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Supplementation of one-carbon (1C) metabolism micronutrients, which include B-vitamins and methionine, is essential for the healthy growth and development of Atlantic salmon ( Salmo salar ). However, the recent shift towards non-fish meal diets in salmon aquaculture has led to the need for reassessments of recommended micronutrient levels. Despite the importance of 1C metabolism in growth performance and various cellular regulations, the molecular mechanisms affected by these dietary alterations are less understood. To investigate the molecular effect of 1C nutrients, we analysed gene expression and DNA methylation using two types of omics data: RNA sequencing (RNA-seq) and reduced-representation bisulphite sequencing (RRBS). We collected liver samples at the end of a feeding trial that lasted 220 days through the smoltification stage, where fish were fed three different levels of four key 1C nutrients: methionine, vitamin B6, B9, and B12. Our results indicate that the dosage of 1C nutrients significantly impacts genetic and epigenetic regulations in the liver of Atlantic salmon, particularly in biological pathways related to protein synthesis. The interplay between DNA methylation and gene expression in these pathways may play an important role in the mechanisms underlying growth performance affected by 1C metabolism.

One of the most crucial biological indicators in tracking long-term variations in the reproductive cycle is sexual development. Scorpaena porcus (Linnaeus, 1758), commonly known as the black scorpionfish, is a small teleost from the family Scorpaenidae. Much is known about its ecology, but data on its reproductive and defense systems are still lacking. Antimicrobial peptides (AMPs), such as piscidins, are integral components of the innate immune system in fish. These peptides exhibit a wide range of activity against bacteria, fungi, viruses, and protozoa and act as the first line of host defense. This study aims to investigate the primary sexual development stages in male and female gonads of black scorpionfish, providing additional knowledge on the reproductive biology of this teleost while evaluating concomitant changes in the expression of a Piscidin-1 antimicrobial peptide. The results show a histological, morpho-structural change from the immature stage to the developing virgin stage. Immunohistochemical analyses show that germinal and somatic cells are strongly reactive to Piscidin-1 in both gonads at an early ontogeny stage. These data suggest that Piscidin-1 may play a key role in the local defense system of scorpionfish gonads at this delicate stage, which is critical for the continuation and maintenance of the species. The present findings are potentially useful for a better understanding of the reproductive cycle of this fish, improving our knowledge of the interaction between the immune system and reproduction.

Embryonic temperature has a lasting impact on muscle phenotype in vertebrates, involving complex molecular mechanisms that encompass both protein-coding and non-coding genes. Circular RNAs (circRNAs) are a class of regulatory RNAs that play important roles in various biological processes, but the effect of variable thermal conditions on the circRNA transcriptome and its long-term impact on muscle growth plasticity remains largely unexplored. To fill this knowledge gap, we performed a transcriptomic analysis of circRNAs in fast muscle of Nile tilapia ( Oreochromis niloticus ) subjected to different embryonic temperatures (24°C, 28°C and 32°C) and then reared at a common temperature (28°C) for 4 months. Nile tilapia embryos exhibited faster development and subsequently higher long-term growth at 32°C compared to those reared at 28°C and 24°C. Next-generation sequencing data revealed a total of 5,141 unique circRNAs across all temperature groups, of which 1,604, 1,531, and 1,169 circRNAs were exclusively found in the 24°C, 28°C and 32°C groups, respectively. Among them, circNexn exhibited a 1.7-fold (log 2 ) upregulation in the 24°C group and a 1.3-fold (log 2 ) upregulation in the 32°C group when compared to the 28°C group. Conversely, circTTN and circTTN_b were downregulated in the 24°C groups compared to their 28°C and 32°C counterparts. Furthermore, these differentially expressed circRNAs were found to have multiple interactions with myomiRs, highlighting their potential as promising candidates for further investigation in the context of muscle growth plasticity. Taken together, our findings provide new insights into the molecular mechanisms that may underlie muscle growth plasticity in response to thermal variation in fish, with important implications in the context of climate change, fisheries and aquaculture., Competing Interests: Author RJ was employed by GenoMar Genetics AS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Rbbani, Murshed, Siriyappagouder, Sharko, Nedoluzhko, Joshi, Galindo-Villegas, Raeymaekers and Fernandes.)

