Concept: Gilt-head bream
In this study, for the first time, both neuropeptides isotocin (IT) and arginine vasotocin (AVT) have been identified and measured in urophysis, the neurohaemal organ of the caudal neurosecretory system of teleost fish. So far, AVT, but not IT, was quantified by radioimmunoassay (RIA) in urophysis of several fish species. We have used high-performance liquid chromatographic assay with fluorescence detection (HPLC-FL) preceded by solid-phase extraction (SPE) and liquid chromatography-electrospray ionization triple-quadrupole tandem mass spectrometry (LC-ESI MS/MS) technique to determine both neuropeptides in urophysis of three fish species. The efficiency of peptide’s SPE extraction was 79-85 %. In HPLC-FL method, the limits of detection (LOD) and quantification (LOQ) were estimated as 1.0 and 3.4 pmol/mL for IT and 0.25 and 2.20 pmol/mL for AVT. In LC-MS/MS method, LOD and LOQ were estimated as 0.4 and 1.2 pmol/mL for IT and 0.06 and 0.2 pmol/mL for AVT. The chromatographic methods are good alternative for RIA, because enable to measure both nonapeptides simultaneously in one sample. In round goby (Neogobius melanostomus), three-spined stickleback (Gasterosteus aculeatus) and sea bream (Sparus aurata), urophysial IT concentrations ranged between 0.056 and 0.678 pmol/mg tissue and AVT concentrations ranged between 0.0008 (or even below detection threshold) and 0.084 pmol/mg tissue.
Spontaneous ovarian tunica albuginea contractility was evaluated in gilthead seabream (Sparus aurata L.) at different phases of the reproductive cycle. Fourteen adult females were sampled from February to November 2012 in a commercial fish farm, and ovaries were removed and processed for histological and contractility analyses. Fish reproductive stages were evaluated on haematoxylin-eosin-stained ovary sections or by simple macroscopic observation of hydrated oocytes in spawning individuals. Tunica albuginea spontaneous contractility was recorded by using ovary wall strips mounted in an organ bath containing modified Ringer’s solution. Ovary macro- and microscopic analyses allowed the identification of three different reproductive conditions: vitellogenesis, spawning and regressing. The gilthead seabream tunica albuginea was capable to contract spontaneously, and significant differences were found in mean contraction amplitude among the three reproductive states, with the highest value recorded in individuals in regressing condition and the lowest in individuals at spawning stage. No differences in mean contractility frequency among the three different groups were found. Possible involvement of spontaneous contractility in facilitating developing follicle advancement towards the ovarian lumen within the ovary and in supporting recovery of regressing ovaries may be hypothesized. The low contractility observed during the final oocyte maturation and spawning phases does not seem to support a role of tunica albuginea during ovulation, which could conversely involve theca cell contraction. Alternatively, possible single instantaneous contractions of tunica albuginea muscle fibres, not detected in the present study, could occur during ovulation in response to neuro-hormonal stimulations; a role of abdominal wall musculature in ovary “squeezing” and consequent release of ovulated eggs cannot be excluded.
The interest in animal personality, broadly defined as consistency of individual behavioural traits over time and across contexts, has increased dramatically over the last years. Individual differences in behaviour are no longer recognised as noise around a mean but rather as adaptive variation and thus, essentially, raw material for evolution. Animal personality has been considered evolutionary conserved and has been shown to be present in all vertebrates including fish. Despite the importance of evolutionary and comparative aspects in this field, few studies have actually documented consistency across situations in fish. In addition, most studies are done with individually housed fish which may pose additional challenges when interpreting data from social species. Here, we investigate, for the first time in fish, whether individual differences in behavioural responses to a variety of challenges are consistent over time and across contexts using both individual and grouped-based tests. Twenty-four juveniles of Gilthead seabream Sparus aurata were subjected to three individual-based tests: feed intake recovery in a novel environment, novel object and restraining and to two group-based tests: risk-taking and hypoxia. Each test was repeated twice to assess consistency of behavioural responses over time. Risk taking and escape behaviours during restraining were shown to be significantly consistent over time. In addition, consistency across contexts was also observed: individuals that took longer to recover feed intake after transfer into a novel environment exhibited higher escape attempts during a restraining test and escaped faster from hypoxia conditions. These results highlight the possibility to predict behaviour in groups from individual personality traits.
There is a constant need to find feed additives that improve health and nutrition of farmed fish and lessen the intestinal inflammation induced by plant-based ingredients. The objective of this study was to evaluate the effects of adding an organic acid salt to alleviate some of the detrimental effects of extreme plant-ingredient substitution of fish meal (FM) and fish oil (FO) in gilthead sea bream diet. Three experiments were conducted. In a first trial (T1), the best dose (0.4%) of sodium butyrate (BP-70 ®NOREL) was chosen after a short (9-weeks) feeding period. In a second longer trial (T2) (8 months), four diets were used: a control diet containing 25% FM (T2-D1) and three experimental diets containing 5% FM (T2-D2, T2-D3, T2-D4). FO was the only added oil in D1, while a blend of plant oils replaced 58% and 84% of FO in T2-D2, and T2-D3 and T2-D4, respectively. The latter was supplemented with 0.4% BP-70. In a third trial (T3), two groups of fish were fed for 12 and 38 months with D1, D3 and D4 diets of T2. The effects of dietary changes were studied using histochemical, immunohistochemical, molecular and electrophysiological tools. The extreme diet (T2-D3) modified significantly the transcriptomic profile, especially at the anterior intestine, up-regulating the expression of inflammatory markers, in coincidence with a higher presence of granulocytes and lymphocytes in the submucosa, and changing genes involved in antioxidant defences, epithelial permeability and mucus production. Trans-epithelial electrical resistance (Rt) was also decreased (T3-D3). Most of these modifications were returned to control values with the addition of BP-70. None of the experimental diets modified the staining pattern of PCNA, FABP2 or ALPI. These results further confirm the potential of this additive to improve or reverse the detrimental effects of extreme fish diet formulations.
