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Fish & Shellfish Immunology 30 (2011) 1087e1094
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Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream Bahram S. Dezfuli a, *, Luisa Giari a, Alice Lui a, Massimo Lorenzoni b, Edward J. Noga c a
Department of Biology & Evolution, University of Ferrara, St. Borsari 46, 44123 Ferrara, Italy Department of Cellular and Environmental Biology, University of Perugia, 06123 Perugia, Italy c Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 St. Hillsborough, Raleigh, NC 27606, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2010 Received in revised form 4 February 2011 Accepted 6 February 2011 Available online 18 February 2011
Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membranebound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P < 0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Sea bream Crustacean ectoparasite Gill cell responses Mast cells Piscidin
1. Introduction One of the most common groups of marine ectoparasites of fish are crustaceans belonging to the order Copepoda. While parasitic copepods are common on cultured and wild marine fish [1], it has only been with the development of semi-intensive and intensive aquaculture that their importance as disease-causing agents has become evident [2e4]. Ergasilids (Ergasilidae) are common copepod parasites of fish, primarily infecting the gills [5]. Apparently, feeding activity and mode of attachment of these copepods are the main reason for host damage and has been addressed in previous studies [6,7]. According to Johnson et al. (2004) [4], outbreaks of disease due to ergasilids are a major source of copepod-induced mortality in brackish and freshwater fish culture of several countries, including Israel [8], Taiwan [9], Japan [10], China [11], the USA [12], South Africa [13] and Hungary [14]. In Italy,
* Corresponding author. Tel.: þ39 0532 455701; fax: þ39 0532 455715. E-mail address:
[email protected] (B.S. Dezfuli). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.02.005
in our study area, in summer 2006, the copepod, Ergasilus sp. induced mortality of 40% of the juvenile sea bream, Sparus aurata, in a semi-intensively cultured fish farm. Subsequently, production of S. aurata has been impaired by annual losses of w5e8% of the juvenile stock each summer since 2007. These losses have been attributed to heavy infections of Ergasilus sp. Understanding S. aurata’s immune response to Ergasilus sp. might allow a better assessment of not only host damage but also provide a better understanding of how this fish defends itself against this parasite, so that potential mitigation strategies may eventually be developed. In the present study, the occurrence of mast cells (MCs), mucous cells and rodlet cells (RCs) was documented, especially near the Ergasilus sp. attachment site. Changes in the number of mast cells and mucous cells have been reported as a response to the attachment of parasitic copepods in both freshwater and marine fish [15e17]. With regard to RCs, their presence within the epithelial tissues of most species of teleost fish examined, has been known for almost 120 years. Their widespread distribution within and across many species of marine and freshwater fish and their morphological
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features are well established, but, the function of these cells continues to be hotly debated [18,19]. Published records on the presence of RCs in relation to copepod ectoparasites are rare, with the only available information referring to Ergasilus sieboldi-infected gills of Abramis brama [17]. The initial response of a host in defending against a parasite/ pathogen is via non-specific immunity [20]. Mast cells have been recognized as important components of non-specific immune defence in many vertebrates [21e23]. Mast cells occur in skin, gills, and digestive tract of several species of fish, and in fish infected with metazoan parasites [19,24e27]. Antimicrobial peptides (AMPs), host defense effector molecules occur in virtually all life forms and are a key component of this defense [28]. Increasing evidence supports their significant role in protecting animals from infections, including fish [22]. Piscidins are the prototypical AMP present in piscine MCs [16,21e23,27,29e31]. Piscidins have potent, broad-spectrum antibacterial and antifungal activity and have strong antiparasitic activity [16,21,27]. With regard to their mechanism of action, piscidins are thought to inhibit the synthesis of the cell wall, nucleic acids, and proteins or even inhibit enzymatic activity [32]. Piscidins were encountered in mast cells of different organs of uninfected fish [23,30,31,33]. Indeed, piscidin 3 was detected in mast cells of infected gills of European sea bass, Dicentrarchus labrax [27] and Latris lineata [16]. The main aim of this investigation was to examine gill cellular responses of S. aurata to Ergasilus sp. infection in relationship to the intensity of infection. We also provide further evidence for the possible importance of piscidin-expressing MCs in host defense against ectoparasitic copepods. We also relate the observations made on the histopathology and ultrastructure of the immune cells of infected gill with their involvement in the gill inflammatory response. 