The growth of the aquaculture industry requires more sustainable and circular economy-driven aquafeed formulas. Thus, the goal of the present study was to assess in farmed gilthead sea bream ( Sparus aurata L.) how different combinations of novel and conventional fish feed ingredients supported proper animal performance in terms of growth and physiological biomarkers of blood/liver/head kidney. A 77-day feeding trial was conducted with three experimental diets (PAP, with terrestrial processed animal protein from animal by-products; NOPAP, without processed animal protein from terrestrial animal by-products; MIX, a combination of alternative ingredients of PAP and NOPAP diets) and a commercial-type formulation (CTRL), and their effects on growth performance and markers of endocrine growth regulation, lipid metabolism, antioxidant defense and inflammatory condition were assessed at circulatory and tissue level (liver, head kidney). Growth performance was similar among all dietary treatments. However, fish fed the PAP diet displayed a lower feed conversion and protein efficiency, with intermediate values in MIX-fed fish. Such gradual variation in growth performance was supported by different biomarker signatures that delineated a lower risk of oxidation and inflammatory condition in NOPAP fish, in concurrence with an enhanced hepatic lipogenesis that did not represent a risk of lipoid liver degeneration.

(Adaptive) radiations have attracted evolutionary biologists for a long time as ideal model systems to study patterns and processes of often rapid speciation. However, whereas a wealth of (sometimes already genome-scale) data is available for host radiations, very few studies target the patterns of diversification in their symbionts, even though they would be excellent models to study symbiont speciation. Our review summarizes what little is known about general patterns of symbiont diversification in often iconic adaptive host radiations and to what extent these patterns are dependent on the evolutionary trajectories of their hosts. We identify research gaps that need to be addressed in the future and discuss the potential of approaches not yet typically used in these study systems, such as epidemiological disease modeling and new omics technologies, for significantly advancing our understanding of these complex eco-evolutionary relationships., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

Early microbial colonization has a profound impact on host physiology during different stages of ontogeny. Although several studies have focused on early bacterial colonization and succession, the composition and role of fungal communities are poorly known in fish. Here, we sequenced the internal transcribed spacer 2 (ITS2) region of fungi to profile the mycobiome associated with the eggs, hatchlings and intestine of Atlantic salmon at various freshwater and marine stages. In most of the stages studied, fungal diversity was lower than bacterial diversity. There were several stage-specific fungal phylotypes belonging to different stages of ontogeny but some groups, such as Candida tropicalis, Saccharomyces cerevisiae, Alternaria metachromatica, Davidiella tassiana and Humicola nigrescens, persisted during successive stages of ontogeny. We observed significant changes in the intestinal fungal communities during the first feeding. Prior to first feeding, Humicola nigrescens dominated, but Saccharomyces cerevisiae (10 weeks post hatch) and Candida tropicalis (12 weeks post hatch) became dominant subsequently. Seawater transfer resulted in a decrease in alpha diversity and an increase in Candida tropicalis abundance. We also observed notable variations in beta diversity and composition between the different farms. Overall, the present study sheds light on the fungal communities of Atlantic salmon from early ontogeny to adulthood. These novel findings will also be useful in future studies investigating host-microbiota interactions in the context of developing better nutritional and health management strategies for Atlantic salmon farming., (© 2024. The Author(s).)

Phenotypic plasticity of metabolism and growth are essential for adaptation to new environmental conditions, such as those experienced during domestication. Epigenetic regulation plays a key role in this process but the underlying mechanisms are poorly understood, especially in the case of hydroxymethylation. Using reduced representation 5-hydroxymethylcytosine profiling, we compared the liver hydroxymethylomes in full-sib Nile tilapia with distinct growth rates (3.8-fold difference) and demonstrated that DNA hydroxymethylation is strongly associated with phenotypic divergence of somatic growth during the early stages of domestication. The 2677 differentially hydroxymethylated cytosines between fast- and slow-growing fish were enriched within gene bodies (79%), indicating a pertinent role in transcriptional regulation. Moreover, they were found in genes involved in biological processes related to skeletal system and muscle structure development, and there was a positive association between somatic growth and 5hmC levels in genes coding for growth factors, kinases and receptors linked to myogenesis. Single nucleotide polymorphism analysis revealed no genetic differentiation between fast- and slow-growing fish. In addition to unveiling a new link between DNA hydroxymethylation and epigenetic regulation of growth in fish during the initial stages of domestication, this study suggests that epimarkers may be applied in selective breeding programmes for superior phenotypes.

