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In this study, adult gilthead seabream (Sparus aurata) were exposed for 21 days to Di-iso-nonylphthalte (DiNP at 15 and 1500 μg kg−1 bw day−1) via the diet. This plastic additive has been recently introduced to replace the di-(2-ethylhexyl)phthalate, the toxicity of which has been demonstrated conclusively both in vivo and in vitro trials. An analysis of a set of biomarkers involved in stress and immune response provides evidence of hepatic toxicity by DiNP in the present study. Both hsp70 and gr mRNA levels were upregulated significantly by DiNP, while plasma cortisol increased only in fish fed with the lowest DiNP dose. The oxidative stress markers g6pdh, glut red, gpx1 and CAT were upregulated by DiNP; gst mRNA was induced by the high dose and gck mRNA was downregulated significantly by the low dose. The mRNA levels of genes involved in the immune response, such as pla2, 5-lox, tnfa and cox2, were upregulated significantly only by the high dose of DiNP, while il1 mRNA increases in both doses. These molecular evidences were complemented with features obtained by Fourier Transform Infrared Imaging (FTIRI) analysis regarding the hepatic distribution of the main biological macromolecules. The FTIRI analysis showed an alteration of biochemical composition in DiNP samples. In particular, the low dose of DiNP induced an increase of saturated and unsaturated lipids and phosphorylated proteins, and a decrease of glycogen levels. The levels of caspase did not change significantly in the study, suggesting that DiNP does not activate apoptosis. Finally, the results also suggested the onset of hepatic oxidative stress and the activation of immune response, adding new knowledge to the already described hepatic DiNP toxicity. [ABSTRACT FROM AUTHOR]

Abstract In the last years, an increasing number of studies reported that food pollution represents a significant route of exposure to environmental toxicants, able to cause mild to severe food illnesses and health problems, including hormonal and metabolic diseases. Pollutants can accumulate in organisms and biomagnify along the food web, finally targeting top consumers causing health and economic problems. In this study, adults of gilthead sea bream, Sparus aurata , were fed with diets contaminated with Bisphenol A (BPA) (4 and 4000 μg BPA kg−1 bw day−1) and Di-isononyl phthalate (DiNP) (15 and 1500 μg DiNP kg−1 bw day−1), to evaluate the effects of the contamination on the muscle macromolecular composition and alterations of its texture. The analysis conducted in the muscle using infrared microspectroscopy, molecular biology and biochemical assays, showed, in fish fed BPA contaminated diets, a decrease of unsaturated lipids and an increase of triglycerides and saturated alkyl chains. Conversely, in fish fed DiNP, a decrease of lipid content, caused by a reduction of both saturated and unsaturated chains and triglycerides was measured. Protein content was decreased by both xenobiotics evidencing a novel macromolecular target affected by these environmental contaminants. In addition, in all treated groups, proteins resulted more phosphorylated than in controls. Calpain and cathepsin levels, orchestrating protein turnover, were deregulated by both xenobiotics, evidencing alterations of muscle composition and texture. In conclusion, the results obtained suggest the ability of BPA and DiNP to modify the muscle macromolecular building, advising this tissue as a target of Endocrine-Disrupting Chemicals (EDCs) and providing a set of biomarkers as possible monitoring endpoints to develop novel OEDC test guidelines. Graphical abstract Unlabelled Image Highlights • BPA and DiNP in the diet affect skeletal muscle macromolecular building. • BPA and DiNP in the diet affect seabream muscle nutritional properties. • Dietary BPA and DiNP administration increases skeletal muscle proteolytic system activity. [ABSTRACT FROM AUTHOR]

Diisononyl phthalate (DiNP) is a plasticizer used to improve plastic performance in a large variety of items which has been reported as an endocrine-disrupting chemical (EDC) in several organisms. The endocannabinoid system (ECS) is a cellular signaling system, whose functionality is tightly involved with reproductive function. The aim of the present study was the assessment of the effects of DiNP on the gonadal ECS and on the reproductive function of male gilthead sea bream Sparus aurata, an important marine aquacultured species in Europe, during the reproductive season. Fish were fed for 21 days with two diets contaminated with different nominal concentrations of DiNP (DiNP LOW at 15 µg DiNP kg−1 bw day−1 and DiNP HIGH at 1500 µg DiNP kg−1 bw day−1), based on the tolerable daily intake (TDI) ruled by the European Food Safety Authority for humans. The transcription of several genes related to the ECS was affected by the DiNP. Specifically, DiNP reduced the levels of endocannabinoids and endocannabinoid-like mediators, concomitant with the increase of fatty acid amide hydrolase (FAAH) activity. At the histological level, DiNP LOW induced the highest occurrence of individuals with regressed testes. Steroidogenesis was affected significantly, since plasma 11-ketotestosterone (11-KT), the main active androgen in fish, was significantly decreased by the DiNP HIGH treatment, while plasma 17β-estradiol (E2) levels were raised, associated with an increase of the gonadosomatic index (GSI). Additionally, the level of testosterone (T) was significantly increased in the DiNP LOW group, however, the same DiNP concentration reduced the levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P). The production of sperm was in general not affected, since spermiation index, sperm density, survival and the duration of forward motility did not exhibit any changes compared to controls. However, computer-assisted sperm analysis (CASA) showed that DiNP reduced the percentage of motile cells. The results clearly suggest a negative effect of DiNP via the diet on the male endocrine system of gilthead sea bream during the reproductive season. [ABSTRACT FROM AUTHOR]

In the last decades, attempts to sustain an environmental friendly aquaculture have encouraged the use of beneficial bacterial, including probiotics, as feed enrichment. The use of probiotics has been extended far beyond finfish aquaculture, since they have been added to experimental and/or commercial diets formulated for invertebrates including Crustaceans, molluscs, echinoderms. The results so far obtained largely demonstrated beneficial effects of probiotics on fish growth, digestive function, immune system modulation, stress resistance and fecundity. Many studies demonstrated the potentiality of probiotic administration to elevate the fish innate defense mechanisms, hence improving survival following exposure to a number of pathogens. Benefits are driven, at least in part, by the modulation of the host microbiota. Probiotic “colonization” is the first key process responsible for increasing welfare. The review will summarize the effects of microbiota changes on welfare, growth and reproduction of both ornamental and commercial species. Reproduction is an energy-costly process that can be successfully faced only when sufficient energy stores are available. Particular emphasis will be given to the effect of probiotics on the interplay between metabolism and reproduction. A number of central energy sensors, hypothalamic neuropeptides, and several peripheral molecular mediators have been implicated in the regulation of the delicate interaction connecting the two processes. The last part of the review condenses recent findings regarding the positive effects of probiotic administration on fish gonads, which deeply support their role towards reproduction, mainly achieved on zebrafish. Indeed, the results presented scientifically support and strongly encourage the large scale use of probiotics in aquaculture practices, suggesting avoidance of pharmaceuticals use. If that becomes well established, a better quality, both health- and environment-wise, could be achieved and supplied to consumers. Statement of relevance Probiotic improvement of growth, wellbeing and reproduction. [ABSTRACT FROM AUTHOR]

Most organisms modulate their reproductive activity responding to day length by the nocturnal release of melatonin by the pineal gland. This hormone is also responsible for synchronizing reproduction with specific external environment stimuli in order to optimize reproductive success. The aim of this study was to establish the effect of melatonin on zebrafish reproduction. Adult females were daily exposed, via water, to two different doses (100 nM and 1 μM) of melatonin. Melatonin led to an increase of the Gonado Somatic Index (GSI) associated with the increase of eggs production, and the raise of gene and protein levels of vitellogenin (VTG) and estradiol receptor a (ERa) in the liver. The ability of melatonin to increase fecundity was consistent with a significant increase of gene transcription of kiss 1, kiss 2, gnrh3, in the brain, and lh in the pituitary, while in the ovary (in class IIIB follicles), with a significant decrease of two genes codifying for intra-ovarian regulators of premature oocyte maturation, the tgfb1 and the bmp15. The reduction in the expression of these two genes was concomitant with the increase of lhr and a modulation of mpra and mprb gene transcription, whose proteins are involved in oocyte maturation. Melatonin also exerted a direct action on follicles as shown by the increase of the oocytes undergoing to germinal vesicle break down (GVBD) and modulated mpr a and b gene expression in the in vitro exposure. These data highlight the effects of melatonin in promoting zebrafish reproduction exerting its effects either in the brain-pituitary and in the gonads. [ABSTRACT FROM AUTHOR]

