Aluminum adjuvant potentiates gilthead seabream immune responses but induces toxicity in splenic melanomacrophage centers.

A key goal of a successful vaccine formulation is the strong induction of persistent protective immune responses without producing side-effects. Adjuvants have been proved to be successful in several species at inducing increased immune responses against poorly immunogenic antigens. Fish are not the exception and promising results of adjuvanted vaccine formulations in many species are needed. In this study, over a period of 300 days, we characterized the apparent damage and immune response in gilthead seabream immunized by intraperitoneal injection with the model antigen keyhole limpet hemocyanin (KLH) alone or formulated with Montanide ISA water-in-oil (761 or 763), or Imject™ aluminum hydroxide (aluminium), as adjuvants. Throughout the trial, external tissue damage was examined visually, but no change was observed. Internally, severe adhesions, increased fat tissue, and hepatomegaly were recorded, but, without impairing animal health. At 120 days post priming (dpp), histopathological evaluations of head-kidney, spleen and liver revealed the presence of altered melanomacrophage centers (MMC) in HK and spleen, but not in liver. Surprisingly, in all aluminium treated fish, classical stains unmasked a toxic effect on splenic-MMC, unequivocally characterized by a strong cell depletion. Furthermore, at 170 dpp transmission electron microscopy confirmed this data. Paradoxically, at the same time powerful immune responses were recorded in most vaccinated groups, including the aluminium treatment. Whatever the case, despite the observed adhesions and MMC depletion, fish physiology was not affected, and most side-effects were resolved after 300 dpp. Therefore, our data support adjuvant inclusion, but strongly suggest that use of aluminium must be further explored in detail before it might benefit the rational design of new vaccination strategies in aquaculture.

Aluminum is a powerful adjuvant in teleost fish despite failing to induce interleukin-1ß release.

Although aluminum salts (Alum) have been extensively used in human vaccination for decades, its mechanism of action is controversial. In fish, the use of Alum as a vaccine adjuvant is scarce and there are no studies aimed at identifying its mechanism of action. In the present study we report that Alum is a powerful adjuvant in the gilthead seabream (Sparus aurata L., Sparidae) and the European seabass (Dicentrarchus labrax L. Moronidae). Thus, Alum increased the specific antibody titers to the model antigen keyhole limpet hemocyanin as the commonly used Freund's adjuvant did in both species. In addition, both adjuvants were able to increase the transcript levels of the gene encoding the major pro-inflammatory mediator interleukin-1ß (Il1b). Strikingly, however, Alum failed to promote Il1b release by seabream leukocytes and even impaired Il1b induction, processing and release in macrophages. However, it increased NADPH oxidase-dependent reactive oxygen species (ROS) production in gilthead seabream leukocytes and purified granulocytes. In addition, Alum promoted gilthead seabream leukocyte death independently of ROS production and caspases, suggesting that damage-associated molecular patterns release from dying cells mediate Alum adjuvant activity. Our results pave the way for future studies aimed at investigating the relevance of danger signals generated by Alum in vivo on its adjuvant activity in order to increase our understanding of the mechanisms of action of Alum in fish vaccines and to help in the design of new adjuvants for aquaculture.

Cimetidine disrupts the renewal of testicular cells and the steroidogenesis in a hermaphrodite fish.

The importance of histamine in the physiology of the testis in mammals and reptiles has been recently shown. Histamine receptors (Hrs) are well conserved in fish and are functional in several fish species. We report here for the first time that histamine and the mRNA of Hrh1, Hrh2 and Hrh3 are all present in the gonad of the hermaphrodite teleost fish gilthead seabream. Moreover, cimetidine, which acts in vitro as an agonist of Hrh1 and Hrh2 on this species, was intraperitoneally injected in one and two years old gilthead seabream males. After three and five days of cimetidine injection, we found that this compound differently modified the gonadal hrs transcript levels and affects the testicular cell renewal and the gene expression of steroidogenesis-related molecules as well as the serum steroid levels. Our data point to cimetidine as a reproductive disruptor and elucidate a role for histamine in the gonad of this hermaphrodite fish species through Hr signalling.

