Skin Of Gilthead Seabream Research Articles

Implication of adipocytes from subcutaneous adipose tissue and fatty acids in skin inflammation caused by λ-carrageenin in gilthead seabream (Sparusaurata)

The role of subcutaneous adipose tissue adipocytes and the effects of fatty acids on carrageenan-induced skin inflammation in gilthead seabream (Sparus aurata) were studied. Fish were injected intramuscularly with phosphate-buffered saline (control) or λ-carrageenin (1%), and skin samples collected at the injection site at 3 and 6 h post-injection (p.i.) were processed for histological study. In addition, the presence and levels of lipid classes, fatty acid methyl esters (FAME) and eicosanoids were evaluated in the skin samples obtained from the injected areas. Histological results indicated an increase in adipocyte area in fish sampled at 3 h p.i. with λ-carrageenin compared to fish in the control group. Furthermore, the frequency of adipocytes between 4500 and 5000 μm2 was increased at 6 h in the λ-carrageenin group compared to the control group. Analysis of lipid classes found that fish injected with λ-carrageenan showed increased free fatty acid (FFA) and sphingomyelin content at 3 and 6 h, respectively, compared to the control group. An increase in saturated fatty acids (SFA), n-6 polyunsaturated fatty acids (PUFA), and a decrease in the values of monounsaturated fatty acids (MUFA), n-3 PUFA and minor fatty acids were observed in fish skin at 6 h after λ-carrageenin injection, with respect to the values obtained in the control group. Regarding the analysis of eicosanoids, an increase in hydroxyeicosatetraenoic acid (5-HETE) was detected in the skin of fish at 6 h post-carrageenin injection compared to the control group. The presented results indicate the contribution of adipocytes and fatty acids in the development and regulation of the inflammatory response triggered by λ-carrageenin in gilthead seabream skin.

Ultrasonography and X-ray micro-computed tomography characterization of the effects caused by carrageenin in the muscle of gilthead seabream (Sparus aurata)

The current work aimed to carry out an in vivo study of the λ-carrageenin-induced inflammation in the skin of gilthead seabream (Sparus aurata). The fish were injected intramuscularly with phosphate-buffered saline (PBS, as control) or λ-carrageenin (1% in PBS), and the injection zone was evaluated by real-time ultrasonography (Vevo Lab, VisualSonics) at 1.5, 3, 6, 12, and 24 h post-injection (p.i.). Results demonstrated that the skin thickness was increased in fish injected with λ-carrageenin and sampled at 1.5, 3, and 6 h p.i. However, the skin thickness of the injected area decreased to the normal values in those fish sampled at 12 and 24 h p.i. In addition, fish injected with λ-carrageenin and analysed at 1.5, 3, and 6 h p.i. showed, in the underlying muscle at the injection place, several hyperechoic small foci surrounded by an anechoic area which were not observed in control fish. Furthermore, the fish were analysed by X-ray micro-computed tomography (micro-CT). The analysis of the micro-CT acquisitions revealed also a dark area in the place of the injection with λ-carrageenin at 1.5, 3, and 6 h. These areas were smaller in fish analysed at longer times (12 h p.i.) and were almost disappeared in fish sampled at 24 h p.i. These areas had an average density of −850 to −115 HU, which did not correspond with any tissue density of the rest of the body. Furthermore, similar dark areas at the injection zones were never observed in control fish. Present results support the use of both non-invasive techniques to study the inflammatory process in fish of commercial interest such as gilthead seabream.