Abstract: The role of lysosomal proteases such as cathepsin B (Ctsb) and one of the paralogs of cathepsin L (Ctsla) during yolk metabolism in fish oocytes is well established. However, the function of Ctsb during embryogenesis, particularly in marine teleosts, has been poorly documented. In this study, the spatio-temporal expression of Ctsb and Ctsla, their enzymatic activities, and the processing of the Ctsb and its cellular localization, was investigated in developing embryos of the killifish (Fundulus heteroclitus). Both fhctsb and fhctsla transcript levels, as well as cathepsin B- and L-like activities, gradually increased in embryos from the 2–4 cell stage up to 7 days post-fertilization. During the morula to gastrula transition an increase of the active FhCtsb single chain form was followed by a rise in cathepsin B activity, which were apparently regulated by post-transcriptional mechanisms. During neurulation, a 8-fold increase in cathepsin B activity was accompanied by a more moderate increase in cathepsin L activity, which was 6-fold enhanced by 7dpf. These increased catalytic activities were well-correlated to changes in the electrophoretic pattern of yolk proteins and a strong expression of fhctsb and its protein product in the yolk syncytial layer. The increase of cathepsin B activity was further correlated with an increment of the relative amount of the FhCtsb single and double chain forms, both active forms of FhCtsb. These results suggest that FhCtsb may be involved in the mechanisms underlying the onset of gastrulation in F. heteroclitus embryos, and may play complementary roles with FhCtsla during yolk metabolism. [Copyright &y& Elsevier]

Public concerns on phthalates distributions in the environment have been increasing since they can cause liver cancer, structural abnormalities and reduce sperm counts in male reproductive system. However, few data are actually available on the effects of Di-(2-ethylhexyl)-phthalate (DEHP) in female reproductive system. The aim of this study was to assess the impacts of DEHP on zebrafish oogenesis and embryo production. Female Danio rerio were exposed to environmentally relevant doses of DEHP and a significant decrease in ovulation and embryo production was observed. The effects of DEHP on several key regulators of oocyte maturation and ovulation including bone morphogenetic protein-15 (BMP15), luteinizing hormone receptor (LHR), membrane progesterone receptors (mPRs) and cyclooxygenase (COX)-2 (ptgs2) were determined by real time PCR. The expressions of BMP15 and mPR proteins were further determined by Western analyses to strengthen molecular findings. Moreover, plasma vitellogenin (vtg) titers were assayed by an ELISA procedure to determine the estrogenic effects of DEHP and its effects on oocyte growth. A significant reduction of fecundity in fish exposed to DEHP was observed. The reduced reproductive capacity was associated with an increase in ovarian BMP15 levels. This rise, in turn, was concomitant with a significant reduction in LHR and mPRβ levels. Finally, ptgs2 expression, the final trigger of ovulation, was also decreased by DEHP. By an in vitro maturation assay, the inhibitory effect of DEHP on germinal vesicle breakdown was further confirmed. In conclusion, DEHP affecting signals involved in oocyte growth (vtg), maturation (BMP15, LHR, mPRs,) and ovulation (ptgs2), deeply impairs ovarian functions with serious consequences on embryo production. Since there is a significant genetic similarity between D.rerio and humans, the harmful effects observed at oocyte level may be relevant for further molecular studies on humans. [ABSTRACT FROM AUTHOR]

Abstract: Zebrafish oocytes growth causes relevant modifications in protein components; in fact, the proteolytic cleavage of vitellogenin, a large sex-specific phospholipoglycoprotein synthesized in the female liver, leads to three main yolk protein components (phosphovitin and lipovitellins 1, 2), present as a complex in the oocyte. FT-IR Microspectroscopy could have the potentiality of monitoring these biochemical changes during the maturation process. Representative spectra for I–II, IIIA, IIIB and IV classes oocytes (Hierarchical Clustering Analysis and Principal Component Analysis) were investigated to find specific vibrational patterns corresponding to different maturation degrees. On going from I–II to IV class oocytes, relevant spectral differences were found with III class exhibiting an intermediate spectroscopic behaviour. In particular, the increase of the convoluted band at 2925cm−1 (CH2 and CH3 stretching modes), as well as of intensity band ratios at 2926/2954cm−1 (ν asym CH2/CH3), 2854/2873cm−1 (ν sym CH2/CH3) and 1452/1392cm−1 (), suggest longer lipidic chains; broadening of Amide I and II bands and absorptions at 1737 (νColipids) and 1157cm−1 (νC:italic>νCydrates) in lipovitellin and vitellogenin spectra, are present in III and IV classes. [Copyright &y& Elsevier]

The aim of this study was to obtain new information on two lysosomal enzymes, cathepsin D (Cat D) and cathepsin L (Cat L) in the Mediterranean mussel, Mytilus galloprovincialis, and to test in a preliminary way if Cat D and Cat L enzymatic activity can be enhanced in response to estrogenic compounds, such as nonylphenol (NP) and estradiol (E2). In addition to assay optimisation, a number of experiments were performed in order to assess Cat D natural seasonality, the effect of captivity on Cat D and Cat L activity, and the response to estrogenic compounds. Exposure experiments were conducted under static conditions; once exposed to NP (300 μg/l) and E2 (1000 μg/l) on day one, mussels were sampled after 7 and 14 days. The main registered effect was a significant increase of Cat D activity in the digestive gland, caused by both NP and E2. The results obtained provide preliminary information on Cat D and Cat L in the Mytilus galloprovincialis; furthermore the Cat D activity enhancement, caused by estrogenic compounds, provides a starting point for further studies designed to evaluate if the Cat D assay in mussels may supply indications on the presence of estrogenic compounds in the environment. [ABSTRACT FROM AUTHOR]

Abstract: In the present study a Lactic Acid Bacteria (LAB) strain Lactobacillus delbrueckii delbrueckii, acting as probiotic, was administered to Dicentrarchus labrax (European sea bass) juveniles for a short (25 days) and a long (59 days) time and the effects of the bacteria on gut colonization in sea bass juveniles cortisol level and growth were evaluated with respect to fish fed on live preys solely (control group). Measures of body weight and standard length (S.L.) were performed to assess juveniles growth, while cortisol, a hormone directly involved in stress responses, was chosen as a stress marker. In addition, the expression of two antagonistic genes involved in muscular growth (IGF-I and myostatin (MSTN) was analyzed through real-time PCR. Fish fed on live preys added with L. delbrueckii delbrueckii showed a high gut colonization by this Lactic Acid Bacteria strain. In the same fish a significantly (p <0.05) lower cortisol level was observed with respect to those fed on live preys solely (control 5.1±0.47 ng/g; group A: 3.6±0.36 ng/g; group B: 4.5±0.48 ng/g). In addition, an increase of IGF-I transcription was observed in fish treated with LAB, being IGF-I mRNA levels six times higher in both treated groups with respect to the control (control: 1±0.01 a.u.; group A: 6.63±0.18 a.u.; group B: 6.16±0.15 a.u.). On the contrary, MSTN mRNA transcription was significantly inhibited in treated groups (control 1±0.02 a.u.; group A: 0.71±0.01 a.u.; group B: 0.26±0.003 a.u.). These results are in agreement with the increase of body weight recorded in this study. Fish fed on LAB showed 81% higher body weight in long treated group and 28% in short treated one with respect to control. [Copyright &y& Elsevier]

