Profile of Innate Immunity in Gilthead Seabream Larvae Reflects Mortality upon Betanodavirus Reassortant Infection and Replication

Historically, gilthead seabream (Sparus aurata) has been considered a fish species resistant to nervous necrosis virus (NNV) disease. Nevertheless, mortality in seabream hatcheries, associated with typical clinical signs of the viral encephalopathy and retinopathy (VER) disease has been confirmed to be caused by RGNNV/SJNNV reassortants. Because of this, seabream larvae at 37 and 86 days post-hatching (dph) were infected by immersion with RGNNV/SJNNV and SJNNV/RGNNV reassortants under laboratory conditions, and mortality, viral replication and immunity were evaluated. Our results show that gilthead seabream larvae, mainly those at 37 dph, are susceptible to infection with both NNV reassortant genotypes, with the highest impact from the RGNNV/SJNNV reassortant. In addition, viral replication occurs at both ages (37 and 86 dph) but the recovery of infective particles was only confirmed in 37 dph larvae,; this value was also highest with the RGNNV/SJNNV reassortant. Larvae immunity, including the expression of antiviral, inflammatory and cell-mediated cytotoxicity genes, was affected by NNV infection. Levels of the natural killer lysin (Nkl) peptide were increased in SJNNV/RGNNV-infected larvae of 37 dph, though hepcidin was not. Our results demonstrate that the seabream larvae are susceptible to both NNV reassortants, though mainly to RGNNV/SJNNV, in an age-dependent manner.     

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 10⁶ CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.     

Ultrasound- and Microwave-Assisted Extractions Facilitate Oil Recovery from Gilthead Seabream (Sparus aurata) By-Products and Enhance Fish Oil Quality Parameters

This research aimed to analyze ultrasound (UAE) and microwave-assisted extraction (MAE) as novel technologies for utilizing gilthead seabream (Sparus aurata) by-products to produce high-quality fish oil for human consumption. The impacts of extraction parameters, namely, temperature, time, solvent-to-solid ratio, and their interactions on the extraction yield, are investigated using response surface methodology (RSM), and a central composite rotatable design. The optimized conditions are 15.47 mL g⁻¹ of solvent-to-solid ratio, 38 min, and 42 °C for UAE and 15.84 mL g⁻¹ of solvent-to-solid ratio, 18 min, and 40 °C for MAE. Under optimal conditions, the maximum extraction yields are 38.40 and 36.70% (g/g) for UAE and MAE, respectively. Both UAE and MAE have significantly higher mass transfer rates (61.70 and 121.58 g h⁻¹, respectively) than Soxhlet extraction (10.78 g h⁻¹). The fatty acid composition, physicochemical, and oxidation analyses of fish oils confirm the suitability of both UAE and MAE for the recovery of high-quality oils from fish processing by-products. The valorized oils mainly include unsaturated fatty acids (≈75%) and are rich in oleic acid. Furthermore, scanning electron microscopy analysis reveals that the key driving force for fast oil extraction is the structural degradation of fish by-products caused by ultrasound and microwave. Practical Applications: Due to environmental and economic viewpoints, the validation of fish oil from fish industry by-products has become a popular research topic recently. Alternative recovery techniques such as ultrasound- (UAE) and microwave-assisted extraction (MAE) protocols may have additional benefits in producing functional oils. Interactive effects of process parameters determine the success of the extraction technique; therefore optimization is a critical approach when applying the extraction protocols. This study shows that UAE and MAE techniques significantly enhanced oil extraction rate from gilthead seabream (Sparus aurota) by-products at lower temperatures and by using lower amounts of solvent. UVA and MAE increase oxidative stability and do not change the fatty acid composition. Hence, the by-product of the gilthead seabream can be a sustainable and food-grade fish oil source and UAE and MAE can be a good alternative to the conventional (Soxhlet) extraction by providing high yield and quality oil.     

