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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]

Sentinel fish hornyhead turbot (Pleuronichthysverticalis) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences.

Ubiquitous exposure to bisphenol A (BPA), an endocrine disruptor (ED), has raised concerns for both human and ecosystem health. Epigenetic factors, including microRNAs (miRNAs), are key regulators of gene expression during cancer. The effect of BPA exposure on the zebrafish epigenome remains poorly characterized. Zebrafish represents an excellent model to study cancer as the organism develops a disease that resembles human cancer. Using zebrafish as a systems toxicology model, we hypothesized that chronic BPA-exposure impacts the miRNome in adult zebrafish and establishes an epigenome more susceptible to cancer development. After a 3 week exposure to 100 nM BPA, RNA from the liver was extracted to perform high throughput mRNA and miRNA sequencing. Differential expression (DE) analyses comparing BPA-exposed to control specimens were performed using established bioinformatics pipelines. In the BPA-exposed liver, 6188 mRNAs and 15 miRNAs were differently expressed (q ≤ 0.1). By analyzing human orthologs of the DE zebrafish genes, signatures associated with non-alcoholic fatty liver disease (NAFLD), oxidative phosphorylation, mitochondrial dysfunction and cell cycle were uncovered. Chronic exposure to BPA has a significant impact on the liver miRNome and transcriptome in adult zebrafish with the potential to cause adverse health outcomes including cancer. [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]

The present study explored alternative strategies to improve the health status and growth metabolism in cultured juvenile fish. In this context the effects of alginic acid administration on the welfare of rainbow trout juveniles were investigated. Both the hepatic gene expression of heat shock protein 70 and the cortisol plasma levels were evaluated in order to estimate the possible beneficial role of alginic acid in improving animal well-being. At the same time the body weight was measured and was correlated with the hepatic expression of insulin-like growth factor I, myostatin, and two isoforms of the thyroid receptor. Finally, improvement of the immune system was investigated by monitoring the expression of innate immune-related genes, such as interleukin1β, interleukin8 and toll-like receptor3, in the spleen. The data obtained demonstrated an improvement in the welfare of rainbow trout juveniles, revealed by growth and innate immune response enhancement by alginic acid administration. These results may be of great importance for the further development of more sustainable aquaculture practices, and can be considered as a biotechnological application to produce a safer product for human health. [ABSTRACT FROM AUTHOR]

We set out to determine whether probiotic addition would improve larval development in the false percula clownfish Amphiprion ocellaris and to determine what molecular responses could be observed in the larvae following probiotic exposure. We supplied the probiotic bacterial strain Lactobacillus rhamnosus IMC 501 to clownfish larvae from the first day posthatch simultaneously by live prey and with addition to rearing water (group 2) and exclusively by live prey (group 3). We observed twofold higher body weight in both clownfish larvae and juveniles when probiotics were supplied via live prey and added to the rearing water. In addition, development was accelerated with metamorphosis occurring 3 days earlier in fingerlings treated with probiotic. Alteration in molecular biomarkers supported the faster growth observation. There was significantly increased gene expression of factors involved in growth and development (insulin-like growth factors I and II, myostatin, peroxisome proliferator-activated receptors α and β, vitamin D receptor a, and retinoic acid receptor γ) when probiotics were delivered via live prey and added to the rearing water. Moreover, probiotic treatment lessened the severity of the general stress response as exhibited by lower levels of glucocorticoid receptor and 70-kDa heat shock protein gene expression. Furthermore, an improvement of skeletal head development was observed, with a 10-20% reduction in deformities for juveniles treated with probiotic. All data suggest a potent effect on development resulting from the administration of lactic acid bacteria to larval clownfish, and this study provides a preliminary molecular entry path into the investigation of mechanisms responsible for probiotic enhancement in fish development. [ABSTRACT FROM AUTHOR]

Glucocorticoids (GCs) are steroid hormones mainly acting as key regulators of body homeostasis and stress responses. Their activities are primarily based on the binding to the GC receptor (GR), a member of the nuclear receptor family, that regulates tissue-specific sets of genes. GCs secretion follows a circadian rhythmicity with a peak linked to the animal's activity phase. In mammals, GCs are also implicated in feeding entrainment mechanisms as internal zeitgeber. Here, we investigated, by means of behavioural and molecular approaches, the circadian clock and its regulation by light and food in wild-type (WT) and null glucocorticoid receptor (gr−/−) zebrafish larvae, juveniles and adults. In both WT and gr−/− larvae and adults, behavioural activity and clock gene expression were entrained to the light–dark (LD) cycle and rhythmic in constant conditions. Differences in the pattern of clock genes' expression indicated a modulatory role of GCs. A significant role of Gr was detected in the feeding entrainment which was absent or markedly dampened in mutants. Furthermore, the expression of two clock-regulated genes involved in glucidic and lipidic metabolism was altered, highlighting the participation of GCs in metabolic processes also in fish. Taken together, our results confirmed the role of GC-mediated Gr signaling in the feeding entrainment in a non-mammalian species, the zebrafish. [ABSTRACT FROM AUTHOR]