This study aimed to expand knowledge on lumpfish stress physiology by investigating the effects of acute stress on primary (i.e., cortisol) and secondary (e.g., metabolites) stress responses, as well as oxidative stress biomarkers, from stress exposure to a recovery phase. The results showed that the lumpfish physiological response to 1 min air exposure is mild, in line with recent studies, and comparable to that described for white sturgeons. Cortisol seems to be the most reliable acute stress biomarker in lumpfish, with a significant increase in plasma 30 min after stress exposure, returning to resting levels 2 h after exposure. In contrast, glucose and lactate were not significantly altered by short-term air exposure. Effects on hepatic energy mobilisation were also detected following the acute stress. This study showed that acute 1 min air exposure seems tolerable, allowing a swift recovery. However, more studies on the impacts of air exposure and repeated acute stressors on lumpfish stress and immune responses are required to develop industry standards for lumpfish health and welfare monitoring.

Dyslipidemia is often associated with unhealthy dietary habits, and many mammalian studies have explored the mode of action of certain bioactive compounds such as β-glucans and n-3 PUFAs to understand their potential to normalize the lipid metabolism. There are only a few investigations that adopted omic approaches to unveil their combined effect on hypercholesterolemia. Zebrafish (Danio rerio) was used as a model organism to reveal the efficacy of Schizochytrium oil and β-glucans (from Euglena gracilis and Phaeodactylum tricornutum) against cholesterol-rich diet induced dyslipidemia. One of the folowing four diets was fed to a particular group of fish: a control high-cholesterol diet, a Schizochytrium oil diet or one of the two diets containing the oil and β-glucan. The plasma HDL, expression of hepatic genes linked to, among others, ferric ion binding and plasma phosphatidylcholines were higher and plasma cholesterol esters and triacylglycerols were lower in the microbial oil-fed fish compared to the fish fed high cholesterol diet. While the fish fed a mix of microbial oil and Euglena β-glucan had lower plasma triacylglycerols and expression of hepatic genes linked to PPAR signaling pathway and enriched biosynthesis of plasma unsaturated fatty acids, the fish fed microbial oil-Phaeodactylum β-glucan combination had lower abundance of triacylglycerols rich in saturated and mono-unsaturated fatty acids and cholesterol esters in the plasma., Competing Interests: Declaration of competing interest Author JD was employed by company SPAROS Lda. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Host-associated microbiota can influence host phenotypic variation, fitness and potential to adapt to local environmental conditions. In turn, both host evolutionary history and the abiotic and biotic environment can influence the diversity and composition of microbiota. Yet, to what extent environmental and host-specific factors drive microbial diversity remains largely unknown, limiting our understanding of host-microbiome interactions in natural populations. Here, we compared the intestinal microbiota between two phylogenetically related fishes, the three-spined stickleback ( Gasterosteus aculeatus ) and the nine-spined stickleback ( Pungitius pungitius ) in a common landscape. Using amplicon sequencing of the V3-V4 region of the bacterial 16S rRNA gene, we characterised the α and β diversity of the microbial communities in these two fish species from both brackish water and freshwater habitats. Across eight locations, α diversity was higher in the nine-spined stickleback, suggesting a broader niche use in this host species. Habitat was a strong determinant of β diversity in both host species, while host species only explained a small fraction of the variation in gut microbial composition. Strong habitat-specific effects overruled effects of geographic distance and historical freshwater colonisation, suggesting that the gut microbiome correlates primarily with local environmental conditions. Interestingly, the effect of habitat divergence on gut microbial communities was stronger in three-spined stickleback than in nine-spined stickleback, possibly mirroring the stronger level of adaptive divergence in this host species. Overall, our results show that microbial communities reflect habitat divergence rather than colonisation history or dispersal limitation of host species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shankregowda, Siriyappagouder, Kuizenga, Bal, Abdelhafiz, Eizaguirre, Fernandes, Kiron and Raeymaekers.)