Effects of dietary n−3/n−6 ratio on lipid metabolism of gilthead seabream (Sparus aurata)

The current study was conducted to evaluate the effects of various dietary n?3/n?6 ratio on growth and lipid metabolism in gilthead seabream (Sparus aurata) juveniles. The fish were fed ad libitum on three isoproteic (52%), isolipidic (14%), and isoenergetic diets (21 MJ/kg), formulated to contain three different n?3/n?6 ratios: a high (3.8), medium (2.5), or low (0.9) ratio. Fish grew from 9.5 ± 0.1 g to 54.2 ± 0.7 g in 12 weeks. Growth rate (2.2% BW/day), voluntary feed intake (2.4 ± 0.1% BM/day), feed conversion ratio (1.38 ± 0.05), and net protein utilization (27.5 ± 1.0%) did not vary among treatments. In contrast, body lipid content and lipid deposition efficiency were significantly reduced in fish fed low n?3/n?6 ratio. Fish fed the highest n?3/n?6 ratio showed the lowest hepatic glucose‐6‐phosphate dehydrogenase (G6PD) activity (p = 0.02). The n?3/n?6 ratio decreased, while DHA/eicosapentaenoic acid (EPA) ratio increased significantly with decreasing dietary n?3/n?6 content. Among n?3 PUFA, the DHA and, particularly, the docosapentaenoic acid (DPA) showed the highest deposition rate both in muscle and liver. The lower deposition rate (<1) of linoleic acid (LA) and linolenic acid (LNA) suggests that a reduction in dietary n?3/n?6 ratio may stimulate their transport to the intermediary metabolism for energy production.     

Probiotics Enhance Bone Growth and Rescue BMP Inhibition: New Transgenic Zebrafish Lines to Study Bone Health

Zebrafish larvae, especially gene-specific mutants and transgenic lines, are increasingly used to study vertebrate skeletal development and human pathologies such as osteoporosis, osteopetrosis and osteoarthritis. Probiotics have been recognized in recent years as a prophylactic treatment for various bone health issues in humans. Here, we present two new zebrafish transgenic lines containing the coding sequences for fluorescent proteins inserted into the endogenous genes for sp7 and col10a1a with larvae displaying fluorescence in developing osteoblasts and the bone extracellular matrix (mineralized or non-mineralized), respectively. Furthermore, we use these transgenic lines to show that exposure to two different probiotics, Bacillus subtilis and Lactococcus lactis, leads to an increase in osteoblast formation and bone matrix growth and mineralization. Gene expression analysis revealed the effect of the probiotics, particularly Bacillus subtilis, in modulating several skeletal development genes, such as runx2, sp7, spp1 and col10a1a, further supporting their ability to improve bone health. Bacillus subtilis was the more potent probiotic able to significantly reverse the inhibition of bone matrix formation when larvae were exposed to a BMP inhibitor (LDN212854).     

Vitamin C Enhances Vitamin E Status and Reduces Oxidative Stress Indicators in Sea Bass Larvae Fed High DHA Microdiets

Docosahexaenoic acid (DHA) is an essential fatty acid necessary for many biochemical, cellular and physiological functions in fish. However, high dietary levels of DHA increase free radical injury in sea bass (Dicentrarchus labrax) larvae muscle, even when vitamin E (α-tocopherol, α-TOH) is increased. Therefore, the inclusion of other nutrients with complementary antioxidant functions, such as vitamin C (ascorbic acid, vitC), could further contribute to prevent these lesions. The objective of the present study was to determine the effect of vitC inclusion (3,600?mg/kg) in high DHA (5?% DW) and α-TOH (3,000?mg/kg) microdiets (diets 5/3,000 and 5/3,000?+?vitC) in comparison to a control diet (1?% DHA DW and 1,500?mg/kg of α-TOH; diet 1/1,500) on sea bass larvae growth, survival, whole body biochemical composition and thiobarbituric acid reactive substances (TBARS) content, muscle morphology, skeletal deformities and antioxidant enzymes, insulin-like growth factors (IGFs) and myosin expression (MyHC). Larvae fed diet 1/1,500 showed the best performance in terms of total length, incidence of muscular lesions and ossification degree. IGFs gene expression was elevated in 5/3,000 diet larvae, suggesting an increased muscle mitogenesis that was confirmed by the increase in the mRNA copies of MyHC. vitC effectively controlled oxidative damages in muscle, increased α-TOH larval contents and reduced TBARS content and the occurrence of skull deformities. The results of the present study showed the antioxidant synergism between vitamins E and C when high contents of DHA are included in sea bass larvae diets.     