Acute inflammatory response in the skin of gilthead seabream (Sparus aurata) caused by carrageenin

Although inflammation is a well-characterized process in mammals, few studies have dealt with the mechanisms involved in this process in fish. The present study evaluated the expression of inflammation-related genes in the skin of fish injected with carrageenin, which has previously been used in inflammatory models in mammals. In our case, fish were injected subcutaneously with PBS (as control) or carrageenin (1%), and skin samples from the injection site were collected 1.5, 3 and 6 h post-injection. The gene expression of inflammatory markers (csfr1, mhc-ii and phox40), several pro-inflammatory cytokines (il1b, tnfa, il6, il8 and il18) and other molecules related (such as myd88 and c-rel) were up-regulated at 1.5 and 3 h in fish injected with carrageenin compared with control levels. By contrast, the gene expression of anti-inflammatory molecules (nlrx1, nlrc5 isoform 1, ctsd and ctss) was down-regulated in fish injected with carrageenin and sampled 3 h post injection, again compared to the gene expression in control fish. According to our results, carrageenin can be considered not only a good stimulator to study skin inflammation in gilthead seabream but also this method might be use to study the modulation of fish inflammatory process caused by internal or external factors.

Implication of mucus-secreting cells, acidophilic granulocytes and monocytes/macrophages in the resolution of skin inflammation caused by subcutaneous injection of λ/κ-carrageenin to gilthead seabream (Sparus aurata) specimens.

To date, the mechanisms of inflammation have been poorly studied in fish of commercial interest, due to the lack of development of appropriate experimental models. The current study evaluated a local inflammation triggered by a polymeric carrageenin mixture (a mucopolysaccharide derived from the red seaweed Chondrus crispus) in the skin of gilthead seabream (Sparus aurata). Fish were injected subcutaneously with phosphate-buffered saline (as control) or λ/κ-carrageenin (1%), and skin samples from the injection sites were collected 1.5, 3 and 6hr post-injection, processed for inclusion in paraplast and stained with haematoxylin-eosin, Alcian blue or periodic acid-Schiff. Furthermore, immunohistochemistry and expression analyses of several cells' markers and proinflammatory genes were also analysed in samples of the injected sites. Microscopic results indicated an increased number of skin mucus-secreting cells and acidophilic granulocytes in the skin of fish studied at 1.5hr and 3hr post-injection with carrageenin, respectively, with respect to the data obtained in control fish. Otherwise, both the gene expression of the non-specific cytotoxic cell marker (granzyme B, grb) and the proinflammatory cytokine (interleukin-1β, il-1β) were up-regulated at 1.5hr in the skin of fish injected with carrageenin compared with the control fish, whilst the gene expression of acidophilic granulocyte markers (NADPH oxidase subunit Phox22 and Phox40, phox22 and phox40) was up-regulated at 3 and 6hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up-regulated at 6hr in the skin of fish injected with carrageenin in comparison with control samples. The present results indicate the chronological participation of two important immune cells involved in the resolution of the inflammation in the skin of gilthead seabream.

Antioxidant Activity in Gilthead Seabream (Sparus aurata L.) Fed with Diet Supplemented with Moringa.

Gilthead seabream is bred mainly in fish farms in the Mediterranean Sea. One important factor responsible for the deterioration of fish quality is lipid oxidation. Moringa oleifera leaves have been described as having high antioxidant content. This work investigates the effect of dietary supplementation with Moringa leaves on the antioxidant activity of seabream. Gilthead seabream specimens were divided into four groups, the control group (fed a commercial diet) and three other groups fed diets enriched with Moringa (5%, 10% and 15%). The antioxidant capacity was measured by assays of free radical scavenging (OH·, H2O2, lipoperoxyl and ABTS), Rancimat test and linoleic acid system in muscle and skin of gilthead seabream, commercial diet, enriched diet and Moringa. Finally, the polyphenol content of Moringa and the fatty acid composition of seabream fed diets with and without Moringa were determined. Results showed an increase in antioxidant activity in gilthead seabream fed with diets enriched with a higher percentage of Moringa; therefore, Moringa could be considered a functional ingredient in diets for fish bred in fish farms and. The antioxidant potential of Moringa leaves could be mainly attributed to the presence of polyphenolic compounds.

Wound-Induced Changes in Antioxidant Enzyme Activities in Skin Mucus and in Gene Expression in the Skin of Gilthead Seabream (Sparus aurata L.)