Characterization of the annual regulation of reproductive and immune parameters on the testis of European sea bass.

The European sea bass, Dicentrarchus labrax L., is a seasonal gonochoristic species, the males of which are generally mature during their second year of life. It has been demonstrated that cytokines and immune cells play a key role in the testicular development. This reproductive-immune interaction might be very important in the sea bass since several pathogens are able to colonise the gonad and persist in this tissue, altering further reproductive functions and spreading disease. This study aims to investigate the reproductive cycle of 1-year European sea bass males by analysing cell proliferation and apoptosis and the expression profile of some reproductive and immune-related genes in the testis, as well as the serum sex steroid levels. Our data demonstrate that, in 1-year-old European sea bass males, the testis undergoes the spermatogenesis process and that the reproductive and immune parameters analysed varied during the reproductive cycle. In the testis, the highest proliferative rates were recorded at the spermatogenesis stage, while the highest apoptotic rates were recorded at the spawning stage. We have also analysed, for the first time in European sea bass males, the serum levels of 17ß-estradiol (E2) and dihydrotestosterone and the gene expression profile of the enzymes implied in their production, determining that at least E2 might be involved in the regulation of the reproductive cycle. Some immune relevant genes, including cytokines, lymphocyte receptors, and anti-viral and anti-bacterial molecules were detected in the testis of naïve European sea bass specimens, and their expression profile was related to the stages of the reproductive cycle, suggesting an important role for the defence of the reproductive tissues.

Antimicrobial response is increased in the testis of European sea bass, but not in gilthead seabream, upon nodavirus infection.

Antimicrobial peptides (AMPs) have a crucial role in the fish innate immune response, being considered a fundamental component of the first line of defence against pathogens. Moreover, AMPs have not been studied in the fish gonad since this is used by some pathogens as a vehicle or a reservoir to be transmitted to the progeny, as occurs with nodavirus (VNNV), which shows vertical transmission through the gonad and/or gonadal fluids, but no study has looked into the gonad of infected fish. In this framework, we have characterized the antimicrobial response triggered by VNNV in the testis of European sea bass, a very susceptible species of the virus, and in the gilthead seabream, which acts as a reservoir, both in vivo and in vitro, and compared with that present in the serum and brain (target tissue of VNNV). First, our data show a great antiviral response in the brain of gilthead seabream and in the gonad of European sea bass. In addition, for the first time, our results demonstrate that the antimicrobial activities (complement, lysozyme and bactericidal) and the expression of AMP genes such as complement factor 3 (c3), lysozyme (lyz), hepcidin (hamp), dicentracin (dic), piscidin (pis) or ß-defensin (bdef) in the gonad of both species are very different, but generally activated in the European sea bass, probably related with the differences of susceptibility upon VNNV infection, and even differs to the brain response. Furthermore, the in vitro data suggest that some AMPs are locally regulated playing a local immune response in the gonad, while others are more dependent of the systemic immune system. Data are discussed in the light to ascertain their potential role in viral clearance by the gonad to avoid vertical transmission.

Flagellin from Marinobacter algicola and Vibrio vulnificus activates the innate immune response of gilthead seabream.

Adjuvants have emerged as the best tools to enhance the efficacy of vaccination. However, the traditional adjuvants used in aquaculture may cause adverse alterations in fish making necessary the development of new adjuvants able to stimulate the immune system and offer strong protection against infectious pathogens with minimal undesirable effects. In this respect, flagellin seems an attractive candidate due to its ability to strongly stimulate the immune response of fish. In the present study, we have evaluated the ability of recombinant flagellin from Marinobacter algicola (MA) and Vibrio vulnificus (Vvul), a non-pathogenic and a pathogenic bacteria, respectively, to stimulate the innate immune system of gilthead seabream (Sparus aurata L.) and compare the effect with that of the classical flagellin from Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, STF). Intraperitoneal injection of MA and Vvul resulted in a strong inflammatory response characterized by increased reactive oxygen species production and the infiltration of acidophilic granulocytes at the injection site. Interestingly, however, only flagellin from MA consistently induced the expression of the gene encoding pro-inflammatory interleukin-1ß. These effects were further confirmed in vitro, where a dose-dependent activation of macrophages and acidophilic granulocytes by MA and Vvul flagellins was observed. In contrast, STF flagellin was found to be less potent in both in vivo and in vitro experiments. Our results suggest the potential use of MA and Vvul flagellins as immunostimulants and adjuvants for fish vaccination.