A variety of antropogenic compounds that have an estrogenic effect, and are known to be present in the environment, shows a significant potential for interference with the health and reproduction of both wildlife and humans. In this review, the effect of estrogenic and antiestrogenic chemicals with widely divergent potencies—nonylphenol (NP), which acts by binding with the estradiol response element, and β-naphthoflavone (β-NF), a dioxin-like compound that exerts its toxic action through the aryl hydrocarbon receptor—was compared with that induced by 17β-estradiol (E2) in a marine teleost, the Gobius niger, under controlled laboratory experiments. The capacity of these compounds to affect the levels of estrogen-regulated proteins such as cathepsin D (CAT D)—in humans, a protein associated with the development of breast cancer, and, in oviparous vertebrates, with reproductive success—was assessed. The results of this study showed that both the estradiol and the higher dose of NP induce CAT D gene expression and its associated activity. On the contrary, β-NF treatments inhibited CAT D gene expression and, at lengthier exposure (96 h), its enzymatic activity. Based on these results, we suggest CAT D as a novel bioindicator of the presence of endocrine-disrupting substances in the environment. The other biomarker assessed in this study is the Heath Shock Protein 70 (HSP70); this protein protects cells against harmful conditions by binding and refolding damaged proteins. Interestingly, HSP70 was found to be affected by all the toxicant compounds employed in the study. The HSP70 gene expression was significantly increased by both NP concentrations and the exposure time of β-NF, with the E2 being the most potent inducer. These data indicate that HSP70 may provide a useful early warning biomarker for studies on the presence of exogenous pollutants in the environment. [Copyright &y& Elsevier]

Simple Summary: Swordfish (Xiphias gladius) are top marine predators which are facing serious threats from overfishing, pollution, and climate change, especially in the Mediterranean Sea. Environmental conditions influence the levels of melatonin-related genes and opsins, which are crucial for regulating important processes like reproduction, growth, and stress responses in fish. Our findings on the expression of these genes in the livers of immature and mature female swordfish indicate a need for more research into their specific functions in different tissues. Mature and immature female swordfish show different gonadal expression patterns of melatonin synthesis-related and opsin genes, indicating that seasonality affects gonadal circadian genes, especially through opsin modulation. The high energy demands of reproduction may cause mature female swordfish to become more susceptible to environmental stressors/changes, potentially affecting their health post-reproduction. The modulation of melatonin and opsin expression may also influence swordfish biology, impacting basal metabolism and stress responses. This study aimed to investigate the hepatic expression patterns of genes involved in melatonin and opsins activity after the reproductive period, comparing mature and immature females. Liver samples of swordfish females, collected from the Central Adriatic Sea, were analyzed to evaluate the expression of a melatonin receptor (mel1b), acetylserotonin O-methyltransferase (asmt), opsins (sws, VA opsin and opsin4), and stress response (sod1, sod2 and hsp4b) genes, while histological analysis focused on hepatic melanomacrophages, melanomacrophage centers, and lipid content. The expression of all genes analyzed did not differ between mature and immature females except for sws, which was significantly downregulated in mature females. The overall health conditions of mature and immature females appeared comparable, as suggested by the histological analysis and the absence of significant differences in the expression of stress response genes. Although the study used a limited number of samples and lacked a comparison between the reproductive and non-reproductive periods, the preliminary results suggest that opsins may play a role in modulating physiological processes beyond reproduction. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) is a lipid signaling system involved in numerous physiological processes, such as endocrine homeostasis, appetite control, energy balance, and metabolism. The ECS comprises endocannabinoids, their cognate receptors, and the enzymatic machinery that tightly regulates their levels within tissues. This system has been identified in various organs, including the brain and liver, in multiple mammalian and non-mammalian species. However, information regarding the sex-specific regulation of the ECS remains limited, even though increasing evidence suggests that interactions between sex steroid hormones and the ECS may ultimately modulate hepatic metabolism and energy homeostasis. Within this framework, we will review the sexual dimorphism of the ECS in various animal models, providing evidence of the crosstalk between endocannabinoids and sex hormones via different metabolic pathways. Additionally, we will underscore the importance of understanding how endocrine-disrupting chemicals and exogenous cannabinoids influence ECS-dependent metabolic pathways in a sex-specific manner. [ABSTRACT FROM AUTHOR]

Diisononyl phthalate, classified as endocrine disruptor, has been investigate to trigger lipid biosynthesis in both mammalian and teleostean animal models. Despite this, little is known about the effects of DiNP exposure at tolerable daily intake level and the possible mechanisms of its toxicity. Probiotics, on the other hand, were demonstrated to have beneficial effects on the organism's metabolism and recently emerged as a possible tool to mitigate the EDC toxicity. In the present study, using a metabolomic approach, the potential hepatic sex-related toxicity of DiNP was investigated in adult zebrafish together with the mitigating action of the probiotic formulation SLAB51, which has already demonstrated its ability to ameliorate gastrointestinal pathologies in animals including humans. Zebrafish were exposed for 28 days to 50 µg/kg body weight (bw)/day of DiNP (DiNP) through their diet and treated with 109 CFU/g bw of SLAB51 (P) and the combination of DiNP and SLAB51 (DiNP + P), and the results were compared to those of an untreated control group (C). DiNP reduced AMP, IMP, and GMP in the purine metabolism, while such alterations were not observed in the DiNP + P group, for which the phenotype overlapped that of C fish. In addition, in male, DiNP reduced UMP and CMP levels in the pyrimidine metabolism, while the co-administration of probiotic shifted the DiNP + P metabolic phenotype toward that of P male and closed to C male, suggesting the beneficial effects of probiotics also in male fish. Overall, these results provide the first evidence of the disruptive actions of DiNP on hepatic nucleotide metabolism and mitigating action of the probiotic to reduce a DiNP-induced response in a sex-related manner. [ABSTRACT FROM AUTHOR]

• Examined gonadal histology of mature Eastern Atlantic Bluefin Tuna during the reproductive and non-reproductive season. • Determined plasma FSH and sex steroid levels with gonadal development in the two reproductive time points. • Characterized the expression profile of genes involved in gametogenesis. • Identified gender-specific signals in the gonads and their seasonal changes. The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as β and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E 2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E 2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences. [ABSTRACT FROM AUTHOR]

The maternal control directing the very first hours of life is of pivotal importance for ensuring proper development to the growing embryo. Thanks to the finely regulated inheritance of maternal factors including mRNAs and proteins produced during oogenesis and stored into the mature oocyte, the embryo is sustained throughout the so-called maternal-to-zygotic transition, a period in development characterized by a species-specific length in time, during which critical biological changes regarding cell cycle and zygotic transcriptional activation occur. In order not to provoke any kind of persistent damage, the process must be delicately balanced. Surprisingly, our knowledge as to the possible effects of beneficial bacteria regarding the modulation of the quality and/or quantity of both maternally-supplied and zygotically-transcribed mRNAs, is very limited. To date, only one group has investigated the consequences of the parentally-supplied Lactobacillus rhamnosus on the storage of mRNAs into mature oocytes, leading to an altered maternal control process in the F1 generation. Particular attention was called on the monitoring of several biomarkers involved in autophagy, apoptosis and axis patterning, while data on miRNA generation and pluripotency maintenance are herein presented for the first time, and can assist in laying the ground for further investigations in this field. In this review, the reader is supplied with the current knowledge on the above-mentioned biological process, first by drawing the general background and then by emphasizing the most important findings that have highlighted their focal role in normal animal development. [ABSTRACT FROM AUTHOR]

Simple Summary: In recent decades, trade of ornamental fish has significantly increased. A rise in demand was observed, especially from top importing countries that contributed majorly to the growth of the market. The destructive fishing methods caused a severe impairment of natural and environmental resources. Thus, there is a need to improve ornamental fish aquaculture, increasing the number of cultured species and limiting wild fish handling and transport stress losses. In this light, the use of microbial feed additives such as probiotics, prebiotics, and synbiotics, could help in improving the immune system and growth as well as increasing reproductive performance in captivity-bred species. Ornamental fish trade represents an important economic sector with an export turnover that reached approximately 5 billion US dollars in 2018. Despite its high economic importance, this sector does not receive much attention. Ornamental fish husbandry still faces many challenges and losses caused by transport stress and handling and outbreak of diseases are still to be improved. This review will provide insights on ornamental fish diseases along with the measures used to avoid or limit their onset. Moreover, this review will discuss the role of different natural and sustainable microbial feed additives, particularly probiotics, prebiotics, and synbiotics on the health, reduction in transport stress, growth, and reproduction of farmed ornamental fish. Most importantly, this review aims to fill the informational gaps existing in advanced and sustainable practices in the ornamental fish production. [ABSTRACT FROM AUTHOR]

