Naphthol phosphate
in 0.1M Tris-HCl (DAKO K699). After staining specimens
were rinsed in distilled water and mounted in Aquamount.

EPIDERMAL CELLS AND THEIR ROLE IN

IMMUNITY
Epithelial cells
The epithelial cells are mechanically injured by the
marginal hooklets of Gyrodactylus derjavini. The large
hamuli do not normally penetrate the epithelium but
numerous minute holes are produced by insertion of the
marginals (Figs. 2-4). In addition, depressions in the
epithelium, presumably caused by the opisthaptor, are
found where these gyrodactylids are fouraging (Fig. 3).
Secretions from the parasite are likely to affect the
epithelium. A recent study (Buchmann 1998b) showed
strong activities of esterases, aminopeptidases and
phosphatases (alkaline and acid) in the intestine of G.
derjavini. As monogeneans regurgitate intestinal
contents (Smyth and Halton 1983) these enzymes are
probably released onto the epithelium where they
contribute to injuring or sensitization of the cell layers.
Thus, SEM-recordings of G. derjavini show release
onto the epithelium of material from the mouth region
(Fig. 4). A corresponding release of unidentified

substances from Gyrodactylus salaris Malmberg

parasitizing the Atlantic salmon was noted by Mo
(1994). It is known that keratinocytes from human skin
produce IL-1 in response to infections with Sarcoptes
scabiei L. (Arlian et al. 1996). Carp epidermal cells are
able to produce an IL-1 like factor (Sigel et al. 1986)
and interestingly, it was demonstrated that IL-1 was
produced in the epidermis of rainbow trout following
infection with G. derjavini (Buchmann and Bresciani
1998). This cytokine has a number of effects on other
cells in the epidermis. In fish, IL-1 induce hyperplasia
(Balm et al. 1995), macrophages are activated by IL-1
(Titus et al. 1991), the response of fish lymphocytes is
potentiated by IL-1 (Hamby et al. 1986, Sigel et al.
1986), and it is known that IL-1 induce mucus secretion
from human goblet cells (Cohan et al. 1991). The
present knowledge therefore allows the suggestion that
IL-1 liberated from epithelial cells upon monogenean
attachment elicit a number of reactions in the fish skin.
The observed hyperplasia in rainbow trout skin
(Buchmann and Bresciani 1998) is probably due to IL1, the mucins in the goblet cells are released (due to IL1 activation) onto the skin where the monogenean is
entangled in the material (Buchmann 1998b),
leucocytes are attracted to the inflammatory site where
macrophages, granulocytes and lymphocytes will exert
their effects. It is known from mammalian hosts that
leukotrienes on some occasion will be produced not
only by leucocytes but also by keratinocytes (Thomsen
1991). As leukotriene LTB4 acts as a potent
chemoattractant for fish leucocytes (Hunt and Rowley
1986) it is not excluded that this factor is involved in
the inflammatory reaction in the fish skin following
monogenean infection. Other cytokines (TNF, IFN) are
produced by human keratinocytes (Bos and Kapsenberg
1993) but a corresponding release in fish epithelia is
still to be investigated.
Mucous cells
The presence of a dense layer of mucous cells
beneath the epithelial cells in the rainbow trout has
prompted suggestions that this cell type could be
involved in fish immunity to ectoparasitic infections
(Pickering 1974, Wells and Cone 1990, Buchmann and
Bresciani 1998). This hypothesis is related to the fact
that mucus and goblet cells from mammalian mucosal
surfaces are known to participate in the host response
against intestinal nematodes (Castro and Harari 1982,
Miller 1987, Rabel et al. 1994, Ishikawa et al. 1994).
The presence in mucus scrapings from fish of several
biologically active substances as immunoglobulin (St.
Louis-Cormier et al. 1984, Rombout et al. 1993,
Buchmann and Bresciani 1998), complement factors
(Vladimirov 1971, Harris et al. 1997, Buchmann and
Bresciani 1998), peptides (Buchmann and Bresciani
1998), lysozyme and proteases (Fletcher 1982,