Isolation of mast cells from the peritoneal exudate of the teleost fish gilthead sea bream (Sparus aurata L.).

Inflammation is the first response of animals to infection or tissue damage. Sparus aurata (Perciformes) was the first fish species shown to possess histamine-containing mast cells at mucosal tissues. We report a separation protocol for obtaining highly enriched (over 95% purity) preparations of fish mast cells in high numbers (5-20 million mast cells per fish). The peritoneal exudate of S. aurata is composed of lymphocytes, acidophilic granulocytes, macrophages and mast cells. We separated the lymphocyte fraction through discontinuous density gradient centrifugation. The remaining cells were cultivated overnight in RPMI-1640 culture medium containing 5% fetal calf serum, which allowed macrophages to adhere to the cell culture flasks. Finally, acidophilic granulocytes were separated from the mast cells though a Magnetic-Activated Cell Separation (MACS) protocol, using a monoclonal antibody against these cells. The purity of mast cells-enriched fractions was analyzed by flow cytometry and by transmission electron microscopy. The functionality of purified mast cells was confirmed by the detection of histamine release by ELISA after stimulation with compound 48/80 and the induction of the pro-inflammatory cytokines IL-1ß and IL-8 following stimulation with bacterial DNA. This fish mast cells separation protocol is a stepping stone for further studies addressing the evolution of vertebrate inflammatory mechanisms.

Seasonal variations of the humoral immune parameters of European sea bass (Dicentrarchus labrax L.).

Seasonal cycles, mainly due to great variations in the light duration and temperature, are important and modulate several aspects of the animal behavior. In the case of poikilotherms animals such as fish this is very relevant. Thus, temperature changes fish immunity and affects disease resistance. We evaluate in this work the season variations of the European sea bass (Dicentrarchus labrax) humoral innate parameters focusing on winter months, at which the culture of this specie is more difficult. Our results showed that not all the innate immune parameters are depressed by low temperatures. Moreover, some of them are more dependent than others to the season and both temperature and photoperiod are operating together.

Fish granulocytes express a constitutively active androgen receptor variant.

In humans, alternative splicing of androgen receptor (AR) is usually involved in some diseases. However, our knowledge about the presence of AR variants in other species and its importance for immunity is scant. Here, we report the identification of a constitutively active AR variant lacking the ligand-binding domain (LBD), ARΔLBD, in the fish gilthead seabream. ARΔLBD is expressed in the testis and the head-kidney (HK), and its expression varies with the reproductive stage and is correlated with plasma testosterone (T). In addition, ARΔLBD is expressed in acidophilic granulocytes (AGs), which are the functional equivalent of mammalian neutrophils, but not in macrophages, and its expression is modulated by both T and immune stimuli. Notably, AR and ARΔLBD were able to interact, being the activity of AR dominant at all concentrations tested of the ligand. These results reveal a new mechanism for the regulation of neutrophil biology in vertebrates and explain the conflicting results that suggest that androgens are less important than AR in human and mouse neutrophil homeostasis.

The effect of 17α-ethynylestradiol on steroidogenesis and gonadal cytokine gene expression is related to the reproductive stage in marine hermaphrodite fish.

Pollutants have been reported to disrupt the endocrine system of marine animals, which may be exposed through contaminated seawater or through the food chain. Although 17α-ethynylestradiol (EE2), a drug used in hormone therapies, is widely present in the aquatic environment, current knowledge on the sensitivity of marine fish to estrogenic pollutants is limited. We report the effect of the dietary intake of 5 µg EE2/g food on different processes of testicular physiology, ranging from steroidogenesis to pathogen recognition, at both pre-spermatogenesis (pre-SG) and spermatogenesis (SG) reproductive stages, of gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. A differential effect between pre-SG and SG specimens was detected in the sex steroid serum levels and in the expression profile of some steroidogenic-relevant molecules, vitellogenin, double sex- and mab3-related transcription factor 1 and some hormone receptors. Interestingly, EE2 modified the expression pattern of some immune molecules involved in testicular physiology. These differences probably reflect a developmental adjustment of the sensitivity to EE2 in the gilthead seabream gonad.