Simple Summary: The vast majority of the world's commercial fish stocks are considered overfished. Among demersal species, in the Mediterranean Sea, the European hake (Merluccius merluccius) is experiencing high fishing pressure, resulting in a critical overexploitation status. The reproductive pattern of the male sex is poorly investigated. In order to provide scientific advice for its sustainable management, this study gave, for the first time in the Central Mediterranean Sea, an overview of the reproductive biology of male European hakes, by using a multidisciplinary approach. The sex ratio throughout the three-year period and by length class showed that males dominated the population from February to June and up to 24 cm, respectively. The reproduction occurred all year round with a seasonal peak in spring/summer. The macroscopic and histological sizes at first maturity were 18.6 cm and 15.4 cm, respectively. The entire reproductive cycle was unequivocally identified by five histological developmental stages of the testis. Finally, the molecular approach allowed us to investigate endocrine regulation from the onset of puberty to the adult lifecycle. The improvement of scientific knowledge about overexploited fishery resources allow us to provide scientific advice for their management and stock protection. By using a multidisciplinary approach, the aim of the study was to characterize, for the first time in the Central Mediterranean Sea (GSA 17), the reproductive biology of males of M. merluccius, currently highly exploited. A multi-year sampling from January 2017 to December 2019 was performed to exhaustively evaluate the sex ratio of the stock, while the 2018 annual sampling was selected to investigate the reproductive pattern of males. Individuals in spawning conditions were found every month, proving that M. merluccius is an asynchronous species, reproducing all year round, with a seasonal reproductive peak in spring/summer, as indicated by GSI. Five gonadal development stages were defined to fully describe the reproductive cycle of males. The macroscopic and histological L50, respectively 18.6 cm and 15.4 cm, were both below the Minimum Conservation Reference Size (MCRS). According to the mRNA levels, fsh and lh played a significant role during spermiation, whereas the gnrhr2a was involved at the beginning of sexual maturity. In the testis, fshr and lhr reached maximum expression levels before the spermiation. The hormonal stimuli of 11-ketotestosterone and its receptor were significantly higher when the specimen was in reproductive activity. [ABSTRACT FROM AUTHOR]

The demand for organic UV filters as active components in sunscreen products has rapidly risen over the last century, as people have gradually realized the hazards of overexposure to UV radiation. Their extensive usage has resulted in their ubiquitous presence in different aquatic matrices, representing a potential threat to living organisms. In this context, the need to replace classic UV filters such as octyl methoxycinnamate (OMC), one of the most popular UV filters reported to be a potential pollutant of aquatic ecosystems, with more environmentally friendly ones has emerged. In this study, using zebrafish, the first in vivo results regarding the effect of exposure to tempol-methoxycinnamate (TMC), a derivative of OMC, are reported. A comparative study between TMC and OMC was performed, analyzing embryos exposed to similar TMC and OMC concentrations, focusing on morphological and molecular changes. While both compounds seemed not to affect hatching and embryogenesis, OMC exposure caused an increase in endoplasmic reticulum (ER) stress response genes, according to increased eif2ak3, ddit3, nrf2, and nkap mRNA levels and in oxidative stress genes, as observed from modulation of the sod1, sod2, gpr, and trx mRNA levels. On the contrary, exposure to TMC led to reduced toxicity, probably due to the presence of the nitroxide group in the compound's molecular structure responsible for antioxidant activity. In addition, both UV filters were docked with estrogen and androgen receptors where they acted differently, in agreement with the molecular analysis that showed a hormone-like activity for OMC but not for TMC. Overall, the results indicate the suitability of TMC as an alternative, environmentally safer UV filter. [ABSTRACT FROM AUTHOR]

Gestational diabetes mellitus (GDM) and small-for-gestational-age (SGA) are two metabolic-related diseases that could affect women during pregnancy. Considering that the chorionic villi (CVs) are crucial structures for the feto-maternal exchange, the alterations in their conformation have been linked to an imbalanced metabolic environment of placenta. In this study, a multidisciplinary approach has been carried out to describe the changes occurring in the placental CVs of GDM and SGA patients. The results revealed higher levels of superoxide dismutase 1 (SOD-1) and catalase (CAT), especially in the GDM placentae, which could be correlated with the hyperglycemic environment characteristic of this pathology. Furthermore, spectroscopy and histologic analyses revealed that both pathologies modify the placental lipid composition altering its structure. However, SGA induces lipid peroxidation and reduces collagen deposition within the CVs. Since the endocannabinoid system (ECS) is involved in placentation and different metabolic activities, the cannabinoid receptor 1 (CB1) and transient receptor potential cation channel subfamily V member 1 (TRPV-1) were analyzed. No changes have been observed either at general or specific levels in the CVs comparing control and pathological samples, suggesting the non-involvement of the cannabinoid system in these two pathologies. [ABSTRACT FROM AUTHOR]

Several in vivo trials have previously demonstrated the beneficial effects of the administration of various probiotic forms on bone health. In this study, we explored the potency of two probiotics, Bacillus subtilis and Lactococcus lactis, alone or in combination with vitamin D (VD), to modulate the transcription of genes involved in the ossification process in a human osteoblast cell line. Genes that mark the "osteoblast proliferation phase", such as RUNX2, TGFB1, and ALPL, "extracellular matrix (ECM) maturation", such as SPP1 and SPARC, as well as "ECM mineralization", such as BGN, BGLAP, and DCN, were all highly expressed in osteoblasts treated with B. subtilis extract. The observed increase in the transcription of the ALPL mRNA was further in agreement with its protein levels as observed by Western blot and immunofluorescence. Therefore, this higher transcription and translation of alkaline phosphatase in osteoblasts treated with the B. subtilis extract, indicated its substantial osteogenic impact on human osteoblasts. Although both the probiotic extracts showed no osteogenic synergy with VD, treatment with B. subtilis alone could increase the ECM mineralization, outperforming the effects of L. lactis and even VD. Furthermore, these results supported the validity of employing probiotic extracts rather than live cells to investigate the effects of probiotics in the in vitro systems. [ABSTRACT FROM AUTHOR]

Simple Summary: The Mediterranean swordfish (Xiphias gladius) stock was affected in last decades from overfish. The evaluation of the cross-talk among metabolism, stress response, immune system and reproduction in immature and mature females could be of great importance. For these reasons, in the present study, by transcriptomic and histological analysis, a deeper insight into the cross-talk among reproduction, metabolism and response to environmental cues across different stages of sexual maturation was provided by the livers of female swordfish. We show that mature females are able to properly reproduce, since they invest a lot of energy in reproduction; however, their detoxification capacity and immune system are compromised as evidenced by transcriptomic and histological approaches. These results suggest that during the reproductive season, mature females may be more susceptible to environmental stress and pollutants than immature ones. Xiphias gladius is an important fishing resource. The Mediterranean stock is affected by overfishing and is declining. In this light, the aim of this study was to evaluate the cross-talk among metabolism, stress response, immune system and reproduction in immature and mature females, coupling histological and transcriptomic approaches. The transcriptome of livers from 3 immature and 3 mature females was analyzed using the Artificial Intelligence RNA-Seq. For the histological analysis, ovary and liver samples were collected from 50 specimens caught during the reproductive season in the Mediterranean Sea. A total of 750 genes were differentially expressed between the livers. The gene ontologtabey analysis showed 91 upregulated and 161 downregulated biological process GO terms. Instead, the KEGG enrichment analysis revealed 15 enriched pathways. Furthermore, the binding occurring between estrogen receptors and aryl hydrocarbon receptor nuclear translocator, upregulated in mature females, could be liable for the inhibition of detoxification pathway. Indeed, at the histological level, mature females showed a higher density and number of melanomacrophage centers, biomarkers of stress. The present findings reveal the cross-talk among response to environmental stressors, metabolism and reproduction, highlighting that mature females invest a lot of energy in reproduction instead of immune response and detoxification. [ABSTRACT FROM AUTHOR]