2. Materials and methods 2.1. Animals Thirty-seven sea bream, S. aurata, measuring 18.16 5.22 cm in total length (mean S.D.) and weighing 73.36 25.23 g (mean S.D.) were obtained from a local semi-intensive fish farm, “Valle Ca’ Zuliani” (Pila di Porto Tolle, Rovigo, Italy) from September to December 2009. On this site, sea bream are artificially reproduced in the hatchery unit and the fry grown on in internal concrete tanks. Once the fry reach between 2 and 5 g in weight, they are then transferred to, and reared in, earthern ponds which are exposed to sea water. It is believed that infection is acquired at this point, directly from the water source. Infected fish can be found all year round but the prevalence and intensity of infection is higher in the summer months. In 2009, the water temperature in the ponds ranged from 3.3 C to 30.3 C (16.6 7.7 C, mean S.D., n ¼ 12) whilst the salinity over the same period was 12.6 ppt to 38.0 ppt (25.5 4.3, mean S.D., n ¼ 12). 2.2. Histology and electron microscopy The fish were transported live to the laboratory where they were anesthetized using MS222 (Sandoz, Basel, Switzerland) and their spinal cords severed. Immediately after euthanasia, the entire gill was placed in a dish of saline solution and, using a steromicroscope, the total number of Ergasilus sp. were counted on the entire gill, including that of the left and right sides of the fish. The infected filaments were then fixed in chilled (4 C) 10% neutral buffered formalin for 8 h. The samples were then paraffin-embedded following standard procedures and 5 mm sections were stained with Giemsa. For light and electron microscopy, infected gill filaments up to 7 mm in diameter were fixed in 2.5% glutaraldehyde in
0.1 M cacodylate buffer for 3 h at 4 C before post-fixing them in 1% osmium tetroxide in the same buffer for 3 h. The specimens were dehydrated through a graded acetone series before being embedded in Epoxy resin (Durcupan ACM, Fluka, Buchs, Switzerland). Semi-thin sections (1.5 mm) were cut on a Reichert Om U 2 ultramicrotome using glass knives and then stained with toluidine blue. Ultra-thin sections (90 nm) were stained with a 4% uranyl acetate solution in 50% ethanol and Reynold’s lead citrate and examined using a Hitachi H-800 electron microscope. For comparative purposes, gills of 11 uninfected sea bream were similarly processed. Light photomicrographs were taken using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). Images of gill sections showing the overall distribution of mast cells and dimensions (150 MCs) were obtained using computerized image analysis software (Nis Elements AR 3.0, Nikon, Tokyo, Japan). 2.3. Immunohistochemistry Anti-piscidin 3 (anti-HAGR) antibody was produced by a commercial laboratory (Bethyl Laboratories, Montgomery, TX) using the company’s standard procedures. Briefly, 2 mg of a 12-mer peptide constituting the C-terminus of piscidin 3 (HAGRSIGRFLTG) was conjugated to keyhole limpet hemocyanin (KLH) using maleimide chemistry, which linked the peptide to KLH via a cysteine added to the N-terminal histidine. The conjugation via the terminal amino acid allows tertiary conformation of the peptide that may be expected to mimic that in the native peptide, thus eliciting anticonformational antibodies important for recognizing the native peptide. The immunogen was mixed with Complete Freund’s Adjuvant (1:1) and the KLH-conjugated peptide was injected into two New Zealand white rabbits biweekly at 5 subcutaneous sites (0.2 mL per site) using the following immunization schedule (100 mg/injection): Days 0, 14, 28 and 42. Thirty ml of antiserum was collected from each rabbit on days 35 and 45. The antisera were pooled and were then affinity-purified using the piscidin fragment conjugated to cyanogen bromide-activated agarose as an immunosorbent (10.5 mg of piscidin fragment was reacted with 15 g of agarose). One hundred and twenty ml of antiserum (two 30 mL bleeds from two rabbits) was loaded onto each affinity column (Uniflow 4, Sterogene, Carlsbad, CA). After washing, the affinitypurified antibody was eluted and concentrated. This method produces greater than 0.1 mg of peptide-specific antibody per mL of antiserum, as determined by recovered