Recombinant TNFα as oral vaccine adjuvant protects European sea bass against vibriosis: insights into the role of the CCL25/CCR9 axis.

Vibrio anguillarum is the main causative agent of vibriosis in cultured sea bass. Unfortunately, available vaccines against this disease do not achieve the desired protection. In this study, to accomplish uptake, processing, and presentation of luminal antigens, a commercial sea bass oral vaccine against V. anguillarum was improved with the addition of recombinant fish-self tumor necrosis factor α (rTNFα), as adjuvant. To explore mechanisms, systemic and local responses were analyzed through serum specific IgM titers, gene expression, lymphocytes spatial distribution in the gut, and in vitro functional assays. We found along the trial, over expressed transcripts of genes encoding cytokines and antimicrobial molecules at the gut of rTNFα supplied group. Orally immunized fish with vaccine alone confer protection against V. anguillarum challenge throughout a short time period. In contrast, adjuvant-treated group significantly extended the response. In both cases, achieved protection was independent of serum IgM. Yet, IgT transcripts were found to increase in the gut of rTNFα-treated fish. More importantly, fish treated with rTNFα showed a dramatic change of their T lymphocytes distribution and localization in gut mucosal tissue, suggesting specific antigen recognition and further intraepithelial T lymphocytes (IEL) activation. To determine the mechanism behind IEL infiltration, we characterized the constitutive and activated pattern of chemokines in sea bass hematopoietic tissues, identifying for the first time in fish gut, an intimate relation between the chemokine ligand/receptor CCL25/CCR9. Ex-vivo, chemotaxis analyses confirmed these findings. Together, our results demonstrate that improved oral vaccines targeting key cytokines may provide a means to selectively modulate fish immune defence.

Testosterone implants modify the steroid hormone balance and the gonadal physiology of gilthead seabream (Sparus aurata L.) males.

Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish; however, many aspects of the role of androgens in adult teleost spermatogenesis remain elusive. We used the in situ forming microparticle (ISM) system containing 1mg of testosterone (T)/kg body weight (T-ISM) in a homogenous population of gilthead seabream at testicular involution stage to study in vivo the effects of T on the sex steroid hormone balance and on the physiology of the gilthead seabream gonad. The levels of T, 11-ketotestosterone (11KT) and 17ß-estradiol (E2) in plasma, gonad and liver were determined in T-ISM implanted specimens after 7, 14, 21 and 28 days. The effect of T-ISM was evaluated on (i) de novo synthesis and metabolism of T in the gonad and liver by measuring the gene expression levels of the main steroidogenic proteins involved, (ii) the progress of spermatogenesis, (iii) the presence of different leukocyte cell types in the gonad, and (iv) the mRNA expression of some genes involved in the leukocyte migratory influx into the gonad and of some immune-relevant molecules. T-ISM implants promote an increase of T up to supra-physiological levels which induce a depletion of E2 levels and maintain the 11KT levels at physiological concentrations. The gene expression profile of some steroidogenic enzymes in gonad and liver ruled out the transformation of T into estrogenic compounds following T-ISM implantation. Moreover, androgens may also be involved in the leukocyte migratory influx, which occurred even when cytokine, chemokine and cell adhesion molecule gene expressions were down-regulated. Moreover, T-ISM implants block germ cell proliferation, although increased dmrt1 gene expression may prevent the complete depletion of germ cells in the gonad. Furthermore, T down-regulated the expression of several tlr genes, which may result in the inhibition of the immune response in the gonad through the impaired ability to recognize and respond to pathogens.

Muscle development and body growth in larvae and early post-larvae of shi drum, Umbrina cirrosa L., reared under different larval photoperiod: muscle structural and ultrastructural study.