CAND2/PMTR1 Is Required for Melatonin-Conferred Osmotic Stress Tolerance in Arabidopsis

Osmotic stress severely inhibits plant growth and development, causing huge loss of crop quality and quantity worldwide. Melatonin is an important signaling molecule that generally confers plant increased tolerance to various environmental stresses, however, whether and how melatonin participates in plant osmotic stress response remain elusive. Here, we report that melatonin enhances plant osmotic stress tolerance through increasing ROS-scavenging ability, and melatonin receptor CAND2 plays a key role in melatonin-mediated plant response to osmotic stress. Upon osmotic stress treatment, the expression of melatonin biosynthetic genes including SNAT1, COMT1, and ASMT1 and the accumulation of melatonin are increased in the wild-type plants. The snat1 mutant is defective in osmotic stress-induced melatonin accumulation and thus sensitive to osmotic stress, while exogenous melatonin enhances the tolerance of the wild-type plant and rescues the sensitivity of the snat1 mutant to osmotic stress by upregulating the expression and activity of catalase and superoxide dismutase to repress H₂O₂ accumulation. Further study showed that the melatonin receptor mutant cand2 exhibits reduced osmotic stress tolerance with increased ROS accumulation, but exogenous melatonin cannot revert its osmotic stress phenotype. Together, our study reveals that CADN2 functions necessarily in melatonin-conferred osmotic stress tolerance by activating ROS-scavenging ability in Arabidopsis.     

Authenticating Production Origin of Gilthead Sea Bream (<Emphasis Type="Italic">Sparus aurata)</Emphasis> by Chemical and Isotopic Fingerprinting

Recent EU legislation (EC/2065/2001) requires that fish products, of wild and farmed origin, must provide consumer information that describes geographical origin and production method. The aim of the present study was to establish methods that could reliably differentiate between wild and farmed European gilthead sea bream (Sparus aurata). The methods that were chosen were based on chemical and stable isotopic analysis of the readily accessible lipid fraction. This study examined fatty acid profiles by capillary gas chromatography and the isotopic composition of fish oil (δ¹³C, δ¹⁸O), phospholipid choline nitrogen (δ¹⁵N) and compound specific analysis of fatty acids (δ¹³C) by isotope ratio mass spectroscopy as parameters that could reliably discriminate samples of wild and farmed sea bream. The sample set comprised of 15 farmed and 15 wild gilthead sea bream (Sparus aurata), obtained from Greece and Spain, respectively. Discrimination was achieved using fatty acid compositions, with linoleic acid (18:2n-6), arachidonic acid (20:4n-6), stearic acid (18:0), vaccenic acid (18:1n-7) and docosapentaenoic acid (22:5n-3) providing the highest contributions for discrimination. Principle components analysis of the data set highlighted good discrimination between wild and farmed fish. Factor 1 and 2 accounted for >70% of the variation in the data. The variables contributing to this discrimination were: the fatty acids 14:0, 16:0, 18:0, 18:1n-9, 18:1n-7, 22:1n-11, 18:2n-6 and 22:5n-3; δ¹³C of the fatty acids 16:0, 18:0, 16:1n-7, 18:1n-9, 20:5n-3 and 22:6n-3; Bulk oil fraction δ¹³C; glycerol/choline fraction bulk δ¹³C; δ¹⁵N; % N; % lipid.     

Assessment of the effects of sex and harvesting season on lipid and fatty acid composition of Sparidae species