affinitypurified antibody. Antibody was greater than 95% IgG, as determined by immunoelectrophoresis using antibodies specific for rabbit IgG, IgM and serum proteins. The titer of the antibodies was determined via ELISA, using the piscidin fragment as the antigen coated onto a microtiter plate. The 12-mer peptide (10 mg mL1 in PBS, pH 7.2e7.5) was coated onto microtiter plates at room temperature for 1 h. The plate was then washed and post-coated with 1% BSA in PBS for 30 min. The plate was washed and then dilutions of antibody in 1% BSA/PBS/0.01% Tween 20 were added, beginning at 1 mg antibody/ mL. After incubation for 1 h, the plate was washed, followed by addition of peroxidase-conjugated goat anti-rabbit IgG (h&l) in 1% BSA/PBS/0.01% Tween 20. After incubation for 1 h, the plate was washed and peroxidase substrate (Tetramethylbenzidene Peroxide Single Reagent [KPL]) was added, incubated for 15 min, and then stopped with 1 N HCl (1:1). The absorbance was then read at 450 nm. The titer was read as the reciprocal of the antibody dilution (dilution of a 1 mg/mL solution) that produced a net optical density of 1.0, compared to a blank (peptide-coated well with no antibody [diluents only] added), which had an OD < 0.1. The ELISA titer of the antibodies used in all assays was approximately 1:18,000. The peptide-specific antibody is less than 1% cross-reactive with unrelated antigens by ELISA, where 1% cross-reactivity means that,
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using the same concentrations of antigen as for the piscidin fragment, 100 times more antibody would be required to produce the same optical density with either free KLH, or a free peptide that shared less than 3 amino acids in the sequence. Piscidin 3-positive cells were detected in gill histological sections using the indirect immunohistochemical method (peroxidase-anti-peroxidase immunocomplex). Briefly, sections (5 mm) were deparaffinised in xylene, rehydrated, then endogenous peroxidase activity and non-specific staining were blocked in 3% H2O2 (10 min) and in goat normal serum (1:20) (30 min). After incubation with the primary antibody (anti-HAGR diluted 1:400) for 3 h at room temperature, sections were incubated for 30 min with goat anti-rabbit immunoglobulins 1:100 (DAKO, Milan, Italy), and then for 30 min with rabbit peroxidase-antiperoxidase 1:200 (DAKO). The sections were then developed using DAB (3,30 -diaminobenzidine 0.04% w/v in TBS 0.05 M, pH 7.4) and H2O2 (0.005%), rinsed and counterstained with Alcian Blue and Harris’s haematoxylin. Non immune rabbit serum and diluent only sections were used as negative controls. Positive control tissue was striped bass (Morone saxatilis Walbaum) intestine. 2.4. Statistical analysis The number of mast cells (MCs) positive to anti-HAGR was counted in the gills via light microscopy, using computerized image analyser software (Nis Elements AR 3.0). For comparative purposes, the cells were screened at 400x magnification in 10 areas of 21,000 mm2 (5 randomly selected areas of primary lamella and 5 randomly selected areas of secondary lamella) for each fish of the three groups: uninfected fish (n ¼ 11), lightly infected fish (3 to 15 parasites per fish, 8.00 4.97, mean SD, n ¼ 7), heavily infected fish (39 to 55 parasites per fish, 46.78 4.89, mean SD, n ¼ 9). The Gaussian distributions (i.e. normality) of the data were assessed
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using the KolmogoroveSmirnov Test. Using the software package STATISTICA 7, ANOVA repeated measures was performed to detect significant differences in the number of the above mentioned cells among groups (uninfected vs. lightly infected vs. heavily infected). A Bonferroni post-hoc test and a P < 0.05 level of significance selected was used. 3. Results In 26 out of 37 S. aurata, the gills were parasitized with Ergasilus sp. (see supplementary Fig. 1); infection intensity ranged from 3 to 55 parasites per fish (26.96 19.20, mean SD, n ¼ 26). Most of the copepods were fully developed ovigerous females. Parasites resided on the external surfaces of hemibranchs (Fig. 1a) and oriented with the mouthparts and trunk toward the gill arch. Most Ergasilus sp. were attached to the first gill arch and fewer to other arches. Some parasites were attached to the middle part of the gill arch (Fig. 1a), but many were observed near the base of the gill filament, in the anterior region. The copepod body lay between the hemibranchs with its major axis parallel to the primary lamella axis (Fig. 1b). Second antennae extended to encircle (Fig. 1b) and increasingly compress the gill filament, which appeared as a thin layer of epithelium and connective tissue overlaying the cartilaginous supporting bar. Most frequently, second antennae disrupted the gill filament (not shown). Erosion, desquamation and necrosis of secondary lamellae were noticed near the site of Ergasilus sp. attachment (Fig. 1c). Branchial hyperplasia was observed on either side of the maxilla and near the copepod mouth. The copepod occluded arteries and caused hemorrhage. Heavily infected gills were more damaged than those of hosts with less Ergasilus sp. Within the parenchyma of the primary lamellae of parasitized gills, large numbers of mast cells (MCs) were seen near the parasite’s attachment site (Fig. 1c). Many MCs were also seen in the