Shi drum specimens were maintained under four different photoperiod regimes: a natural photoperiod regime (16L:8D), constant light (24L), equal durations of light and dark (12L:12D) and a reduced number of daylight hours (6L:18D) from hatching until the end of larval metamorphosis. Specimens were then kept under natural photoperiod conditions until 111 days post-hatching. Muscle and body parameters were studied. During the vitelline phase, there was little muscle growth and no photoperiod effects were reported; however, a monolayer of red muscle and immature white muscle fibres were observed in the myotome. At hatching, external cells (presumptive myogenic cells) were already present on the surface of the red muscle. At the mouth opening, some presumptive myogenic cells appeared between the red and white muscles. At 20 days, new germinal areas were observed in the apical extremes of the myotome. At this stage, the 16L:8D group (followed by the 24L group) had the longest body length, the largest cross-sectional area of white muscle and the largest white muscle fibres. Conversely, white muscle hyperplasia was most pronounced in the 24L group. Metamorphosis was complete at 33 days in the 24L and 12L:12D groups. At this moment, both groups showed numerous myogenic precursors on the surface of the myotome as well as among the adult muscle fibres (mosaic hyperplastic growth). The 16L:8D group completed metamorphosis at 50 days, showing a similar degree of structural maturity in the myotome to that described in the 24L and 12L:12D groups at 33 days. When comparing muscle growth at the end of the larval period, hypertrophy was highest in the 16L:8D group, whereas hyperplasia was higher in the 24L and 16L:8D groups. At 111 days, all groups showed the adult muscle pattern typical of teleosts; however, the cross-sectional area of white muscle, white muscle fibre hyperplasia, body length and body weight were highest in the 24L group, followed by the 12L:12D group; white muscle hypertrophy was similar in all groups. Larval survival was higher under natural photoperiod conditions compared to all the other light regimes.

17α-Ethynylestradiol alters the immune response of the teleost gilthead seabream (Sparus aurata L.) both in vivo and in vitro.

There is increasing public attention concerning the effect of endocrine disruptor chemicals (EDCs) on the immune system. One important group belonging to EDCs are the environmental estrogens. Commonly found in the effluents in wastewater treatment plants, 17α-ethynylestradiol (EE(2)) which is used in contraceptive pills, is an endocrine disruptor with strong estrogenic effects. This study aims to investigate the capacity of EE(2) to modulate in vivo and in vitro the innate immune response of the gilthead seabream (Sparus aurata L.), a teleost species of great commercial value. For this purpose, adult specimens were bath-exposed to EE(2) (0, 5 and 50 ng/L) and then immunized with hemocyanin in the presence of the adjuvant aluminum. The results indicate that, after 15 days of EE(2)-exposure, the disruptor was able to inhibit in a dose-dependent manner the induction of interleukin-1ß (IL-1ß) gene expression, but did not significantly alter the specific antibody titer. To shed light on the role played by EE(2) into seabream immune response, leukocytes were exposed in vitro to several concentrations of EE(2) (0, 0.5, 5, 50 and 500 ng/ml) for 3, 16 and 48 h and the production of reactive oxygen intermediates, the phagocytic activity and the gene expression profile of these cells were analyzed. EE(2) was seen to inhibit both cellular activities and to alter the immune gene expression profile in primary macrophages. Thus, low concentrations of EE(2) increase the mRNA levels of IL-1 ß, IL-6, tumour necrosis factor α and tumour growth factor ß in non-activated macrophages. In contrast, EE(2) treatment of activated macrophages resulted in the decreased expression of pro-inflammatory genes and the increased expression of genes encoding anti-inflammatory and tissue remodeling/repair enzymes. Taken together, our results suggest that EE(2) might alter the capacity of fish to appropriately respond to infection although it does not behave as an immunosuppressor.

Aquaporin-4 immunoreactivity in Müller and amacrine cells of marine teleost fish retina.