This study investigated the antioxidant enzyme activities in the skin mucus of gilthead seabream (Sparus aurata L.) at 3 and 7 days post-wounding (dpw). The expression levels of the genes that encode stress proteins (grp170, grp94, grp75, sod and hsp70) and skin regeneration-related proteins (tf, igf1, tgfb1, der1, apo1 and erdj3) in the skin also were determined. Mucus and skin samples were obtained from the left and right flanks of non-wounded and wounded fish. In both flanks of the wounded fish, catalase and glutathione reductase activities in the skin mucus increased (p < 0.05) at 3 and 7 dpw (100 ± 31% and 111 ± 25%, respectively), whereas superoxide dismutase activity increased (p < 0.05) only at 7 dpw (135 ± 15%). The expression levels of stress proteins in the skin of the wounded flank of the wounded fish mainly increased at 7 dpw (grp170 increased to 288 ± 85%, grp94 to 502 ± 143%, grp75 to 274 ± 69%, sod to 569 ± 99%, and hsp70 increased to 537 ± 14%) (p < 0.05). However, the expression levels of the tissue regeneration-related genes varied depending on the flank investigated, on the experimental time, and on the gene studied. To the best of our knowledge, this is the first work to determine the effect of a wound in different skin parts of the same fish.

Carvacrol, Thymol, and Garlic Essential Oil Promote Skin Innate Immunity in Gilthead Seabream (Sparus aurata) Through the Multifactorial Modulation of the Secretory Pathway and Enhancement of Mucus Protective Capacity

One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as they constitute a physical and biochemical shield against environmental and pathogenic threats, comprising elements from both the innate and acquired immunity. In the present study, the modulation of the skin transcriptional immune response, the bacterial growth capacity in skin mucus, and the overall health condition of gilthead seabream (Sparus aurata) juveniles fed a dietary supplementation of garlic essential oil, carvacrol, and thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish fed the phytogenic-supplemented diet revealed the regulation of genes associated to cellular components involved in the secretory pathway, suggesting the stimulation, and recruitment of phagocytic cells. Genes recognized by their involvement in non-specific immune response were also identified in the analysis. The promotion of the secretion of non-specific immune molecules into the skin mucus was proposed to be involved in the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not affected by the phytogenics supplementation, the regulation of genes coding for oxidative stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol appear to promote the gilthead seabream skin innate immunity and the mucus protective capacity, decreasing its susceptibility to be colonized by pathogenic bacteria.

Skin Pigmentation in Gilthead Seabream (Sparus aurata L.) Fed Conventional and Novel Protein Sources in Diets Deprived of Fish Meal.

Simple SummaryIntensive farming of carnivorous fish species relies on the use of feeds, where fish meals have represented, for a long while, ideal sources of protein to ensure optimal growth, health and quality to cultured fish. Due to the gap between the demand for these commodities by the growing aquaculture industry and the unsustainability of further exploitation of the alieutic resources, their levels of inclusion in fish feeds have been mostly replaced by terrestrial plant protein-rich derivatives and conventional animal processed proteins. Recently, novel ingredients (i.e., insects and microalgae) have been proposed to this end. While the impact of different alternative proteins on fish growth and health has been studied, limited information exists on the effects of such dietary changes on quality traits of cultured fish such as skin pigmentation. The present study was aimed at assessing the pattern of yellow pigmentation of the skin in gilthead seabream fed various alternative protein sources (vegetable ingredients, insects, poultry by-product meal, red swamp crayfish and marine microalgae) included in different proportions in fishmeal-free diets, in order to evaluate new feed formulations on the basis of their coloring capacity, as intense skin coloration have been associated with high-quality of farmed fish products.The pattern of yellowish pigmentation of the skin was assessed in gilthead seabream (Sparus aurata) fed for 12 weeks iso-proteic (45%) and iso-lipidic (20%) diets deprived of fish meal and containing either a blend of vegetable protein-rich ingredients or where graded levels of the vegetable protein blend were replaced by insect (Hermetia illucens—10%, 20% or 40%) pupae meal, poultry by-product meal (20%, 30% or 40%), red swamp crayfish meal (10%) and marine microalgae (Tisochrysis lutea and Tetraselmis suecica—10%) dried biomass. Digital images of fish fed diets differing in protein sources were analyzed by means of an automatic and non-invasive image analysis tool, in order to determine the number of yellow pixels and their dispersion on the frontal and lateral sides of the fish. The relationship between the total carotenoid concentration in the diet and the number of yellow pixels was investigated. Test diets differently affected gilthead seabream skin pigmentation both in the forefront and the operculum, due to their carotenoid content. The highest yellow pixels’ number was observed with the diet containing microalgae. Fish fed poultry by-product meal were characterized by the lowest yellow pixels’ number, diets containing insect meal had an intermediate coloring capacity. The vegetable control, the microalgae mix diet and the crayfish diet had significantly higher values of yellow pixels at both inspected skin sites.