Positive effects of probiotics on fish reproduction have been reported in several species. In the present study, 40 male European eels were weekly treated with recombinant hCG for 9 weeks and with three different concentrations (10 3 , 10 5 , and 10 6 CFU/mL) of probiotic Lactobacillus rhamnosus IMC 501 (Sinbyotec, Italy). The probiotics were daily added to the water from the sixth week of the hCG treatment. Males from the treated and control groups were sacrificed after 1, 2, and 3 weeks of probiotic treatment (seventh–ninth weeks of hCG treatment); at this point, sperm and testis samples were also collected. Sperm volume was estimated, and motility was analyzed by computer-assisted sperm analysis software. Alternations in transcription of specific genes involved in reproductive process such as activin , androgen receptors α and β ( arα and arβ ), progesterone receptor 1 ( pr1 ), bone morphogenetic protein 15 ( bmp15 ), and FSH receptor ( fshr ) were analyzed in the testis. After 2 weeks of probiotic treatment, sperm production and sperm motility parameters (percentage of motile cells and percentage of straight-swimming spermatozoa) were increased in the European eel treated with 10 5 CFU/mL compared to controls or to the other probiotic doses. These changes were associated with increases in messenger RNA expression of activin , arα, arβ, pr1 , and fshr . Conversely, after 3 weeks, activin and pr1 expression decreased. No significant changes were observed on bmp15 expression throughout the duration of the treatment with 10 5 CFU/mL dose. The lowest and highest probiotic dose (10 3 and 10 6 CFU/mL, respectively) inhibited the transcription of all genes along all the experiment, except for arα and arβ after 1 week of probiotic treatment when compared to controls. The changes observed by transcriptomic analysis and the sperm parameters suggest that a treatment with L rhamnosus at 10 5 CFU/mL for 2 weeks could improve spermatogenesis process in Anguilla anguilla. [ABSTRACT FROM AUTHOR]

Highlights: [•] Marine flatfish exposed to municipal wastewater effluent. [•] Investigated associations among gene expression, higher biological responses and exposure. [•] Fish exposed to effluent showed expression changes in genes involved in metabolism. [•] As well as stress responses, xenobiotic metabolism, and steroid synthesis. [•] Significant correlations found between the expression of 22 genes and other responses. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 106 CFU g−1 feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic–host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response. [Copyright &y& Elsevier]

Abstract: The potential of common sole Solea solea (Linnaeus, 1758) for consistent production in recirculating systems is limited by the huge losses usually observed during the early stages of larval development, mainly due to pathogen infections and insufficient nutritional balance. In the hope that use of a suitable probiotic strain may counter these difficulties, in this study we isolated Enterococcus faecium IMC 511 from common sole brood stock and utilized it as probiotic candidate during fish feeding. After a daily administration (twice a day) to sole larvae through live feeds, the intestinal presence of E. faecium IMC 511 was estimated on days 10, 30, and 50 post hatch, and the effects on intestinal microbial load, fish survival, welfare, and growth were evaluated by morphometric and molecular approaches by examining relative body weight, total length, and gene expression of myostatin. In particular, the effect on animal welfare was assessed through analyses of cortisol levels and 70kDa Heat Shock Protein gene expression. The results showed that while the Vibrio populations of the intestine of S. solea larvae were reduced in a significant way by the probiotic E. faecium IMC 511, survival was not affected. 70kDa Heat Shock Protein and myostatin gene expression were significantly reduced in probiotic fed larvae with respect to the control group. It is of note that E. faecium IMC 511 seemed to be predominant at day 50 post hatching, probably due to stabilization of the intestinal microbiota, a factor that, together with the lower levels of HSP70, could improve animal well-being and growth. Cortisol levels significantly increased after 10days of treatment in probiotic fed larvae, while on days 30 and 50 no significant differences were observed among all experimental groups. Considered together, the microbiological data and the molecular and morphometric results suggest that 50days of probiotic treatment can improve common sole larval growth. Also, it is hoped that these results offer a useful starting point for elucidating the pathways involved in S. solea growth fostered by E. faecium IMC 511 administration. [Copyright &y& Elsevier]