Aquaporins (AQPs) are membrane proteins that facilitate water transport across biological membranes and are essential for the proper function of neural tissue. Although AQPs have been extensively studied in mammalian retina, their presence in lower vertebrate retina is less frequently characterized. AQP4 expressed in mammalian and chick Müller cells plays a major part in maintaining retinal homeostasis. In this study, we examined the immunoreactivity of AQP4 in the adult retina of gilthead sea bream (Sparus aurata-teleost fish), during light and dark adaptation. The AQP4 expression was detected in Müller cell somas at the inner nuclear layer and in the end-feet processes near the vitreoretinal border. Moreover, AQP4 was also evident in cone photoreceptor cells and in a GABAergic subpopulation of amacrine cells (AQP4-ACs). Four different types of AQP4-ACs were characterized based on their morphology and dendrite stratification. Interestingly, a stronger AQP4 immunoreactivity was observed in the inner nuclear layer during dark adaptation, accompanied by a significant increment in AQP4-ACs cell size. Hence, AQP4 may play an important role in water distribution in the teleost fish retina.

TLR agonists extend the functional lifespan of professional phagocytic granulocytes in the bony fish gilthead seabream and direct precursor differentiation towards the production of granulocytes.

Neutrophils are major cells participants in innate host responses. They are short-lived leukocytes, although microbial products activate intracellular signaling cascades that prolong their survival by inhibiting constitutive apoptosis. To gain insight into the phylogeny of this important cell type, we examined the ability of toll-like receptor agonists to extend the lifespan of gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils. The results obtained demonstrated that apoptosis was also the default state of seabream acidophilic granulocytes and that toll-like receptor agonists were able to dramatically extend their functional lifespan (up to 10 days) by inhibiting apoptosis and inducing a long lasting activation of phagocytic and respiratory burst activities, together with the expression of genes coding for several proinflammatory molecules. This process was independent on contaminating cells and interleukin-1ß production. In addition, the results showed that p38 mitogen-activated protein kinase, but not nuclear factor κB, c-Jun terminal kinase or phosphatidylinositol 3-kinase, was involved in the inhibition of acidophilic granulocyte apoptosis following toll-like receptor engagement. Finally, stimulation of head kidney hematopoietic precursor cells with toll-like receptor agonists promoted their terminal differentiation to acidophilic granulocytes. These results demonstrated that the extension of neutrophil lifespan by microbial products is conserved in lower vertebrates although the magnitude of the response is much higher in fish.

Digestive enzymatic profile of Dentex dentex and response to different dietary formulations.

Digestive physiology of on-growing common dentex (Dentex dentex), including protease, amylase and lipase activity in stomach, pyloric caeca, anterior and posterior intestine, was evaluated. The influence of dietary macronutrient balance on these digestive processes was also assessed. Four experimental diets with different protein:lipid:carbohydrate ratios (43/16/28; 43/24/4; 38/19/28 and 38/24/13) were formulated. The highest activity for acid proteases was located in the stomach at pH 1.5. Alkaline proteolytic activities showed the highest values in the pyloric caeca and posterior intestine at pH 8.5-9.0. Dentex showed substantial amylase activity in the pyloric caeca and posterior intestine. Lipase activity was higher in the pyloric caeca, anterior and posterior intestine and was not detected in the stomach. Feed composition influenced alkaline protease activity in the anterior and posterior intestine and was higher for the diet with less protein and more carbohydrates. Enhanced amylase activity was observed in the pyloric caeca and posterior intestine in those groups fed on higher carbohydrate and lower lipid level diets. High dietary carbohydrate levels produced the highest lipase activity but this only occurred in the anterior intestine. We can conclude that the digestive tract of dentex adapts well to protein digestion and possesses a high potential for digesting the other dietary macronutrients, too. Dietary carbohydrate content seems to induce changes in protease, amylase and lipase activity.

Vaccination of larvae of the bony fish gilthead seabream reveals a lack of correlation between lymphocyte development and adaptive immunocompetence.