In the present study, the lipid amount and fatty acid profile of different Sparidae species, including gilthead seabream, juvenile gilthead seabream, annular seabream, white seabream, common two-banded seabream were evaluated. Fish were seasonally collected from Köyceğiz Lagoon (Muğla, South Western Turkey) from June 2018 to June 2019 and after collection, the sex of each specimen was recorded. According to the results of the study, the highest lipid amount was found in female annular seabream individuals as 8.09 ± 0.78% in November and the lowest lipid amount was found in male juvenile gilthead seabream as 0.98 ± 0.12% in March. Palmitic acid and oleic acid were determined as the most abundant SFA and MUFA for all species, respectively. The highest value of DHA, which was the predominant PUFA was assessed as 15.33 ± 0.26% in female white seabream in November whereas the lowest value (3.83 ± 0.36%) was found in gilthead seabream in December. The n-6/n-3 ratio was determined between 0.27 ± 0.00 (for male common two-banded seabream in July)-1.20 ± 0.03 (for male gilthead seabream in December) and it followed within the range of healthy values for all species. As a conclusion, it was found that values of lipid and fatty acid profiles among the examined Sparidae species vary among the season of collection. The results of the study gave the seasonal nutritional values of four economically-important Sparidae species that being rich in healthy polyunsaturated fatty acids such as DHA, have beneficial in human nutrition.     

Abnormal Response of the Proliferation and Differentiation of Growth Plate Chondrocytes to Melatonin in Adolescent Idiopathic Scoliosis

Abnormalities in the melatonin signaling pathway and the involvement of melatonin receptor MT2 have been reported in patients with adolescent idiopathic scoliosis (AIS). Whether these abnormalities were involved in the systemic abnormal skeletal growth in AIS during the peripubertal period remain unknown. In this cross-sectional case-control study, growth plate chondrocytes (GPCs) were cultured from twenty AIS and ten normal control subjects. Although the MT2 receptor was identified in GPCs from both AIS and controls, its mRNA expression was significantly lower in AIS patients than the controls. GPCs were cultured in the presence of either the vehicle or various concentrations of melatonin, with or without the selective MT2 melatonin receptor antagonist 4-P-PDOT (10 µM). Then the cell viability and the mRNA expression of collagen type X (COLX) and alkaline phosphatase (ALP) were assessed by MTT and qPCR, respectively. In the control GPCs, melatonin at the concentrations of 1, 100 nM and 10 µM significantly reduced the population of viable cells, and the mRNA level of COLX and ALP compared to the vehicle. Similar changes were not observed in the presence of 4-P-PDOT. Further, neither proliferation nor differentiation of GPCs from AIS patients was affected by the melatonin treatment. These findings support the presence of a functional abnormality of the melatonin signaling pathway in AIS GPCs, which might be associated with the abnormal endochondral ossification in AIS patients.     

Effect of freezing on quality of sea bass and gilthead sea bream

Freezing is an efficient method of fish preservation. The aim of the present work was to examine the impact of freezing in fatty acid composition and in the in vitro inhibitory activity of sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) fillet lipids against platelet activating factor (PAF). The Bligh and Dyer extraction method and the counter‐current distribution method were used to obtain total, polar and neutral lipids. The fatty acid analysis conducted using the internal standard method and the biological assay on washed rabbit platelets took place calculating the in vitro inhibitory activity of fish lipids against 2.5 × 10^?11 M of PAF. No statistical changes (p<0.05) occurred in fatty acid content of fresh and thawed gilthead sea bream, while fatty acid amount in thawed sea bass was significantly higher (p<0.05) compared to fresh fish. Total lipids of both thawed fish species exhibited stronger anti‐thrombotic activity compared to fresh fish. Freezing preserved fish quality and reinforced the anti‐thrombotic properties of fish oils, since even after 6 months of freezing, fish oils preserve their nutritional value in terms of protecting against cardiovascular diseases. Practical applications: Fish fillets contain high amount of unsaturated lipids that may easily undergo lipid oxidation. Freezing and frozen storage prevent such oxidative changes so fish quality is retained. Fatty acids and PAF‐antagonists in fish are of major importance since they contribute to the nutritional value of fish. The practical application of this work lies on the evaluation of the nutritional value of fish in terms of cardio protection by examining the impact of freezing on the levels of fatty acids and PAF‐antagonists in aquacultured fish fillets.     