Fig. 1. Gross lesion, wet mount and histological sections of Sparus aurata gills parasitized with Ergasilus sp. (a) Removal of the operculum shows the presence of several parasites (arrows) on the external surface of the gill hemibranchs. The copepods are attached mainly to the middle and basal part of gill filament, bar ¼ 5 mm. (b) Wet mount of a gill filament showing the copepod with second antennae (arrows) encircling the gill filament, bar ¼ 100 mm. (c) Histological section of gill infected with Ergasilus sp. Within the primary lamella, several mast cells (arrows) are visible; asterisk shows the copepod mouthpart, arrowheads indicate desquamation and necrosis of the gill tissue, bar ¼ 20 mm. (d) Micrograph of several mast cells (arrows) in vicinity of a capillary; note the eccentric nuclei and granules in the cytoplasm of these cells, bar ¼ 10 mm.
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primary lamellae in close contact with a capillary, or in the surrounding interstitium (Figs. 1c and d, 3c and d), with some MCs in the lumen of the vessel (Figs. 2f, 4a and c). In both parasitized and uninfected fish, the mast cells in the primary lamellae were basal to the mucous cells (Fig. 3a) and rodlet cells. Mast cells (major axis 11.10 1.96 mm, mean SD, n ¼ 150) were irregular in shape, with an eccentric, polar nucleus (Figs. 1d, 4a, c and d); their cytoplasm was filled with numerous large, electron-dense, membrane-bound granules (Figs. 1d, 4d and e). Transmission electron microscopy revealed that the cytoplasm of MCs of the interstitium contained very few mitochondria, an inconspicuous Golgi apparatus and many homogeneous and electron-dense granules (Figs. 3c and 4d). In contrast, all mast cells inside blood vessels seemed to be immature and had well-developed rough endoplasmic reticulum (Fig. 4c), many free ribosomes, several spherical-round mitochondria, low numbers of electrondense granules (Fig. 4a and c) and some electron-lucid vesicles
(Fig. 4c). In the vast majority of infected gills, several mast cells were inside the gill blood vessels on the capillary endothelium (Fig. 4b). In some vessels, the mast cells seemed to be detaching from the vessel wall (Fig. 4a and c) and moving toward the center of the lumen. In several instances mast cells were present inside the interstitium, in the vicinity of a capillary (Fig. 3d). However, MC degranulation was not observed in either uninfected or infected tissues. Fig. 2a is a negative control and showing absence of piscidin 3 immunoreactivity in infected gill of sea bream. Gill sections treated with anti-piscidin 3 antibody showed immunopositive MCs (Fig. 2bef). The number of gill mast cells positive to anti-HAGR per 21,000 mm2 was 6.49 4.33 (mean S.D., n ¼ 110 areas) in uninfected fish, 4.64 2.94 (mean S.D., n ¼ 70 areas) in lightly infected fish and 15.00 10.84 (mean S.D., n ¼ 90 areas) in heavily infected fish. Heavily infected fish had significantly more piscidin 3-positive MCs than both lightly infected and uninfected fish
Fig. 2. Immunohistochemical staining of S. aurata gills. (a) Negative control showing absence of piscidin 3 immunoreactivity in infected gill of sea bream, bar ¼ 20 mm. (b) Immunohistochemical localization of piscidin 3 in uninfected gill of S. aurata treated with anti-piscidin 3 antibody. Positive mast cells (arrows) are visible in the primary lamella, secondary lamellae (arrowheads) and inside the lumen (curved arrow) of a capillary, bar ¼ 20 mm. (c) Many immunopositive mast cells (arrowheads) are present within the epithelium of primary lamella and also inside the secondary lamellae; arrows show copepod appendages and asterisk indicates mouthpart of Ergasilus sp., bar ¼ 50 mm. (d) High magnification of Fig. 2c; note numerous immunopositive cells (arrow heads) in primary/secondary lamellae; arrow shows copepod appendage, bar ¼ 20 mm. (e) Piscidin 3-positive immunostaining of mast cells (arrows) in primary/secondary lamellae of infected gill far from the copepod body, bar ¼ 20 mm. (f) Some immunopositive mast cells (arrowheads) within the primary lamella in close vicinity to a capillary and two mast cells (arrows) inside the vessel lumen of parasitized gill, far from the copepod, bar ¼ 20 mm.