Fish eggs are released and embryos hatch into a pathogenically hostile environment, at a time when their immunological capacity is severely limited. Although the eggs are initially protected by the envelope as well as by several innate and adaptive immune substances, which are transferred to eggs during fish vitellogenesis, it seems that young specimens depend fundamentally on their innate defence mechanisms. Here we show in the gilthead seabream, an immunologically tractable teleost fish model, that the first lymphocyte marker genes, those coding for the two subunits for the recombination activating gene, were detected by RT-PCR around 21-27 days post-hatching (dph). In addition, the transcripts coding for the alpha and beta subunits of the T-cell receptor and the light and heavy chains of immunoglobulin M were detected at 27-48 dph. However, most innate immune genes analyzed were already expressed at hatching, including those coding for the toll-like receptors, pro- and anti-inflammatory molecules, antiviral and antibacterial factors, and phagocyte markers. Using the information from the gene expression study, we also examined the achievement of immunocompetence by analyzing the protection induced by a bacterin against the pathogenic bacterium Photobacterium damselae subsp. piscicida. The results show that vaccination of young larvae of this species by either immersion or oral routes resulted in increased susceptibility to infection of the specimens, and point to the lack of correlation between the achievement of immunocompetence and detection of the adaptive immunity markers.

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration.

BACKGROUND: Leukocytes are found within the testis of most, if not all, mammals and are involved in immunological surveillance, physiological regulation and tissue remodelling. The testis of seasonal breeding fish undergoes a regression process. In the present study, the second reproductive cycle (RC) of the protandrous seasonal teleost fish, gilthead seabream, was investigated and the presence of leukocytes analysed. Special attention has been paid to the testicular degenerative process which is particularly active in the last stage of the second RC probably due to the immediacy of the sex change process. METHODS: Sexually mature specimens (n = 10-18 fish/month) were sampled during the second RC. Some specimens were intraperitoneally injected with bromodeoxyuridin (BrdU) before sampling. Light and electron microscopy was used to determine the different stages of gonadal development and the presence of leukocytes and PCR was used to analyse the gene expression of a testis-differentiating gene and of specific markers for macrophages and B and T lymphocytes. Immunocytochemistry and flow cytometry were performed using a specific antibody against acidophilic granulocytes from the gilthead seabream. Cell proliferation was detected by immunocytochemistry using an anti-BrdU antibody and apoptotic cells by in situ detection of DNA fragmentation. RESULTS: The fish in the western Mediterranean area developed as males during the first two RCs. The testis of all the specimens during the second RC underwent a degenerative process, which started at post-spawning and was enhanced during the testicular involution stage, when vitellogenic oocytes appeared in the ovary accompanied by a progressive increase in the ovarian index. However, only 40% of specimens were females in the third RC. Leukocytes (acidophilic granulocytes, macrophages and lymphocytes) were present in the gonad and acidophilic granulocyte infiltration occurred during the last two stages. At the same time DMRT1 gene expression decreased. CONCLUSIONS: The results demonstrate that innate and adaptive immune cells are present in the gonads of gilthead seabream. Moreover, the whole fish population underwent a testicular degenerative process prior to sex change, characterized by high rates of apoptosis and necrosis and accompanied by an infiltration of acidophilic granulocytes and a decrease in DMRT1 levels.

Distribution of the professional phagocytic granulocytes of the bony fish gilthead seabream (Sparus aurata L.) during the ontogeny of lymphomyeloid organs and pathogen entry sites.

Although it is believed that fish fry depend fundamentally on their innate defence mechanisms, the ontogeny of fish innate immune cells is poorly understood. In the present study, we have used a specific monoclonal antibody against acidophilic granulocytes (AGs), the main professional phagocytic cell type of the bony fish gilthead seabream, to study their localization during the development of the main lymphomyeloid organs, namely the head kidney, spleen and thymus, and of the two major portals for pathogen entry, namely the gills and intestine. AGs were observed in the posterior intestine and in the blood earlier than in the haematopoietic kidney (21 vs. 27 days post-hatching, dph). AGs were observed scattered between other cells of the haematopoietic lineage in the head kidney of larvae, but were grouped around the blood vessels of this organ in juveniles and adults, where they were also much more numerous. In the spleen and in the thymus, AGs were observed much later (62 dph) and appeared scattered. AGs were also observed in the gill lamella and the posterior intestine near the anus throughout development.