Skin Mucus Fatty Acid Composition of Gilthead Sea Bream (Sparus Aurata): A Descriptive Study in Fish Fed Low and High Fish Meal Diets

Terrestrial protein and lipid sources are commonly used as substitutes for marine fishery-derived raw ingredients in fish diets. However, their use is related with several side-effects on marine fish performance, health, or disease resistance. Physical barriers of the skin, gills, and gut constitute the primary defense mechanism of fish. Skin mucus mucosal mucins, water, proteins, ions, and lipids determine the physical, chemical, and protective characteristics of skin mucus. Very little is known about the influence of diet composition on fish skin mucus fatty acid profile. Gilthead sea bream skin mucus contained 10% of total lipids (TL), which consisted of 50–60% neutral (NL) and 40–50% polar lipids (PL) fractions. Σn−3 long chain polyunsaturated fatty acids (LC-PUFA) deposition was preferential in the NL fraction, whereas Σn−6LC-PUFA accumulation was similar in both lipid classes. Docosahexaenoic acid (DHA; 22:6n−3) was the main LC-PUFA stored in skin mucus (14% TL) in relation to eicosapentaenoic acid (EPA; 20:5n−3) (2–3% TL) and arachidonic acid (ARA; 20:4n−6) (2% TL). This study denotes the importance of DHA as component of skin mucus lipids compared to other essential fatty acids, such as EPA and ARA, as well as importance of maintaining an adequate Σn−3/ Σn−6 ratio, regardless of dietary intake.

Chronic wounds alter the proteome profile in skin mucus of farmed gilthead seabream

BackgroundSkin and its mucus are known to be the first barrier of defence against any external stressors. In fish, skin wounds frequently appear as a result of intensive culture and also some diseases have skin ulcers as external clinical signs. However, there is no information about the changes produced by the wounds in the mucosae. In the present paper, we have studied the alterations in the proteome map of skin mucus of gilthead seabream during healing of experimentally produced chronic wounds by 2-DE followed by LC-MS/MS. The corresponding gene expression changes of some identified skin proteins were also investigated through qPCR.ResultsOur study has successfully identified 21 differentially expressed proteins involved in immunity and stress processes as well as other metabolic and structural proteins and revealed, for the first time, that all are downregulated in the skin mucus of wounded seabream specimens. At transcript level, we found that four of nine markers (ighm, gst3, actb and krt1) were downregulated after causing the wounds while the rest of them remained unaltered in the wounded fish. Finally, ELISA analysis revealed that IgM levels were significantly lower in wounded fish compared to the control fish.ConclusionsOur study revealed a decreased-expression at protein and for some transcripts at mRNA levels in wounded fish, which could affect the functionality of these molecules, and therefore, delay the wound healing process and increase the susceptibility to any infection after wounds in the skin of gilthead seabream.

Dorso-ventral skin characterization of the farmed fish gilthead seabream (Sparus aurata).