Buchmann: Fish skin immunity and monogeneans

Figs. 2-4. Scanning electron micrographs of Gyrodactylus derjavini. Fig. 2. Marginal hooklets penetrating epithelial cells on a
rainbow trout fin (pectoral fin). Fig. 3. Depression in the epithelium of a rainbow trout tail fin parasitized by G. derjavini. Fig. 4.
G. derjavini on a rainbow trout tail fin. Note the release of materials from the mouth region.. Scanning electron microscopy was
performed on specimens of G. derjavini parasitizing fins of rainbow trout by fixing in 2.5% cacodylate buffered glutaraldehyde
for 24 h, post-fixing in osmium tetroxide, whereafter specimens were critical point dried, sputtered with gold and studied in a
Jeol scanning electron microscope. Scale bars: Fig. 2 = 10 m; Figs. 3, 4 = 100 m.

Yano 1996), varying carbohydrates (Buchmann and

Bresciani 1998) and lectins (Yano 1996) add further to
the suggestion that mucus affects survival of invading
monogeneans. The release of the goblet cell contents
due to IL-1 production by injured epithelial cells could
connect these two cell types with monogenean invasion.
Cells in the inflammatory reactions
Following injuries or parasitic invasion of the fish
epidermis inflammatory reactions occur with infiltration
of various cell types including neutrophils and
macrophages in rainbow trout (Finn and Nielsen 1971)
and, in addition, eosinophils and basophils in carp
(Cross and Matthews 1993). This corresponds well to
mammalian reactions to Sarcoptes scabiei infections
(Arlian et al. 1994b). Activation of leucocytes elicit the
production of leukotrienes as LTB4, a substance known
to induce migration in fish neutrophils and eosinophils
(Hunt and Rowley 1986). Both macrophages and
neutrophils from fish are known to secrete IL-1
(Verburg-van Kemenade et al. 1995) which again will
affect other macrophages and fish lymphocytes (Hamby
et al. 1986, Secombes and Fletcher 1992). TNF and INF
have been shown to regulate mucus production in
goblet cells from the human colon (Jarry et al. 1992).
As TNF-like molecules are present in rainbow trout
(Ahne 1993) and related molecules are produced by
cells in the inflammatory reaction (Alexander and
Ingram 1992, Verburg-van Kemenade et al. 1996) it is
possible that they participate in the increased mucus
production already involving IL-1. These cytokines all
affect the different leucocytes which probably have
important functions in the reactions to monogeneans.
Thus, Hardie et al. (1994) found that TNF in interaction
with macrophage activating factor (MAF) positively
affects rainbow trout macrophage respiratory burst
activity. Such events are highly antiparasitic in vitro
(Whyte et al. 1989) and it cannot be excluded that the
reactive oxygen metabolites released in these processes
will affect the monogeneans. The demonstration of a
delayed type hypersensitivity in fish against the
protozoan parasites Cryptobia salmositica (Thomas and
Woo 1990) and Ichthyophthirius multifiliis (Sin et al.
1996) suggests that a similar response could be
activated in rainbow trout against monogeneans. Such a
reaction would involve release of macrophage migration
inhibition factor (MIF) from sensitized lymphocytes at
the site of infection. Thereby the reactive cells would be
concentrated near the invading pathogen. However,
such a reaction in rainbow trout against monogeneans
has not yet been demonstrated.
SPECIFIC ANTIBODIES
In mammalians infected with Sarcoptes scabiei
serum antibody titers increase significantly after infec-