Simple Summary: Microplastic accumulation in aquatic environments could represent a potential risk to wildlife health and survival. Their transfer along the trophic web could threaten ecosystem stability. Over the years, the common cuttlefish (Sepia officinalis) has represented an important resource for the fishery of the Adriatic Sea. In this study, for the first time, we assessed the presence of microplastics within cuttlefish eggs, more precisely in embryos and yolk. The presence of microplastics has already been described in common cuttlefish adult specimens. Our findings highlighted how this species is subjected to microplastic exposure starting from early-life stages. The possible effects of microplastic accumulation on embryo health and development should be investigated considering that the reproductive strategy of this species consists of adults laying eggs in separate batches during the single spawning season at the end of which both males and females die. Therefore, population stability primarily depends on the survival and correct development of embryos. Once they reach the aquatic environment, microplastics (MPs) are accidentally ingested by aquatic biota, thus entering the food chain with possible negative effects. The present study investigated, for the first time, MP presence in cuttlefish (Sepia officinalis) eggs and their association with embryonic development. Cuttlefish eggs were sampled from four different sites along the Marche region (Senigallia, Ancona, Numana, and San Benedetto del Tronto). Embryo and hatchling biometric parameters were evaluated and the internal structural integrity was examined through histological analysis. MPs were detected and characterized in embryos and yolk samples. MPs were identified in all sites (size < 5 µm), however, their presence has not been associated with an impairment of either embryo or hatchling internal structures. Noteworthy, the highest number of MPs (in both yolk and embryo samples) were found in Numana (37% of the total amount), where the lowest hatchling size was observed. On the other hand, the highest embryo mantle length was associated with the lowest number of MPs detected (9%) in Ancona. Overall, only MP fragments and sphere types (74 and 26%, respectively) were observed, and the most frequent polymers were Polyvinyl chloride (52%), Polypropylene, and Cellulose acetate (15% both). Further studies are needed to assess the possible MP effects on the yolk quality and assimilation. [ABSTRACT FROM AUTHOR]

Probiotics are live microorganisms that confer several beneficial effects to the host, including enhancement of bone mineralization. However, probiotic action on bone regeneration is not well studied and therefore we analysed various effects of probiotic treatment on the caudal fin regeneration of zebrafish. Morphological analysis revealed an increased regenerated area with shorter and thicker lepidotrichia segments after probiotic treatment. Fourier transform infrared spectroscopy imaging analysis highlighted the distribution of phosphate groups in the regenerated fins and probiotic group showed higher amounts of well-crystallized hydroxyapatite. At the midpoint (5 days post amputation) of regeneration, probiotics were able to modulate various stages of osteoblast differentiation as confirmed by the upregulation of some key marker genes such as runx2b, sp7, col10a1a, spp1 and bglap, besides suppressing osteoclast activity as evidenced from the downregulation of ctsk. Probiotics also caused an enhanced cell cycle by regulating the expression of genes involved in Retinoic acid (rarga, cyp26b1) and Wnt/β-catenin (ctnnb1, ccnd1, axin2, sost) signaling pathways, and also modulated phosphate homeostasis by increasing the entpd5a levels. These findings provide new outlooks for the use of probiotics as a prophylactic treatment in accelerating bone regeneration and improving skeletal health in both aquaculture and biomedical fields. [ABSTRACT FROM AUTHOR]

Preeclampsia is a human pregnancy-specific disease characterized by abnormal placentation that usually presents with maternal hypertension and proteinuria. The main hallmark of preeclampsia, impaired trophoblast migration, and the subsequent disruption of uterine arteries remodeling lead to several molecular alterations in the placental compartments with those occurring in the chorionic villi being of the utmost importance. Given the essential role of the endocannabinoid system during preimplantation and trophoblast migration, we have combined the histological and hyperspectral imaging analyses to shed light on the involvement of two cannabinoid receptors in the macromolecular alterations related to preeclampsia. The results obtained by immunohistochemistry showed a significant increase in the protein levels of cannabinoid receptor 1 (CB1) in the preeclamptic chorionic villi. However, no changes were reported regarding transient receptor potential vanilloid 1 (TRPV-1) levels either in the bulk placental samples or chorionic villi when comparing control and preeclamptic patients. Histological analysis and Fourier-transform infrared spectroscopy (FTIRI) showed an increase in collagen deposition together with higher levels of lipid peroxidation and phosphorylated compounds in the pathological villi. Since CB1 enhancement has been described as promoting fibrosis and oxidative stress in several tissues, we proposed that the higher receptor abundance in preeclampsia could be triggering similar molecular effects in preeclamptic term placentas. [ABSTRACT FROM AUTHOR]

The widespread use of plastics determines the inevitable human exposure to its by-products, including microplastics (MPs), which enter the human organism mainly by ingestion, inhalation, and dermal contact. Once internalised, MPs may pass across cell membranes and translocate to different body sites, triggering specific cellular mechanisms. Hence, the potential health impairment caused by the internalisation and accumulation of MPs is of prime concern, as confirmed by numerous studies reporting evident toxic effects in various animal models, marine organisms, and human cell lines. In this pilot single-centre observational prospective study, human breastmilk samples collected from N. 34 women were analysed by Raman Microspectroscopy, and, for the first time, MP contamination was found in 26 out of 34 samples. The detected microparticles were classified according to their shape, colour, dimensions, and chemical composition. The most abundant MPs were composed of polyethylene, polyvinyl chloride, and polypropylene, with sizes ranging from 2 to 12 µm. MP data were statistically analysed in relation to specific patients' data (age, use of personal care products containing plastic compounds, and consumption of fish/shellfish, beverages, and food in plastic packaging), but no significant relationship was found, suggesting that the ubiquitous MP presence makes human exposure inevitable. [ABSTRACT FROM AUTHOR]

Probiotics are live microorganisms that confer several beneficial effects to the host, including enhancement of bone mineralization. However, probiotic action on bone regeneration is not well studied and therefore we analysed various effects of probiotic treatment on the caudal fin regeneration of zebrafish. Morphological analysis revealed an increased regenerated area with shorter and thicker lepidotrichia segments after probiotic treatment. Fourier transform infrared spectroscopy imaging analysis highlighted the distribution of phosphate groups in the regenerated fins and probiotic group showed higher amounts of well-crystallized hydroxyapatite. At the midpoint (5 days post amputation) of regeneration, probiotics were able to modulate various stages of osteoblast differentiation as confirmed by the upregulation of some key marker genes such as runx2b, sp7, col10a1a, spp1 and bglap, besides suppressing osteoclast activity as evidenced from the downregulation of ctsk. Probiotics also caused an enhanced cell cycle by regulating the expression of genes involved in Retinoic acid (rarga, cyp26b1) and Wnt/β-catenin (ctnnb1, ccnd1, axin2, sost) signaling pathways, and also modulated phosphate homeostasis by increasing the entpd5a levels. These findings provide new outlooks for the use of probiotics as a prophylactic treatment in accelerating bone regeneration and improving skeletal health in both aquaculture and biomedical fields. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Abstract: The interactions between the endogenous gut microbiota and the fish host are integral in mediating the development, maintenance and effective functionality of the intestinal mucosa and gut associated lymphoid tissues (GALTs). These microbial populations also provide a level of protection against pathogenic visitors to the gastrointestinal (GI) tract and aid host digestive function via the production of exogenous digestive enzymes and vitamins. Manipulation of these endogenous populations may provide an alternative method to antibiotics to control disease and promote health management. Applications of probiotics for Mediterranean teleosts can stimulate immune responses, enhance growth performance, feed utilisation, digestive enzyme activities, antioxidant enzyme activities, gene expression, disease resistance, larval survival, gut morphology, modulate GI microbiota and mediate stress responses. Although considerably less information is available regarding prebiotic applications for Mediterranean teleosts, prebiotics also offer benefits with regards to improving immune status and fish production. Despite the promising potential benefits demonstrated in current literature, obtaining consistent and reliable results is often difficult due to our incomplete understanding of indigenous fish GI microbiota and their subsequent host interactions which mediate and drive both localised and systemic host immunological responses. Additionally, the probiotic and prebiotic (biotics) mechanisms which mediate host benefits at the mucosal interface are poorly understood. Future studies focused on these interactions utilising gnotobiotic techniques should provide a better understanding of how to extract the full potential of biotic applications to promote immune function of Mediterranean teleosts. [ABSTRACT FROM AUTHOR]