The skin is the first barrier of defence in fish, protecting against any external stressor and preserving the integrity and homeostasis of the fish body. The aim of this study was to characterise gilthead seabream skin by isolating cells and studying the cell cycle by flow cytometry, to study the skin histology by scanning electron microscopy and the transcription level of some immune-relevant genes by RT-PCR. Furthermore, the results obtained from samples taken from the dorsal and the ventral part of the specimens are compared. No differences were observed in the cell cycle of cells isolated from the dorsal and ventral zones of the skin or in the gene expression of the genes studied in both epidermal zones. However, the epidermis thickness of the ventral skin was higher than that of the dorsal skin, as demonstrated by image analysis using light microscopy. Besides, scanning electron microscopy pointed to a greater cell size and area of microridges in the apical part of the dorsal epidermal cells compared with ventral skin epidermal cells. This study represents a step forward in our knowledge of the skin structure of an important farmed teleost, gilthead seabream, one of the most commonly farmed fish worldwide. Furthermore, for functional characterization, experimental wounds were carried out comparing the wound healing rate between the dorsal and ventral regions of skin over the time. The results showed higher ratio of wound healing in the ventral region, whose wounds were closed after 15 days, compared to dorsal region of skin. Taking into account all together, this study represents a step forward in our knowledge of the skin structure and skin regeneration of an important farmed teleost, gilthead seabream, one of the most commonly farmed fish worldwide.

Chronic stress impairs the local immune response during cutaneous repair in gilthead sea bream (Sparus aurata, L.)

Scale removal in fish triggers a damage-repair program to re-establish the lost epidermis and scale and an associated local immune response. In mammals, chronic stress is known to delay wound healing and to modulate the cutaneous stress axis, but this is unstudied in teleost fish the most successful extant vertebrates. The present study was designed to test the hypothesis that chronic stress impairs cutaneous repair in teleost fish as a consequence of suppression of the immune response. The hypothesis was tested by removing the scales and damaging the skin on one side of the body of fish previously exposed for 4 weeks to a chronic crowding stress and then evaluating cutaneous repair for 1 week. Scale removal caused the loss of the epidermis although at 3days it was re-established. At this stage the basement membrane was significantly thicker (p=0.038) and the hypodermis was significantly thinner (p=0.016) in the regenerating skin of stressed fish relative to the control fish. At 3days, stressed fish also had a significantly lower plasma osmolality (p=0.015) than control fish indicative of reduced barrier function. Chronic stress caused a significant down-regulation of the glucocorticoid receptor (gr) in skin before damage (time 0, p=0.005) and of star at 3 and 7days (p<0.05) after regeneration relative to control fish. In regenerating skin key transcripts of cutaneous repair, pcna, colivα1 and mmp9, and the inflammatory response, tgfβ1, csf-1r, mpo and crtac2, were down-regulated (p<0.05) by chronic stress. Irrespective of chronic stress and in contrast to intact skin many hyper pigmented masses, putative melanomacrophages, infiltrated the epidermis of regenerating skin. This study reveals that chronic stress suppresses the local immune response to scale removal and impairs the expression of key transcripts of wound healing. Elements of the stress axis were identified and modulated by chronic stress during cutaneous repair in gilthead seabream skin.

Invitro cytokine profile revealed differences from dorsal and ventral skin susceptibility to pathogen-probiotic interaction in gilthead seabream.

Skin is the first barrier of defense on fish, which is crucial to protection against different stressors, including pathogens. Skin samples obtained from dorsal and ventral part of Sparus aurata specimens were incubated with Photobacterium damselae subsp. piscicida (a pathogen for this fish species), with Shewanella putrefaciens Pdp11 (a probiotic bacteria isolated from healthy gilthead seabream skin) or with both bacteria. The gene expression profile of nine cytokines (il1b, tnfa, il6, il7, il8, il15, il18, il10 and tgfb) was studied by qPCR in all the skin samples. The present findings revealed different patterns of cytokine profile in dorsal and ventral skin of gilthead seabream, which could be related to the influence and susceptibility to a possible infection.

Determination of sulfadiazine, trimethoprim, and N(4) -acetyl-sulfadiazine in fish muscle plus skin by Liquid Chromatography-Mass Spectrometry. Withdrawal-time calculation after in-feed administration in gilthead sea bream (Sparus aurata L.) fed two different diets.