tion although this factor is considered of minor importance (Arlian et al. 1994a). Production in fish of specific
serum immunoglobulin against gill monogeneans has
been seen (Vladimirov 1971, Buchmann 1993).
However, gill tissue is far more fragile and blood filled
than fish skin whereby gill parasite antigens are more
likely to come in direct contact with host blood. In
addition, recent immunocytochemical experiments with
whole worms have shown that rainbow trout
immunoglobulins from infected fish do not bind to the
tegument of G. derjavini (Buchmann 1998a) corresponding to the lack of antibody binding of sciaenid
fishes to monogenean antigens (Thoney and Burreson
1988). Some studies (e.g. Scott 1985) found that the
acquired protection of fish against gyrodactylids was
limited to few weeks. This supports that the protection
mainly is based on non-specific reactions. However,
this does not exclude that antibodies could bind to
molecules in the parasite intestine or other internal
structures. In addition, it is known from studies on a
parasitic ciliate (Ichthyophthirius multifiliis) infecting
the epidermis of a range of freshwater fishes that serum
antibody titres are raised following infection (Clark et
al. 1988, Clark and Dickerson 1997). In that system the
specific humoral response is considered protective and
shows that specific antibodies indeed are produced in
response to parasites invading the epidermis.
COMPLEMENT AND NON-SPECIFIC
HUMORAL FACTORS
Recently complement from rainbow trout was seen to
bind to and kill G. derjavini (Buchmann 1998a), which
corresponds to the lethal effect of salmon complement
to G. salaris (Harris et al. 1997). As complement factors
are produced not only by host liver cells but also by
macrophages (Lappin and Whaley 1993, Dalmo et al.
1997) this system is probably an important factor in the
response to ectoparasites. The glycocalyx of G. derjavini is composed of various carbohydrates consisting
of galactose derivatives, lactose derivatives and prominent mannose derivatives in the cephalic duct openings.
They are easily binding lectins and activate the
alternative complement pathway (Buchmann 1998a).
This corresponds partly to the response of rainbow trout
to Cryptobia salmositica. In that system both the
classical antibody dependent and the alternative
pathway are involved in the protective immunity (Woo
1996). The presence of carbohydrates in the glycocalyx
of monogeneans (Smyth and Halton 1983, Buchmann
1998a) leads to the suggestion that lectins in fish mucus
could contribute to the host response against
monogeneans. Fish mucus is a rich source of lectins
(Yano 1996). Their function is elusive (Arason 1996)
but could take part in the non-specific defence against
external pathogens. The observed binding of mucus to

Buchmann: Fish skin immunity and monogeneans

Fig. 5. Schematic illustration of the hypothetical network of cellular and humoral interactions involved in the response of the fish
epithelium against infections with monogeneans. Abbreviations: Mo monogenean parasite, E epithelial cell, M mucous
cell, Leu leucocyte, T T-lymphocyte, B B-lymphocyte.

the parasite surface (Buchmann 1998b) could be due to

lectins. No immediate injury of the parasite tegument is
caused by mucus binding. However, incubation of G.
derjavini in mucus scrapings from rainbow trout is
lethal within few hours but that effect could be caused
by the complement content.
A number of other non-specific molecules in fish
epidermis could play a role in the response to monogeneans. Lysozyme is present in high amount in fish
epidermis (Fletcher 1982, Lie et al. 1989, Alexander
and Ingram 1992) but does not seem to correlate with
resistance to monogenean infection (Buchmann and
Bresciani 1998). Proteases are present in fish epithelia
(Hjelmeland et al. 1983, Braun et al. 1990) but their
effects on monogeneans are unknown.
Peptides as somatostatin and ACTH have been
detected in mucus scrapings. Their possible antiparasitic
role remains undetermined but as monogeneans are
known to posses peptidergic innervations of their
nervous systems (Reuter 1987, Maule et al. 1990,
Halton et al. 1993, Cable et al. 1996) an effect on

parasite behaviour is not to be excluded. It is known

from amphibians that skin secretions contain a vast
amount of bioactive peptides and defensins (Lazarus
and Attila 1993) and it is possible that monogeneans are
affected by secretions of fish skin. Thus, cestodes
respond actively to somatostatin (Sukhdeo et al. 1984).
Besides being of general importance these nonspecific reactions are suggested to play a role in the
varying susceptibility of different salmonids to
gyrodactylids which has been documented during the
last years (Bakke et al. 1990, Bakke and MacKenzie
1993, Buchmann and Uldal 1997).
LYMPHOCYTES
Generally infiltration with B-lymphocytes is weak in
the inflammatory reactions to ectoparasites in mammalian skin. However T-lymphocytes are important
factors involved in the reaction to these infections
(Arlian et al. 1997) and do generally play an important
role in skin immunity (Bos and Kapsenberg 1993). In