Abstract: In the present study, we investigated whether the use of Enterococcus faecium IMC 511 as a probiotic can modulate neuroendocrine system responses during the larval rearing of Solea solea; to this end, the gene expression patterns of proopiomelanocortin (POMC), endocannabinoid receptor 1A (CB1A), and thyroid receptor alpha (TRα) were quantified, and whole-body cortisol levels were measured. Probiotic treatment up-regulated transcription of all selected genes and cortisol concentrations on day 10 post hatch (ph), while on day 30 ph experimental groups showed significantly lower levels of both POMC and CB1A compared to those of the control group. These changes were no longer evident on day 60 ph, when POMC, CB1A, TRα gene expression and cortisol titers were found to be similar in all experimental groups. Our results suggest that metabolic responses to probiotic treatment can be modulated through the activation of genes selected for functional interaction between the hypothalamic-pituitary-thyroid (HPT) axis and the melanocortin and the endocannabinoid systems. Furthermore, the observed (30 ph) down-regulation of both POMC and CB1A gene expression coupled with up-regulation of TRα mRΝΑ levels suggest the activation of a compensatory mechanism that promotes growth and development and perhaps modulates food intake. [Copyright &y& Elsevier]

Abstract: Melatonin is the hormonal mediator of photoperiodic information to the central nervous system in vertebrates and allows the regulation of energy homeostasis through the establishment of a proper balance between energy intake and energy expenditure. The aim of this study was to evaluate the role of melatonin in appetite central control analyzing the involvement of this hormone in the regulation of feeding behavior in the zebrafish Danio rerio. For this purpose, the effect of two different melatonin doses (100nM and 1μM) administered for 10days, via water, to zebrafish adults was evaluated at both physiological and molecular level and the effect of melatonin was considered in relation to the most prominent systems involved in appetite regulation. For the first time, in fact, melatonin control of food intake by the modulation of leptin, MC4R, ghrelin, NPY and CB1 gene expression was evaluated. The results obtained indicate that melatonin significantly reduces food intake and the reduction is in agreement with the changes observed at molecular level. A significant increase in genes codifying for molecules involved in feeding inhibition, such as leptin and MC4R, and a significant reduction in the major orexigenic signals including ghrelin, NPY and CB1 are showed here. Taken together these results support the idea that melatonin falls fully into the complex network of signals that regulate food intake thus playing a key role in central appetite regulation. [Copyright &y& Elsevier]

A 42-day study was conducted to determine the effect of incorporating dietary low molecular weight sodium alginate (LMWSA), extracted from the brown algae, Undaria pinnatifida and Macrocystis pyritera , and Pediococcus acidilactici MA 18/5 M (PA), Lallemand Animal Nutrition S.A., Blagnac, France, on growth performance, antioxidant defense activity, intestinal lysozyme gene (LYZ) expression, histo-morphology, microbiota, and digestive enzymes activity of Asian sea bass (Lates calcarifer) juveniles. Six experimental diets were formulated including: Diet (1) a basal diet (Control), Diet (2) 5 g LMWSA kg−1 diet, Diet (3) 10 g LMWSA kg−1 diet, Diet (4) 0.9 × 107 CFU PA g−1 diet, Diet (5) 5 g LMWSA kg−1 diet + 0.9 × 107 CFU PA g−1 diet, and Diet (6) 10 g LMWSA kg−1 diet + 0.9 × 107 CFU PA g−1 diet were fed to Asian sea bass, L. calcarifer (12.0 ± 0.2 g). The results showed that fish fed PA alone (Diet 4) and the combination of both supplements (Diet 5) had the greatest weight gain. Fish fed Diet 6 and those fed Diet 1 (Control) had the highest and lowest villus height, apparent villus surface and crypt depth, respectively. Fish fed diets administered with PA (Diet 4) or synbiotics (Diets 5 and 6) showed higher total viable and lactic acid bacteria counts than all the other groups. The evaluated digestive enzymes activities including total protease, trypsin, lipase, and α-amylase remarkably increased by administration of LMWSA or its combination with PA. Moreover, liver antioxidant enzymes activities including superoxide dismutase, catalase, and Glutathione S-transferase pronouncedly enhanced following the administration of LMWSA or its combination with PA. Supplementing diet with blends of 10 g kg−1 of LMWSA and PA (Diet 6) more pronouncedly enhanced c-type and g-types LYZ expression in comparison with those fed 5 g kg−1 of LMWSA and PA (Diet 5). Based on the results obtained, it can be claimed incorporating diet with LMWSA and PA separately or in symbiotic form had promising results as functional feed additives in juvenile Asian sea bass L. calcarifer. • Combined administration of sodium alginate [LMWSA] and pediococcus acidilactici [PA] improved growth performance. • Supplementing aqua-feeds with LMWSA and PA increased lactic acid bacteria levels in the intestine. • Combined administration of LMWSA and PA improved the histo-architectural structure of the intestine. • Dietary administration of LMWSA and PA enhanced the antioxidant defence in Asian sea bass. [ABSTRACT FROM AUTHOR]