A metabolomic study has been performed to identify sensitive and robust biomarkers of malnutrition in farmed fish, using gilthead sea bream (Sparus aurata) as a model. The metabolomic fingerprinting of serum from fasted fish was assessed by means of ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. More than 15,000 different m/z ions were detected and Partial Least Squares-Discriminant analysis allowed a clear differentiation between the two experimental groups (fed and 10-day fasted fish) with more than 90% of total variance explained by the two first components. The most significant metabolites (up to 45) were elucidated on the basis of their tandem mass spectra with a broad representation of amino acids, oligopeptides, urea cycle metabolites, L-carnitine-related metabolites, glutathione-related metabolites, fatty acids, lysophosphatidic acids, phosphatidylcholines as well as biotin- and noradrenaline-related metabolites. This untargeted approach highlighted important adaptive responses in energy and oxidative metabolism, contributing to identify robust and nutritionally-regulated biomarkers of health and metabolic condition that will serve to assess the welfare status of farmed fish.
Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papilloma-like lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead seabream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not completely characterized yet. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papilloma- and polyomaviruses termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics such as an intron within the L1 gene and, as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought.
The long-term goal of this research project is to set up efficient protocol that can be used to develop a standardized approach for vitrification of marine fish spermatozoa. In particular, the aim of the present study was to develop a vitrification protocol for sea bream (Sparus aurata) spermatozoa. To draw up the protocol, we tested two different dilution media (1% NaCl and Mounib medium), three different vitrification devices (loops, drops and cut straws), different cryoprotectants (CPs) and three different equilibration times (30, 60 and 120 s). The effect of the different vitrification procedures on spermatozoa quality was checked by measuring spermatozoa motility rate and viability, mitochondrial membrane potential and the fertilizing ability of both fresh and post-thawed gametes. The best result was obtained by dropping directly into liquid nitrogen 20 μl of spermatozoa suspension (drop-wise method) diluted with Mounib buffer containing 10% Me2SO + 10% glycerol. The addition of a mixture of anti-freezing proteins, AFPI and AFPIII, to Mounib buffer significantly increases the spermatozoa quality following vitrification so confirming the usefulness of AFPs in improving the quality of gametes subjected to the vitrification process. The present study proves that vitrification offers an alternative to conventional sperm cryopreservation also in this species.
Medical plants could be used as a prophylactic method in aquaculture because they are considered safe and so very promising alternatives to the use of chemicals. The aim of the present work was to examine the effects of dietary fenugreek (Trigonella foenum graecum) seeds administered for 8 weeks on the metabolic and immune status of gilthead seabream (Sparus aurata L.). Four experimental groups were designated: one receiving a basal diet (control) and three fed powdered fenugreek seeds incorporated in the fish feed at 1%, 5% and 10%. The results show that significant decreases in aspartate aminotransferase, creatine kinase, potassium and the albumin/globulin ratio were detected in the serum of fish fed 10% fenugreek compared with the values recorded in control fish. As regards the immune status, fish fed the 5% supplemented diet had higher haemolytic complement and peroxidase activities than the control fish whilst antiprotease activity was higher in fish fed the 1% fenugreek level respect to control fish and the fish fed the highest fenugreek supplementation rate. Interestingly, the results also revealed a significant enhancement of most of the cellular immune parameters studied, especially in fish fed the highest level of fenugreek (10%). However, the bacteriostatic activity of serum against fish pathogenic and non-pathogenic bacteria was non-affected to any significant extent in fish fed the supplemented diets. Overall, the results suggest that the high level of dietary fenugreek tested in this work (10%) did not negatively affect any of the metabolic parameters measured in serum but increased some of them. In addition, the inclusion of fenugreek seeds in the gilthead seabream diet at 5% or 10% improved the humoral and cellular immune activities, respectively. Further studies are needed to better understand the effects of this natural product, which may be suitable for use as a feed additive in fish aquaculture.
The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested.
Microplastics are well-documented pollutants in the marine environment that result from production or fragmentation of larger plastic items. The knowledge about the direct effects of microplastics on immunity, including fish, is still very limited. We investigated the in vitro effects of microplastics [polyvinylchloride (PVC) and polyethylene (PE)] on gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) head-kidney leucocytes (HKLs). After 1 and 24 h of exposure of HKLs with 0 (control), 1, 10 and 100 mg mL-1 MPs in a rotatory system, cell viability, innate immune parameters (phagocytic, respiratory burst and peroxidase activities) and the expression of genes related to inflammation (il1b), oxidative stress (nrf2, prdx3), metabolism of xenobiotics (cyp1a1, mta) and cell apoptosis (casp3) were studied. Microplastics failed to affect the cell viability of HKLs. In addition, they provoke very few significant effects on the main cellular innate immune activities, as decrease on phagocytosis or increase in the respiratory burst of HKLs with the highest dose of microplastics tested. Furthermore, microplastics failed to affect the expression of the selected genes on sea bass or seabream, except the nrf2 which was up-regulated in seabream HKLs incubated with the highest doses. Present results seem to suggest that continue exposure of fish to PVC or PE microplastics could impair fish immune parameters probably due to the oxidative stress produced in the fish leucocytes.