fish B-cells are recognized as immunoglobulin positive

lymphocytes. Due to the lack of T-cell markers fish Tcells are often referred to as immunoglobulin-negative
lymphocytes. However, recently molecular evidence
was presented for the existence of a T-cell receptor in
rainbow trout (Partula et al. 1995) and the occurrence in
fish of B- and T-cell equivalents are now accepted. The
role of lymphocytes in the response of fishes to
monogenean invasion is still unknown. It is accepted
that Langerhans cells (LC) of the mammalian epidermis
take up antigen from the external pertubator and migrate
through the dermal lymphatics to the draining lymph
nodes. In this region the antigen/MHC complex on the
LC surface is presented to T-cells (Bos and Kapsenberg
1993, Stingl et al. 1993). Future work should establish
if similar events occur in fish skin. It was suggested by
Graves et al (1985) that non-specific cytotoxic cells are
responsible for the protective response of fish to skininvading ciliates. Whether a corresponding cell type has
effects on monogeneans should be elucidated.
CONCLUSIONS
The demonstration of strong anti-monogenean
factors in the activated fish skin has prompted a
hypothetical model for the skin immune system in fish
(Fig. 5). This is based on the present knowledge of the
anatomy of the fish skin, the findings of inflammatory
cells in the skin, the demonstration of cytokines,
complement, immuno-globulin and peptides in the skin
and mucus. Research in mammalian systems has
established links between several factors in the skin and

some parallels seem to exist in fish. The activation of

the fish skin epidermis by monogeneans (mechanically
and/or chemically) causes production of cytokines (IL-1
and possibly TNF and INF ) from the epithelial cells
(and leucocytes). The IL-1 (and TNF and INF) affects
the mucous cells and causes mucus secretion. The
leucocytes (and possibly epithelial cells) subsequently
produce leukotrienes, IL-1, INF and TNF with a range
of effects on granulocytes and macrophages. Cytokines
direct the leucocytes to the site of inflammation and
noxious substances including reactive oxygen
metabolites are released by the leucocytes affecting the
monogenean. The secreted mucus and cellular
liberations include complement factors reacting with the
glycocalyx of the monogenean. In addition, released
peptides could affect worm behaviour. Antibodies may
or may not bind to the worm but it is entangled in
mucus containing complement, lectins and C-reactive
proteins. Involvement of specific B and T cells is
possible but even without this specificity the nonspecific responses in the epidermis would create a
microenvironment hostile to the parasites. This would
lead to either a killing of the monogeneans or an escape
to less hostile surfaces (uninfected naive fish, fish tank
bottom or less reactive sites on the host).
Acknowledgements. This study was supported by the Danish
Agricultural and Veterinary Research Council, the Danish
Ministry of Food, Agriculture and Fisheries and the Elisabeth
and Knud Petersen Foundation. The author is indebted to Dr.
Jos Bresciani, Section of Zoology, Royal Veterinary and
Agricultural University, for assistance with the scanning
electron microscopic recordings.

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Received 23 December 1997

Accepted 28 July 1998

May 3 5, 1999
Course:

Evolution of Parasites and of Host-parasite Relationships

(volution des parasites et des relations htes-parasites)
Paris, France
Contact:

Dr. Marie-Claude Durette-Desset or Prof. Jean-Lou Justine

Laboratoire de Biologie Parasitaire, Helminthologie, Protistologie
Musum National dHistoire Naturelle,
61 rue Buffon,
75231 Paris cedex 05, France

Phone: +33 1 40 79 35 03
Fax: +33 1 40 79 34 99
e-mail: mcdd@mnhn.fr or justine@mnhn.fr
Programme: http://www.mnhn.fr/ens/ed/07-justi.htm