In aquatic animals, the mucosal barrier is the first line of innate immune defence against external chemicals and pathogens. In this study, the effects of dietary
Moringa oleifera
leaf (MOL) ...supplementation on skin and gill mucosal immunity, antioxidants and stress responses were evaluated in seabream (
Sparus aurata
) fingerlings exposed to hydrogen peroxide (H
2
O
2
)
. A total of 144 specimens (10.11 ± 0.41 g) were divided into four treatments (three replicates per treatment contained 12 specimens each) and fed a non-supplemented control diet or a 1, 2.5 or 5% MOL-supplemented diet. After three weeks of feeding, six specimens from each aquarium were sampled for blood, mucus and tissues. The other six fish in each aquarium were subjected to H
2
O
2
exposure. The results revealed that MOL did not negatively affect either cortisol or glucose levels. MOL supplementation significantly (
P
< 0.05) improved skin mucosal immunity-related characteristics, including phosphatase, peroxidase and lysozyme activity and IgM levels. Additionally, MOL upregulated the expression of antioxidant genes (
sod
and
cat
), an anti-inflammatory gene (
tgf-β
), tight junction protein genes (
occludin
and
zo-1
),
c3,
and
igm
in both the skin and gills. However, H
2
O
2
exposure significantly (
P
< 0.05) increased both cortisol and glucose levels and disrupted skin mucosal immune function by significantly (
P
< 0.05) decreasing phosphatase, peroxidase, protease, antiprotease and lysozyme activity and IgM levels. H
2
O
2
exposure severely decreased the mRNA levels of the studied genes. MOL dietary supplementation at the 5% level successfully attenuated the negative effects of H
2
O
2
on the mucosal immune response in both the skin and gills. In conclusion, dietary MOL supplementation at the 5% level is recommended to improve
S. aurata
mucosal immune function under both normal and stress conditions. Additionally, exposure to H
2
O
2
disrupts the mucosal immunity of fish. This contributes knowledge on the routes involved in mucosal innate immunity and could help to understand the fish resistance against chemicals exposure.
Graphical abstract
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 6 hr 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 1.5 hr and 3 hr 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 1.5 hr 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 6 hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up‐regulated at 6 hr 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.
This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate‐buffered saline (control) in gilthead seabream (Sparus aurata). Skin mucus, ...serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6 hr post‐injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrio anguillarum and Photobacterium damselae, and total immunoglobulins compared with those of the control group. However, the carrageenin‐injected fish sampled at 6 hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6 hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin‐1β and prostaglandin D synthase 1 were upregulated at 1.5 and 3 hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6 hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ‐carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.
This work aimed to carry out an in vivo study of the skin healing process in gilthead seabream (Sparus aurata) after being experimentally wounded. Firstly, the structure of normal skin was studied by ...real‐time ultrasonography (Vevo Lab, VisualSonics) and light microscopy. Besides this, experimental wounds were made on the left flank of each fish with a circular biopsy punch (8 mm diameter) below the lateral line. The healing process was assessed on live fish at 0, 6, 11 and 23 days post‐wounding using the real‐time ultrasonography in B‐mode and Power Doppler mode (Vevo 3100 FUJIFILM, VisualSonics). Through the ultrasonography images, both the skin structure and the evolution of the changes that wounds originated in the surrounding tissues were studied in vivo over time. Concomitantly, the pattern of neovascularization in the wounded area was followed during the healing process and it was demonstrated that, although the neovascularization started very early after the skin damage, it was increased in wounded areas from day 11 post‐wounding onwards. The results obtained proved the utility and power of using ultrasounds in fish to evaluate in vivo complex biological processes in real time, which are difficult to study by other methodologies. The present data shed some light on the reparation of external injuries in aquatic vertebrates.
In recent years, the increasing use of fish as new animal models in scientific research and the growth of fish farming (mainly for human consumption) have highlighted the need for advanced technology ...to deepen our knowledge of fish biology. Hence, the present study was carried out to radiologically analyse the whole body of gilthead seabream (Sparus aurata) specimens using X‐ray computed tomography (CT). Images were acquired in an Albira SPECT/PET/CT tri‐modal preclinical‐scanner. Segmentation, measurements and three‐dimensional reconstruction were made using the Carestream Molecular imaging Albira CT system in conjunction with Pmod, AMIDE and Amira software packages. The results showed that the density values of gilthead seabream are in the range −700 to +2500 HU for the whole body. We also determined the density ranges that topographically coincide with the swim bladder, soft tissues, fat, skin and skeleton. This work describes, validates and demonstrates the application of a fully automated image analysis technique to study and quantify fish body composition, whether segmented or as a whole. In addition, the basis for applying this image technique in other in vivo studies is established.