Glyphosate is a component of commonly used herbicides for controlling weeds in crops, gardens and municipal parks. There is increasing awareness that glyphosate-based herbicides, in addition to acting on plants, may also exert toxicity in wildlife and humans. In this study, male and female adult zebrafish were exposed to 700 µg/L of glyphosate (GLY), for 28 days. We used the metabolomic approach and UHPLC-ESI-MS to analyze liver samples to investigate the adverse effects of glyphosate on hepatic metabolism. The impact of GLY was found to be sex-specific. In female, GLY exposure affected purine metabolism by decreasing the levels of AMP, GMP and inosinic acid, consequently increasing uric acid levels with respect to the control (CTRL). Exposure to GLY also caused a decrease of UMP levels in the pyrimidine metabolism pathway. In male, GLY exposure decreased the aminoadipic acid within the lysine degradation pathway. Transcript analysis of genes involved in stress response, oxidative stress and the immune system were also performed. Results demonstrated an increased stress response in both sexes, as suggested by higher nr3c1 expression. However, the hsp70.2 transcript level was increased in female but decreased in male. The results demonstrated reduced sod1, sod2, and gpx1a in male following exposure to GLY, indicating an impaired oxidative stress response. At the same time, an increase in the cat transcript level in female was observed. mRNA levels of the pro-inflammatory interleukins litaf and cxcl8b.1 were increased in female. Taken together, the results provide evidence of disrupted nucleotide hepatic metabolism, increased stress inflammatory response in female and disruption of oxidative stress response in male. [ABSTRACT FROM AUTHOR]

Abstract: The effects of administration of the immunomodulator Ergosan, an algal extract containing alginic acid, in juvenile rainbow trout (Oncorhynchus mykiss) exposed to AquaVac™ vaccination, were tested. Juveniles treated with Ergosan, 95 days after the beginning of first solid feeding and control fish fed solely on commercial diet, were vaccinated by immersion in AquaVac™ solution. The time-course of the effects of vaccination on liver immunorelated gene modulation and on the tolerance to stress manipulation connected with the vaccination was investigated. Liver and plasma sampling was performed at the following times: T =pre-vaccination, T0=5min, T1=2h, T2=8h, T3=24h, T4=48h and T5=72h post-vaccination. Interleukin-1β (IL-1β), interleukin-8 (IL-8), tumor necrosis factor α 2 (TNF alpha 2) and heat shock protein 70 (Hsp70) gene expression in trout liver was monitored by real-time PCR using Acidic Ribosomal Phosphoprotein P0 (ARP) as internal standard. The evaluation of the plasma cortisol levels was performed by EIA. In AquaVac™-vaccinated fish, both the gene expression of Hsp70 and the plasma cortisol levels during the time-course were significantly (P <0.05) lower in Ergosan-treated fish with respect to control, indicating the positive role of Ergosan on handling stress tolerance. This study also demonstrated the stimulatory properties of Ergosan on cytokine genes expression involved in innate immune response: liver IL-1β, IL-8 and TNF alpha 2 gene expression was significantly (P <0.05) higher in trout fed on Ergosan compared to control, indicating a positive role of this feed additive in improving the immune responsiveness to AquaVac™ vaccine. [Copyright &y& Elsevier]

Abstract: The present study investigates, for the first time, critical processes in early life history in captive rearing of the lemonpeel angelfish Centropyge flavissimus. Video recordings of courtship behaviors in the 300 L spawning tank showed several courtship behaviors to be similar to those described for wild fish, but courtship activities associated with harem maintenance were not observed in the captive fish. Typical courtship activity was seen on every day of observation, but gamete release occurred on only 7 of 10 observation days. The reason for the lack of gamete release was not obvious. Tank spawning began when temperature and photoperiod conditions were changed to spring and reached a maximum peak (in terms of egg number) during the summer, when the temperature was 28 °C. Egg production and fertilization rate remained relatively constant day to day and spawning continued for months at a time as long as temperature and photoperiod were maintained at optimum levels. Once the embryo hatched (15 h post fertilization) and the yolk sac stage was reabsorbed (48 h post hatching), larvae were ready to feed. Cultured Parvocalanus sp. nauplii or size-sieved wild zooplankton were suitable first food. Proper environmental conditions included rearing the larvae at 28 °C. This study provides the first data on the influence of temperature and first food offered on larval survival in the lemonpeel angelfish. [Copyright &y& Elsevier]

This investigation was performed to assess the influences of the host‐associated probiotic Bacillus altitudinis B61‐34b (BAA) on growth, immune response and disease resistance to Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus). Fish (25.50 ± 0.52 g fish−1) were distributed into 15 aquaria (150 litres tank−1) at density of 20 fish tank−1. The fish were fed basal diets with BAA inclusions in different concentrations: 0 (BAA1—Control), 106 (BAA2), 107 (BAA3), 108 (BAA4) and 109 (BAA5) CFU ml−1 for 8 weeks. A completely randomized design with three replications was used. The results indicated that BAA administration generated greater (p < 0.05) skin mucus and peroxidase activities compared to the control, with maximum levels recorded in BAA4 and BAA5. Higher serum immunities, such as serum lysozyme, serum peroxidase, complement, phagocytosis and respiratory burst activities were observed in the BAA4 and BAA5 dietary groups compared to the control, BAA1, BAA2 and BAA3 diets. The relative percentage of survival, growth performance and FCR were also significantly higher (p < 0.05) in the BAA4 and BAA5 groups. In conclusion, dietary supplementation of B. altitudinis at 108 CFU ml−1 can improve Nile tilapia's growth performance, skin mucus and serum immunities, and disease resistance. [ABSTRACT FROM AUTHOR]

Being on the top of the food chain, tunas are subjected to significative phenomena of bioaccumulation of conservative contaminants such as Potential Toxic Elements (PTEs). In this study, Cd, Pb and Fe levels in muscle of Mediterranean bluefin tuna (Thunnus thynnus) were determined by atomic absorption spectroscopy (GFAAS), both in wild and farmed groups, to investigate the safety and the quality of this fish as seafood. A total of 68 samples were collected, wild samples (n = 30) from Sardinia island (Italy) and farmed samples (n = 38) from an aquaculture fish farm in Malta. Mean values, expressed as mg kg−1 wet weight, were found as 0.014 (wild) and 0.02 (farmed) for Cd; 0.11 (wild) and 0.03 (farmed) for Pb and 13 (wild) and 7 (farmed) for Fe. Relationships between metal concentrations and biometric parameters were evaluated and a comparison between the levels of metals of wild and farmed groups has also been conducted. No statistically significant difference between the two groups was found for Cd, with 99% of samples below the EU limit. The difference for Pb levels were statistically significant, with wild samples showing concentrations more than four times higher than the farmed ones, but with 98% of samples below the EU limit. The levels of Fe were significantly lower in the farmed group with respect to wild specimens, although samples of both groups could be considered good products for the intake of this element. On the base of the recommended tolerable weekly intakes, samples of this study can be considered a safe seafood. • Pb and Fe levels are statistically higher in wild ABFTs compared to farmed ones. • 99% of ABFTs resulted a safe food compared to the legal limits for Cd and Pb. • Wild and farmed ABFTs can be considered good products for the intake Fe. [ABSTRACT FROM AUTHOR]

In fish, like other organisms, the lack of balance between the production of reactive oxygen species (ROS) and antioxidant defense system (so-called oxidative stress) can cause DNA hydroxylation, protein denaturation, lipid peroxidation, apoptosis, and ultimately cell damage. To improve the antioxidant defense capability, different approaches such as the administration of synthetic antioxidants were practiced. During the past years, dietary approaches have been suggested as a promising way of increasing the antioxidant defense activity in different fish and shellfish species. Among them, microbial feed additives (including pre-, pro-, and synbiotics) showed promising effects in terms of affecting antioxidant enzymes activities. Their mechanism of action in influencing the antioxidant system is not fully understood. The present review briefly discussed the antioxidant defense activity in fish, influencing factors with special focus on dietary approaches and microbial feed additives. In addition, the proposed mechanism of action of microbial feed additives on the antioxidant system has been discussed. [ABSTRACT FROM AUTHOR]