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Fig. 3. Electron micrographs of cells from E. seiboldi-infected gill. (a) In the sub-epithelial position of the primary lamella, note the presence of mast cells (arrowheads), a rodlet cell (curved arrow) adjacent to a mast cell, and mucous cells (arrows), bar ¼ 4.6 mm. (b) Two rodlet cells (arrows) in the inner part of a primary lamella in the vicinity of some mucous cells (curved arrows), bar ¼ 2.1 mm. (c) Several mast cells (arrows) in the interstitium in close contact with a capillary (asterisk); the cytoplasm of mast cells is filled with many homogenous, electron-dense granules, bar ¼ 3.0 mm. (d) Few mast cells (arrows) in the interstitium in vicinity of a capillary (asterisk), bar ¼ 3.1 mm.
Fig. 4. Electron micrographs of infected gill cells. (a) Three mast cell (arrowheads) inside the blood vessel of a primary lamellae and one mast cells (arrow) in the interstitium are evident, scale bar ¼ 3.3 mm. (b) Mast cells (arrowheads) on the capillary endothelium of a gill vessel, bar ¼ 3.2 mm. (c) High magnification of an immature mast cell inside the vessel; note well-developed rough endoplasmic reticulum (arrows), many free ribosomes, few electron-dense granules (arrowheads) and some electron-lucent vesicles (curved arrows), bar ¼ 0.8 mm. (d) A mature mast cell; the cytoplasm is filled with numerous electron-dense granules; note the eccentric nucleus (arrow), bar ¼ 1.1 mm.
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(ANOVA, P < 0.05). But, there was no significant difference in number of piscidin 3-positive MCs lightly infected fish versus uninfected fish (P > 0.05). Mast cells immunopositive for piscidin 3 were observed both close to the copepod (Fig. 2c and d) and far away from the parasite (Fig. 2e, f). In both uninfected and parasitized gills, piscidin 3-positive mast cells were observed in the primary lamellae (Fig. 2bed), as well as in the apex, middle and basal part of the secondary lamellae (Fig. 2d and e). Piscidin-positive mast cells were also adjacent to capillaries and in some instances, within them (Fig. 2b and f). Rodlet cells were seen adjacent to mast cells and mucous cells. RCs were ovoid, had a basal nucleus, and a very distinct sub-plasmalemmar capsule (Fig. 3b). The cytoplasm contained several rodlets. Rodlet cells were observed in sub-epithelial positions of the primary lamellae (Fig. 3b), and rarely in secondary lamellae. In both uninfected and parasitized gills, no piscidin 3 immunoreaction was seen in RCs and mucous cells. 4. Discussion Parasitic copepods naturally occur on wild fish. With regard to cultured fish, the action of these crustaceans may result in decreased flesh quality, mortality and a need for increased drug treatment, adding as much as 6% to production costs [34e36]. Previous studies have reported that the mode of attachment and feeding activity of parasitic copepods were the main reasons of damage to the gills [6,7]. Indeed, it was mentioned that ergasilid copepod infections can reduce gill function, resulting in decreased growth rate due to impaired respiration, morbidity and often substantial mortality in brackish water fish [11,13,14]. Mortality due to E. sieboldi has been reported in [37,38]. In our study area, low mortality (w2%) of young S. aurata due to Ergasilus sp. has been noticed in winter during the past few years (Dezfuli unpublished data). Nonetheless, in the semi-intensive farm where we sampled, since 2007, there has been an annual loss of w5e8% of the juvenile stock of sea bream each summer, attributed to heavy infections of Ergasilus sp.. At present, economic losses due to this copepod are primarily from the costs of treatments, implemented management strategies, reduced growth rates and carcass downgrading at harvest. With reference to the gills, this is a major route of pathogen entry after epithelial damage [39]. Attachment to the gill can result in severe local damage such as inflammation, infiltration, and hyperplasia of rodlet cells, mucous cells and mast cells [16,30,40e43]. Herein, in gill of sea bream parasitized with Ergasilus sp., some RCs were encountered. The same finding has been reported in infected gill of A. brama [17] and in parasitized gills of D. labrax [24]. Rodlet cells are exclusive to fish and represent an inflammatory cell type closely linked to other piscine inflammatory cells [44e47]. There are many studies that consistently report an association between RC proliferation and the presence of a variety of parasites [45e49]. In our current study in S. aurata, fish heavily infected with Ergasilus