Cranial Base Synchondrosis Lacks PTHrP-Expressing Column-Forming Chondrocytes

The cranial base contains a special type of growth plate termed the synchondrosis, which functions as the growth center of the skull. The synchondrosis is composed of bidirectional opposite-facing layers of resting, proliferating, and hypertrophic chondrocytes, and lacks the secondary ossification center. In long bones, the resting zone of the epiphyseal growth plate houses a population of parathyroid hormone-related protein (PTHrP)-expressing chondrocytes that contribute to the formation of columnar chondrocytes. Whether PTHrP⁺ chondrocytes in the synchondrosis possess similar functions remains undefined. Using Pthrp-mCherry knock-in mice, we found that PTHrP⁺ chondrocytes predominantly occupied the lateral wedge-shaped area of the synchondrosis, unlike those in the femoral growth plate that reside in the resting zone within the epiphysis. In vivo cell-lineage analyses using a tamoxifen-inducible Pthrp-creER line revealed that PTHrP⁺ chondrocytes failed to establish columnar chondrocytes in the synchondrosis. Therefore, PTHrP⁺ chondrocytes in the synchondrosis do not possess column-forming capabilities, unlike those in the resting zone of the long bone growth plate. These findings support the importance of the secondary ossification center within the long bone epiphysis in establishing the stem cell niche for PTHrP⁺ chondrocytes, the absence of which may explain the lack of column-forming capabilities of PTHrP⁺ chondrocytes in the cranial base synchondrosis.     

Peripheral Skin Temperature and Circadian Biological Clock in Shift Nurses after a Day off

The circadian biological clock is essentially based on the light/dark cycle. Some people working with shift schedules cannot adjust their sleep/wake cycle to the light/dark cycle, and this may result in alterations of the circadian biological clock. This study explored the circadian biological clock of shift and daytime nurses using non-invasive methods. Peripheral skin temperature, cortisol and melatonin levels in saliva, and Per2 expression in pubic hair follicle cells were investigated for 24 h after a day off. Significant differences were observed in peripheral skin temperature and cortisol levels between shift and daytime nurses. No differences in melatonin levels were obtained. Per2 maximum values were significantly different between the two groups. Shift nurses exhibited lower circadian variations compared to daytime nurses, and this may indicate an adjustment of the circadian biological clock to continuous shift schedules. Non-invasive procedures, such as peripheral skin temperature measurement, determination of cortisol and melatonin in saliva, and analysis of clock genes in hair follicle cells, may be effective approaches to extensively study the circadian clock in shift workers.     

Fatty acid composition of wild and cultivated gilthead seabream (Sparus aurata) and sea bass (Dicentrarchus labrax)

Total lipid content and fatty acid composition were determined for wild and cultivated gilthead seabream and sea bass. Fatty acid analyses were carried out by gas chromatography‐ mass spectrometry. Respective total lipid content of flesh in cultivated gilthead seabream and sea bass were 1.7‐5.0‐times those of wild samples. Palmitic acid (C16:0) and oleic acid (C18:1n‐9) were the major fatty acids respectively among the saturated fatty acids and monounsaturated fatty acids of each fish species. It is noteworthy that both linoleic acid (C18:2n‐6) and arachidonic acid (C20:4n‐6) were predominant in total n‐6 polyunsaturated fatty acids in the respective cultivated and wild types. Eicosapentaenoic acid (C20:5n‐3) and docosahexaenoic acid (C22:6n‐3) amounts were significantly higher in flesh of cultivated fish than in wild fish.     

Melatonin Receptor Genes in Vertebrates

Melatonin receptors are members of the G protein-coupled receptor (GPCR) family. Three genes for melatonin receptors have been cloned. The MT1 (or Mel1a or MTNR1A) and MT2 (or Mel1b or MTNR1B) receptor subtypes are present in humans and other mammals, while an additional melatonin receptor subtype, Mel1c (or MTNR1C), has been identified in fish, amphibians and birds. Another melatonin related orphan receptor, GPR50, which does not bind melatonin, is found exclusively in mammals. The hormone melatonin is secreted primarily by the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone acts systemically in numerous organs. In the brain, it is involved in the regulation of various neural and endocrine processes, and it readjusts the circadian pacemaker, the suprachiasmatic nucleus. This article reviews recent studies of gene organization, expression, evolution and mutations of melatonin receptor genes of vertebrates. Gene polymorphisms reveal that numerous mutations are associated with diseases and disorders. The phylogenetic analysis of receptor genes indicates that GPR50 is an outgroup to all other melatonin receptor sequences. GPR50 may have separated from a melatonin receptor ancestor before the split between MTNR1C and the MTNR1A/B ancestor.