Early feeding (started during gut metamorphosis and establishment of the hypothalamic–pituitary–interrenal axis) with probiotic-supplemented diets, besides modifying the intestinal microflora, evoked profound effects on the physiology of fish larvae. Using rotifers and Artemia as living vectors, the autochthonous bacterium Lactobacillus delbrueckii delbrueckii or a multispecies probiotic formulation (autochthonous Lactobacillus fructivorans+ Lactobacillus plantarum from human feces) were orally administered to sea bass and gilthead sea bream larvae, respectively. The treatments enhanced larval rearing (significantly increased body weight, decreased cortisol levels, and improved stress response compared with controls) and the immune system. In sea bass, the probiotic raised intestinal T cells, in keeping with increased total body TcR-β transcripts, and increased acidophilic granulocytes concomitant to lower transcription of pro-inflammatory genes ( IL-1-β, TGF-β, IL-10, Cox-2). In sea bream, the multispecies probiotic formulation raised intestinal immunoglobulin (Ig+) cells and acidophilic granulocytes mainly belonging to the MAb G7+ phagocytic population. These results point to stimulatory actions of probiotics on the gut immune system that are devoid of harmful effects and that correlate with improvement of fry survival. [ABSTRACT FROM AUTHOR]

Gut microbiota plays a fundamental role in maintaining host's health by controlling a wide range of physiological processes. Administration of probiotics and manipulation of photoperiod have been suggested as modulators of microbial composition and are currently undergoing an extensive research in aquaculture as a way to improve health and quality of harvested fish. However, our understanding regarding their effects on physiological processes is still limited. In the present study we investigated whether manipulation of photoperiod and/or probiotic administration was able to alter microbial composition in zebrafish larvae at hatching stage. Our findings show that probiotic does not elicit effects while photoperiod manipulation has a significant impact on microbiota composition. Moreover, we successfully predicted lipid biosynthesis and apoptosis to be modulated by microbial communities undergoing continuous darkness. Interestingly, expression levels of caspase 3 gene (casp3) and lipid-related genes (hnf4a, npc1l1, pparγ, srebf1, agpat4 and fitm2) were found to be significantly overexpressed in dark-exposed larvae, suggesting an increase in the occurrence of apoptotic processes and a lipid metabolism impairment, respectively (p < 0.05). Our results provide the evidence that microbial communities in zebrafish at early life stages are not modulated by a short administration of probiotics and highlight the significant effect that dark photoperiod elicits on zebrafish microbiota and potentially on health. [ABSTRACT FROM AUTHOR]

A 7‐week study was conducted to investigate the effect of different fasting and re‐feeding regimes on compensatory growth and some physiological parameters of juvenile Siberian sturgeon (Acipenser baerii). Fish (46.5 ± 0.5 g) were fed on a diet (containing 450 g/kg crude protein and 20 MJ/kg digestible energy) according to four feeding regimes in triplicate including: control group (C, fed everyday), W1 (2 weeks of feeding followed by 1 week of fasting and 4 weeks of re‐feeding), W2 (1 week of feeding followed by 2 weeks of fasting and 4 weeks of re‐feeding) and W3 (3 weeks of fasting followed by 4 weeks of re‐feeding). The fasted groups including W1 (119.6 ± 2.1 g), W2 (118.0 ± 1.7 g) and W3 (108.5 ± 4.8) significantly lost their weights during fasting phase and did not attain the final weight of the C (137.3 ± 1.7 g) after re‐feeding phase. The re‐feeding phase increased the specific growth rate in the fasted groups compared to the C (p <.05). After the fasting phase, concentrations of T3, T4, glucose, total protein and triglyceride in plasma of fasted groups were decreased, but levels of total cholesterol, aspartate aminotransferase, alkaline phosphatase and lactate dehydrogenase increased compared to the C. After re‐feeding phase, except for glucose level, all mentioned metabolites were restored in the plasma of W1 group, but total protein level and aspartate aminotransferase concentrations in plasma were not restored in W2 and W3 groups. Overall, our finding demonstrated 4 weeks of re‐feeding was too short to induce full compensatory growth in A. baerii juveniles. [ABSTRACT FROM AUTHOR]

Mercury (Hg) is a well-known toxic element, diffused in the environment, especially in the Mediterranean Sea which is rich in cinnabar deposits. Mercury bioaccumulation in fish is of great concern, especially for top-level aquatic predators (e.g., shark, tuna, swordfish) and above all for species of large human consumption and high nutritional value. This work aimed to determine Hg concentrations in farmed and wild Atlantic Bluefin tuna (Thunnus thynnus) caught in the Mediterranean area in order to evaluate the level of Hg bioaccumulation. selenium (Se) content was also determined, since this element is an antagonist of mercury toxicity. Mercury and Se were analysed by atomic absorption spectrometry after microwave digestion of the samples. Hg content in farmed tuna was below the legal limit (1 mg/kg, wet weight, w.w.) for all specimens (0.6 ± 0.2 mg/kg), whereas the wild ones had a content over the limit (1.7 ± 0.6 mg/kg); Se concentration was higher in farmed specimens (1.1 ± 0.9 mg/kg) compared to wild ones (0.6 ± 0.3 mg/kg). A safe seafood could show a Se/Hg ratio >1 and a health benefit value (HBVSe) > 0: farmed tuna had higher values than the wild specimens (Se/Hg 5.48 vs. 1.32; HBVSe 11.16 vs. 0.29). These results demonstrate that for Hg, there is a better risk/benefit ratio in farmed T. thynnus. making it safer than wild tuna. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is an abundant environmental contaminant and studies have shown the presence of BPA in the urine of over 90% of population tested in Canada and USA. In addition to its reported harmful effects, there is concern for its transgenerational effects. For a compound to induce transgenerational effect, an epigenetic mark should be mitotically and meiotically stable without reprogramming in primordial germ cells and post fertilization embryos. In the present study, female zebrafish were treated with an environmental dose (20 μg/L) of BPA and then crossed with untreated males. To assess epigenetic effects, transcript levels of several genes involved in female reproduction were measured in adult and in 24 hpf embryos up to F3 generation. Exposure to BPA affected adult female fertility up to F2 generation. In F0, F1 and F2 ovaries transcript levels for several genes involved in reproduction, including esr, star, lhcgr and fshr were affected. To investigate epigenetic mechanisms of gene expression modulation, we studied promoter DNA methylation. Among genes involved in gonadal differentiation, amh transcript level was reduced in 24 hpf embryos, up to the F3 generation. Variation in amh transcript level was associated with hyper-methylation of its promoter and changes in H3K4me3/H3K27me3 enrichment, coherent with gene silencing. The findings provide evidence for transgenerational effects of BPA in zebrafish and demonstrate that amh is susceptible to stable epigenetic alterations. Transgenerational effects of BPA on female reproductive physiology. Unlabelled Image • BPA's effects show a transgenerational transmission via maternal route. • Transgenerational transmission involves both histone modification and DNA methylation. • Amh hypermethylation shows a stable transmission throughout generations. [ABSTRACT FROM AUTHOR]

Abstract Endometriosis is a chronic gynaecological disease characterised by the presence of endometrial cells in extra-uterine regions. One of the main factors impacting on the fertility of women affected by endometriosis is the poor oocyte quality. Granulosa Cells (GCs) regulate oocyte development and maintain the appropriate microenvironment for the acquisition of its competence; hence, the dysregulation of these functions in GCs can lead to severe cellular damages also in oocytes. In this study, luteinized GCs samples were separately collected from both ovaries of women affected by Unilateral Ovarian Endometriosis and analysed by infrared and Raman microspectroscopy. The spectral data were compared with those of GCs from women with diagnosis of tubal, idiopathic or male infertility (taken as control group). The coupling of these two spectroscopic techniques sheds new light on the alteration induced by this pathology on GCs metabolism and biochemical composition. In fact, the study revealed similar biochemical modifications in GCs from both ovaries of women affected by unilateral ovarian endometriosis, such as the alteration of the protein pattern, the induction of oxidative stress mechanisms, and the deregulation of lipid and carbohydrate metabolisms. These evidences suggest that unilateral endometriosis impairs the overall ovarian functions, causing alterations not only in the ovary with endometriotic lesions but also in the contralateral "healthy" one. Graphical Abstract Unlabelled Image Highlights • FTIRM and RMS highlighted changes induced by UOE on GCs biochemical composition. • GCs from ovary with UOE and the contralateral one showed the same impairment. • Unilateral endometriosis impairs the overall ovarian functions. [ABSTRACT FROM AUTHOR]