This study presents a depletion study for sulfadiazine and trimethoprim in muscle plus skin of gilthead sea bream (Sparus aurata L.). N(4) -acetyl-sulfadiazine, the main metabolite of sulfadiazine (SDZ), was also examined. The fish were held in seawater at a temperature of 24-26°C. SDZ and trimethoprim (TMP) were administered orally with medicated feed for five consecutive days at daily doses of 25mg SDZ and 5mg TMP per kg of fish body weight per day. Two different diets, fish oil- and plant oil-based diets, were investigated. Ten fish were sampled at each of the days 1, 3, 5, 6, 8, 9, 10, and 12 after the start of veterinary medicine administration. However for the calculation of the withdrawal periods, sampling day 1 was set as 24 h after the last dose of the treatment. Fish samples were analyzed for SDZ, TMP, and acetyl-sulfadiazine (AcSDZ) residues by liquid chromatography-mass spectrometry. SDZ and TMP concentrations declined rapidly from muscle plus skin. Considering a maximum residue limit of 100μg/kg for the total of sulfonamides and 50μg/kg for TMP residues in fish muscle plus skin, the withdrawal periods of the premix trimethoprim-sulfadiazine 50% were calculated as 5 and 6days, at 24-26 °C, in fish oil (FO) and plant oil (PO) groups, respectively. The investigation of this work is important to protect consumers by controlling the undesirable residues in fish.

Arsenic Induced Inflammation and Apoptosis in Liver, Head-Kidney and Skin of Gilthead Seabream (Sparus aurata)

Arsenic is a metal with strong impact on the aquatic environment but its effects on marine fish immunity is little known. In this study, we have evaluated the regulation in the expression of genes encoding important acute-phase proteins (ceruloplasmin, transferrin and vitellogenin), antimicrobial peptides (b-defensin, hepcidin and histone 2B) and apoptosis (caspases 3, 8 and 9) in the skin, liver and head-kidney (HK) of the marine gilthead seabream (Sparus aurata L.) after waterborne As acute exposure for 2 or 10 days. Ceruloplasmin and vitellogenin transcription was significantly increased at 2 days on skin whilst the transferrin gene was down-regulated in HK and skin after 2 and 10 days, respectively. Concerning the antimicrobial peptides, b-defensin and hepcidin were firstly up-regulated in the HK and later they were up-regulated in the skin as well as the histone 2B gene. Finally, apoptosis was induced in all the tissues after As-exposure as indicated by the up-regulation of caspase genes. The data obtained provide an approach for elucidating the different molecular mechanisms induced by arsenic toxicity in marine fish.

Dietary administration of β-1,3/1,6-glucan and probiotic strain Shewanella putrefaciens, single or combined, on gilthead seabream growth, immune responses and gene expression

It is widely known that β-glucans and probiotic bacteria are good immunostimulants for fish. In the present work we have evaluated the dietary effect of β-1,3/1,6-glucan (isolated from Laminarina digitata) and Pdp 11 (Shewanella putrefaciens, probiotic isolated from gilthead seabream skin), single or combined, on growth, humoural (seric level of total IgM antibodies and peroxidase and antiprotease activities) and cellular innate immune response (peroxidase and phagocytic activities of head-kidney leucocytes), as well as the expression of immune-related genes in gilthead seabream (Sparus aurata). Four treatment groups were established: control (non-supplemented diet), Pdp 11 (109 cfu g−1), β-1,3/1,6-glucan (0.1%) and β-1,3/1,6-glucan + Pdp 11 (0.1% and 109 cfu g−1, respectively). Fish were sampled after 1, 2 and 4 weeks of feeding. Interestingly, all supplemented diets produced increments in the seabream growth rates, mainly the Pdp 11-suplemented diet. Overall, Pdp 11 dietary administration resulted in decreased serum IgM levels and peroxidase activity. However, the seric antiprotease activity was increased in fish fed with both supplements together. Furthermore, β-1,3/1,6-glucan and combined diet increased phagocytic activity after 2 or 4 weeks. At gene level, IL-1β and INFγ transcripts were always up-regulated in HK but only the interleukin reached significance after 4 weeks in the group fed with β-glucan. On the contrary, IgM gene expression tended to be down-regulated being significant after 1 week in seabream specimens fed with β-glucan or β-glucan plus Pdp 11. These results suggest that β-1,3/1,6-glucan and Pdp 11 modulate the immune response and stimulates growth of the gilthead seabream, one of the species with the highest rate of production in Mediterranean aquaculture.

Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata)

Mucosal surfaces of fish, including skin, gill and gut, contain numerous immune substances poorly studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to identify and characterize for the first time different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata). To do this, the levels of total immunoglobulin M, several enzymes and proteins (peroxidase, lysozyme, alkaline phosphatase, esterases, proteases and antiproteases), as well as the bactericidal activity against opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum, Photobacterium damselae) and non-pathogenic bacteria (Escherichia coli, Bacillus subtilis) were measured in the skin mucus and compared with those found in the serum. This study demonstrates that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. In addition, skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, while human bacteria can even grow more in the presence of mucus. The results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system with potential application for the aquaculture.

Infiltration and activation of acidophilic granulocytes in skin lesions of gilthead seabream, Sparus aurata, naturally infected with lymphocystis disease virus

Light, ultrastructural and immunocytochemical investigations were carried out on the skin of gilthead seabream, Sparus aurata L., naturally infected with lymphocystis iridovirus, to assess pathology and host cellular responses. Of 220,000 young seabream examined, 32,400 (14.7%) had clinical signs of lymphocystis and within 6 months of disease appearance, 45% of clinically affected fish had died. A subsample of 20 S. aurata (80.0 ± 12.5 mm total length, mean ± S.D.), including 10 with lymphocystis on the skin and 10 clinically normal, were examined via immunohistochemistry. Affected skin displayed macroscopic, wart-like clusters of hypertrophic fibroblasts which arose from the dermis and were covered by the epithelium. Clusters were encountered on the head, trunk and fins, but there was no evidence of visceral lymphocystis. The lymphocysts were surrounded by numerous granular cells that were positive for the antimicrobial peptide (AMP) piscidin 3 and underwent intense degranulation. To identify the type of granular cells involved in this viral disease, a double immunohistochemical staining with the monoclonal antibody G7 (mAb G7), which is specific for seabream acidophilic granulocytes (AGs), and with anti-histamine (as a marker for mast cells, MCs) was applied to the skin sections of the 10 clinically normal fish and 10 fish with lymphocystis. In infected skin, the number of G7-positive cells (i.e., AGs) (18.5 ± 10.5, mean number of cells per 20,000 μm 2 ± S.D.) was significantly higher compared to their density in uninfected skin (1.4 ± 2.2) ( t test, p < 0.01). Notably, the AGs that infiltrated the skin lesions of infected animals were found to be degranulated and to produce the pro-inflammatory cytokine interleukin-1β. No histamine-positive granular cells (i.e., MCs) were encountered in the lymphocystis lesions. The present study shows the response of skin to lymphocystis disease virus (LCDV) and provides evidence that AGs, but not MCs, are recruited and activated in response to this skin infection.

Gilthead seabream ( Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10 8 cfu g −1 Pdp11, 10 8 cfu g −1 51M6 or with 0.5 × 10 8 cfu g −1 Pdp11 plus 0.5 × 10 8 cfu g −1 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.

Flumequine depletion from muscle plus skin tissue of gilthead seabream (Sparus aurata L.) fed flumequine medicated feed in seawater at 18 and 24 °C

We examined flumequine depletion from muscle plus skin of gilthead seabream held in seawater at 18 and 24 °C. Seven groups of 10 fish each were sampled at intervals ranging from 24 to 168 h after in-feed administration of flumequine at 35 mg/kg/day for 5 days. Muscle plus skin tissue samples were analyzed for flumequine by high-performance liquid chromatography and fluorescence detection (HPLC-SFD). Parent flumequine concentrations declined rapidly from muscle plus skin after dosing with elimination half-lives of t 1/2 =22.14 and 21.43 h at 18 and 24 °C, respectively. Withdrawal periods for the maximum residue limit (MRL) of 600 μg/kg flumequine in muscle plus skin at 95% tolerance limit were 106.08 and 75.84 h at 18 and 24 °C, respectively, after treatment.