The effect of the dietary incorporation of drumstick,
Moringa oleifera
, leaf meal (MOL; 0, 5, 10 and 15%) on the growth, feed utilization, some skin mucus and systemic immune parameters and ...intestinal immune-related gene expression in gilthead seabream (
Sparus aurata
) specimens. The experiment lasted 4 weeks. The results revealed that MOL can be incorporated in
S. aurata
diet up to 10% with no significant negative effect on growth and feed utilization. However, there was a significant decrease with MOL at a level of 15% after 2 weeks of feeding. The systemic immune status of fish fed with the different levels of MOL showed an improvement in head kidney leucocyte phagocytosis, respiratory burst and peroxidase activities. Also, serum humoral components, including protease, ACH
50
and lysozyme activities and IgM level, increased with MOL inclusion especially at the 5% level. MOL at 5% improved skin-mucosal immunity such as protease, antiprotease, peroxidase and lysozyme activities. Moreover, the feeding of MOL revealed an upregulation of the intestinal mucosal immunity genes (
lyso
and
c3
), tight junction proteins (
occludin
and
zo-1
) and anti-inflammatory cytokines (
tgf-β
) with a downregulation of pro-inflammatory cytokine (
tnf-α
). Therefore, it is recommended to incorporate MOL in
S. aurata
diets at a level of 5% for the best immune status or 10% for the high growth performance and acceptable immune surveillance.
Graphical abstract
ᅟ
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.
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•The intramuscular injection of λ-carrageenin to seabream induced inflammation.•The skin thickness increased until 6 h post-injection (p.i.) and decreased to the normal values in 12 h.•In the underlying muscle at the injection place several hyperechoic small foci surrounded by an anechoic area were observed.•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.•The micro-CT acquisitions revealed a dark area in the place of the injection with λ-carrageenin visible for 6 h.
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.
In an attempt to control the proliferation of the pathogenic bacterium Vibrio parahaemolyticus in gilthead seabream (Sparus aurata), the immunostimulant effect of lysate and ToxA from this bacterium ...was evaluated. Fish were intraperitoneally injected twice (first injection, day 1 of the experiment; second injection, day 7) and sampled after one week (on days 8 and 15). Afterwards, all fish specimens were experimentally infected with V. parahaemolyticus and mortality was recovered for 1 week. Fish injected with lysate, ToxA and phosphate buffer saline (control) showed 100%, 50% and 0% survival, respectively, when challenged with the pathogen. Skin mucus immune parameters and immune-related gene expression in skin and spleen were also evaluated. The results showed that mucus immune parameters were enhanced in the lysate and ToxA groups compared with the values obtained for fish from the control group. Expression of IL-1β, TNF-α, C3 and IgM genes was significantly up-regulated in the lysate and ToxA groups, principally after infection with the bacterium. Interestingly, TLR5 gene expression increased in fish immunized with lysate. The most prominent histological characteristic in gut from infected fish was the presence of a great number of intraepithelial leucocytes as well as inflammation of the submucosa, while severe hydropic degeneration and hemosiderosis were detected in liver from infected fish. Injection of lysate or ToxA had a protective effect against the deleterious consequences of subsequent infection with V. parahaemolyticus in gut and liver. The findings underline the potential of lysate and ToxA as potent preventive antigens against this kind of vibriosis.
•The immunostimulant effects of lysate and ToxA from Vibrio parahaemolyticus have been evaluated.•Fish immunized with lysate and ToxA had 100% and 50% of survival, respectively, when challenged with the pathogen.•Immune parameters in skin mucus were enhanced by intraperitoneal immunization.•Injection of lysate or ToxA to fish has a protective effect on the deleterious effects caused by V. parahaemolyticus.•Lysate and ToxA could be considered as potent preventive antigens to vibriosis for gilthead seabream.
Changes in different immune activities in the skin mucus of gilthead seabream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) specimens exposed to a constant light–dark photoperiod ...(12 h L:12 h D) were studied. Samples were collected at 08:00 (light on), 14:00, 20:00 (light off), 02:00, and again at 08:00 to determine immunoglobulin M (IgM) levels, several enzymes related to the immune system, and bactericidal activity. IgM levels were higher during the day in seabream and reached a minimum value at 20:00, but it was hardly affected in sea bass. No significant variations were recorded in the levels of protease and antiprotease. Peroxidase reached its maximum level in seabream at 02:00, the same time that it reached its minimum level in sea bass. Lysozyme showed little variation in seabream, but it was significantly lower at 14:00 than during the rest of the cycle in sea bass. Finally, different interspecific variations on bactericidal activity against Vibrio harveyi were recorded. The findings demonstrate that the immune parameters present in skin mucus of these important fish species are affected by the light–dark cycle and that there are substantial interspecies differences.
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