sp. had significantly more piscidin 3-positive mast cells than either lightly infected or uninfected fish. These findings are somewhat similar to that of Andrews et al. [16], who reported that mast cells (MCs) were absent in healthy gills of L. lineata, while numerous MCs, many of which were piscidin-3 positive, were seen in gills infected with the copepod Chondracanthus goldsmidi. However, in our previous study on D. labrax, we noticed that while there was no significant difference in the number of mast cells positive for anti-piscidin 3 antibody in gills parasitized with a monogenean compared to uninfected gills, the intensity of the antibody reaction strongly suggested that there was greater expression of piscidin 3 in the MCs of infected fish [27]. Thus, the
cumulative evidence suggests that piscidin-expressing MCs may play an important role in defending against this important group of parasites, but the method in which this expression occurs varies with either the specific host, parasite, or both. Innate immunity of vertebrates relies on different type of cells, and one of the most common is mast cells [50]. Mast cells are present in most species of teleost and in a variety of tissues (e.g., alimentary canal, gills and skin) [51,52]. Mast cells are motile [19,53] and have been recognized in regions of active inflammatory response especially due to bacterial and parasitic infections [25,54,55]. Gill is one of the tissues first exposed to environmental challenges and pathogens, so an ability to mount an immune defense is crucial [16,51]. In S. aurata, MCs were documented inside, and/or in close proximity to capillaries and they were observed throughout the loose connective tissue of the gill arch. With reference to the presence of mast cells in loose connective tissue some authors have suggested a resident population for these cells [51,56]. In all vertebrates, mast cells likely are strategically positioned at perivascular sites to regulate inflammatory responses [57]. The close association of mast cells with the capillary endothelial cells suggests that they may migrate across the endothelium [51,58]. Furthermore, mast cells migrate within the gill microcirculation, which may facilitate rapid movement throughout the gill tissue [58]. These observations support the suggestion that the gill is primed with the innate immune system, enabling an immediate response to pathogenic or parasitic organisms [59,60]. Several records have documented degranulation of mast cells in response to exposure to a variety of known degranulating agents and pathogens with release of their contents [25,51e53,58]. However, in gills of S. aurata, we never observed evidence of degranulation in mast cells. The functional similarity between mammalian and fish mast cells has stimulated studies into identifying the contents of granules in fish MCs. According to Murray et al. [51], mast cells could be directly involved in the destruction of pathogens and they provide further evidence for their multifunctional role in teleosts. Investigations have shown that mast cells of fish intestine and gills are involved in the production of specific AMPs, namely pleurocidin [51] and piscidins [21,23,30,33,61]. Piscidins have potent, broadspectrum antifungal and antibacterial activity and recently been found to have strong antiparasitic activity and were reported in gills of fish infected with three protistan ectoparasites [21], gills parasitized with a copepod [16], and gills infected with a monogenean [27]. The occurrence of antimicrobial peptide piscidin 3 in site of Ergasilus sp. infection might be a combined immune response resulting from both the copepod’s attachment and feeding activities and the threat of pathogens entering through the disruption of the gill lamella. The same assumption was adopted by Andrews et al. [16] on piscidin 3 expression in mast cells of gill of the striped trumpeter infected with a copepod. Our data are in agreement with the suggestion that piscidins play an important role in the nonspecific immune defense of many teleosts [33].
Acknowledgments We are indebt to Samantha Squerzanti, Silvia Fabbri, and Andrea Margutti from the University of Ferrara for technical assistance. This study was supported by grants from the Italian Ministry of the University and Scientific Research and Technology. Conflict of interest The authors declare that there is no conflict of interest.
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Appendix. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.fsi.2011.02.005.
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