A previously established gas chromatography-mass spectrometry method was applied for the determination of the fatty acid composition in the muscles of Atlantic bluefin tuna (Thunnus thynnus L). The fish were collected in the Mediterranean Sea and cultivated for several months. The goal of the study was to demonstrate the feasibility of these measurements and to improve knowledge about the influence of size and gender on the fatty acid composition. Although no significant differences were found with respect to gender, males and females showed different correlations of fatty acid composition with weight. In the males, a statistically significant linear correlation was found for 14:0, 16:0, 18:1n9, 20:1n9, 22:1n9 (positive correlation), and for 20:4n6, 20:5n3 22:6n3 (negative correlation). In the females, a statistically significant polynomial correlation for specific fatty acids was highlighted: specimens with medium size showed the lowest content of 20:1n9 and 22:1n9 and the highest content of 20:4n6, 20:5n3, and 22:6n3. Therefore, males smaller than 150 kg and females from 150 to 250 kg appeared to have the highest greater nutritional value. This study demonstrated the practicality of the analytical methodology and enhanced the knowledge of the influence of size on the fatty acid composition of Atlantic bluefin tuna muscle, reporting a newly established gender difference and representing a starting point to produce the best nutritional characteristics for farmed tuna. [ABSTRACT FROM AUTHOR]

Abstract The increasing manufacture of plastics and their mismanagement has turned plastic into a ubiquitous waste in the marine environment. Among all the substances conforming the plastic items, the effects of a dietary Bisphenol A (BPA) and Di-isononyl phthalate (DiNP) have been evaluated in adult male gilthead sea bream, focusing on their effects in the modulation of the Endocannabinoid System (ECS). In zebrafish, the ECS has been recently chosen as a new target for the activity of some Endocrine Disrupting Chemicals (EDC), since it represents a complex lipid signaling network essential for the well-being of the organisms. The results obtained in gilthead seabream showed that BPA and DiNP altered the structure and the biochemical composition of liver, increasing the presence of lipids and triglycerides and decreasing the glycogen and phospholipids. Moreover, the addition of BPA or DiNP in the gilthead sea bream diet altered the levels of endocannabinoids (EC) and EC-like mediators in the liver. These alterations were also associated to changes at the transcriptomic level of genes involved in lipid biosynthesis and ECS metabolism. At the central level, both BPA and DiNP reduced the expression of the endocannabinoid receptor type I (cnr1) and the neuropeptide Y (npy) as well as the levels of the endocannabinoid Anandamide (AEA), suggesting a downregulation of appetite. The results herein reported highlighted the negative effects of chronic dietary exposure to DiNP or BPA on ECS functions and lipid metabolism of male gilthead sea bream liver, showing a similar disruptive activity of these contaminants at metabolic level. Moreover, the novelty of the biomarkers used evidenced possible innovative endpoints for the development of novel OEDCS test guidelines. Highlights • Dietary exposure to DiNP and BPA modified the ECS signaling in liver and brain of adult gilthead sea bream. • Dietary exposure to DiNP and BPA induced biochemical and morphological changes in the liver. • Dietary exposure to DiNP and BPA altered the triglyceride biosynthesis. • DiNP and BPA exert similar disruptive action on seabream metabolism. [ABSTRACT FROM AUTHOR]

Endocrine disrupting chemicals (EDCs) are known to disrupt normal metabolism and can influence the incidence of obesity in animals and humans. EDCs can exert adverse effects at low concentrations, often in a non-monotonic dose-related fashion. Among EDCs, Bisphenol A (BPA) is extensively used in the production of polycarbonate plastic, and is among the most abundant contaminants in the world. Diethylene glycol dibenzoate (DGB), an approved alternative to phthalates in the production of plastic and latex products, however, is less abundant and its effects are almost completely unknown. The aim of this study is to provide information on the hepatic effects of BPA and DGB on lipid metabolism, and investigate possible links between these contaminants and the increased incidence of obesity. In the present study, we exposed zebrafish to three different BPA doses (5; 10; 20 μg/L) and five different doses of DGB (0.01; 0.1; 1; 10; 100 μg/L) for a period of 21 days, and investigated transcript levels for genes involved in lipid metabolism as well as measuring liver content of phosphates, lipids and proteins. The results demonstrate disruptive effects of BPA and DGB on lipid metabolism in a non-monotonic dose-related fashion. The lowest dose of BPA increased the storage of triglycerides and promoted fatty acid synthesis, while the highest concentration promoted de novo lipogenesis and cholesterologenesis. Exposure to DGB was also found to affect lipid metabolism leading to increased lipid production and mobilization in a non-monotonic dose-related fashion. Analysis of BPA and DGB by FT-IR revealed that exposure to both compounds lead to changes in the biochemical composition of liver. The findings provide a support for the hypothesis that BPA and DGB may be among the environmental contaminants with obesogenic property. [ABSTRACT FROM AUTHOR]

Atlantic Bluefin Tuna Thunnus thynnus (ABFT) is considered one of the most important socio-economic species but there is a lack of information on the physiological and molecular processes regulating its growth and metabolism. In the present study, we focused on key molecules involved in growth process. The aim of the present study was to associate molecular markers related to growth with canonical procedures like morphological measurements such as curved fork length (CFL) and round weight (RWT). The ABFT specimens (n = 41) were organized into three different groups A, B and C according to their age. The molecular analysis of liver samples revealed that igf1 , igf1r and mTOR genes, involved in growth process, were differentially expressed in relation to the age of the fish. In addition, during the analyzed period, faster growth was evident from 5 to 8 years of age, after that, the growth rate decreased in terms of length yet increased in terms of adipose tissue storage, as supported by the higher fat content in the liver. These results are useful in expanding basic knowledge about the metabolic system of ABFT and provide new knowledge for the aquaculture industry. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA), a widely used chemical to produce polycarbonate plastics, has become an ubiquitous pollutant due to its extensive use. Its endocrine disrupting properties have been documented in several studies, as well as its potential to induce metabolic and reproductive impairments at environmentally relevant concentrations. Recent insights highlighted the role of the Endocannabinoid System (ECS) in energy homeostasis and lipid metabolism. In fact, disruption of the ECS may induce metabolic alterations among other effects. Thus, the main objective of this study was to investigate the disruptive effects of environmentally relevant concentrations of BPA on the ECS of female zebrafish liver and brain. Adult female zebrafish were exposed for 3 weeks to three different concentrations of BPA (5 μg/L; 10 μg/L; 20 μg/L). We observed changes in the expression of a number of genes involved in the Anandamide (AEA) and 2-Arachidonoylglycerol (2-AG) metabolism in the liver and brain, as well as altered levels of endocannabinoids and endocannabinoid-like mediators. These changes were associated with greater presence of hepatic lipid vacuoles, following exposure to the highest concentration of BPA (20 μg/L) tested, although there were no changes in food intake and in the expression of the molecular markers for appetite. The overall results support the hypothesis that exposure to BPA induced changes in the central and hepatic ECS system of adult female zebrafish causing the increase of the area covered by lipids in the liver at the highest concentration tested, but not via food intake. [ABSTRACT FROM AUTHOR]

Due to the undesirable consequences associated with antibiotics use, researchers and food producers have studied alternative feeding, for the control of fish diseases and animal welfare improvement. However, the beneficial properties of RNA yeasts extract in aquaculture have been barely considered. The present study investigated the beneficial properties of RNA yeast extract from Kluyveromyces fragilis on survival rate, weight and length as well as on molecular pathways involved in growth, immuno-system and oxidative stress using zebrafish (Danio rerio) as an experimental model. The yeast extract has been administrated to zebrafish at three different concentrations (60-180-300 ppm) via zooplankton (Artemia salina) for 21 days. Results highlighted yeast extract RNA capability to enhance growth and to improve larvae survival rate in a dose-dependent manner. In fact, gene expression data showed the ability of the RNA yeast extract to up-regulate genes involved in growth and to restore stress-related condition due to the early larval development coupled with the RNA yeast extract administration. In addition, gene expression showed that RNA yeast extract acts as inflammatory-reducer, but did not enhance the immune response. Histological analysis showed that all three treated groups displayed a thicker gut epithelium, higher intestinal crypts coupled with higher enterocytes lengths compared to control group. In conclusion, these findings provide a large gene network through which yeast RNA extract acts and, by inducing transcriptional changes, modulate the physiological control of growth and inflammatory response coupled with the increase in length and dry weight, together with a higher survival ratio and an intestinal architecture amelioration. [ABSTRACT FROM AUTHOR]