A Western diet elevates the circulating lipoprotein and triglyceride levels which are the major risk factors in cardiovascular disease (CVD) development. Consumption of long-chain omega-3 fatty acids can stall the disease progression. Although these fatty acids can significantly impact the intestine under a hypercholesterolemic condition, the associated changes have not been studied in detail. Therefore, we investigated the alterations in the intestinal transcriptome along with the deviations in the plasma lipids and liver histomorphology of zebrafish offered DHA- and EPA-rich oil. Fish were allocated to 4 dietary treatments: a control group, a high cholesterol group and microbial oil groups with low (3.3%) and high (6.6%) inclusion levels. We quantified the total cholesterol, lipoprotein and triglyceride levels in the plasma. In addition, we assessed the liver histology, intestinal transcriptome and plasma lipidomic profiles of the study groups. The results suggested that higher levels of dietary microbial oil could control the CVD risk factor indices in zebrafish plasma. Furthermore, microbial oil-fed fish had fewer liver vacuoles and higher mRNA levels of genes involved in β-oxidation and HDL maturation. Analyses of the intestine transcriptome revealed that microbial oil supplementation could influence the expression of genes altered by a hypercholesterolemic diet. The plasma lipidomic profiles revealed that the higher level of microbial oil tested could elevate the long-chain poly-unsaturated fatty acid content of triglyceride species and lower the concentration of several lysophosphatidylcholine and diacylglycerol molecules. Our study provides insights into the effectiveness of microbial oil against dyslipidemia in zebrafish., Competing Interests: JD was employed by SPAROS Lda. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor SM declared a past collaboration with the author VK., (Copyright © 2023 Gora, Rehman, Dias, Fernandes, Olsvik, Sørensen and Kiron.)

Alginate oligosaccharides (AOS) are natural bioactive compounds with anti-inflammatory properties. We performed a feeding trial employing a zebrafish ( Danio rerio ) model of soybean-induced intestinal inflammation. Five groups of fish were fed different diets: a control (CT) diet, a soybean meal (SBM) diet, a soybean meal+β-glucan (BG) diet and 2 soybean meal+AOS diets (alginate products differing in the content of low molecular weight fractions - AL, with 31% < 3kDa and AH, with 3% < 3kDa). We analyzed the intestinal transcriptomic and plasma metabolomic profiles of the study groups. In addition, we assessed the expression of inflammatory marker genes and histological alterations in the intestine. Dietary algal β-(1, 3)-glucan and AOS were able to bring the expression of certain inflammatory genes altered by dietary SBM to a level similar to that in the control group. Intestinal transcriptomic analysis indicated that dietary SBM changed the expression of genes linked to inflammation, endoplasmic reticulum, reproduction and cell motility. The AL diet suppressed the expression of genes related to complement activation, inflammatory and humoral response, which can likely have an inflammation alleviation effect. On the other hand, the AH diet reduced the expression of genes, causing an enrichment of negative regulation of immune system process. The BG diet suppressed several immune genes linked to the endopeptidase activity and proteolysis. The plasma metabolomic profile further revealed that dietary SBM can alter inflammation-linked metabolites such as itaconic acid, taurochenodeoxycholic acid and enriched the arginine biosynthesis pathway. The diet AL helped in elevating one of the short chain fatty acids, namely 2-hydroxybutyric acid while the BG diet increased the abundance of a vitamin, pantothenic acid. Histological evaluation revealed the advantage of the AL diet: it increased the goblet cell number and length of villi of the intestinal mucosa. Overall, our results indicate that dietary AOS with an appropriate amount of < 3kDa can stall the inflammatory responses in zebrafish., Competing Interests: Author JD was employed by company SPAROS Lda., RP by CEVA and KM by Kemin Aquascience. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rehman, Gora, Abdelhafiz, Dias, Pierre, Meynen, Fernandes, Sørensen, Brugman and Kiron.)

Background: Circular RNAs (circRNAs) are covalently closed-loop RNAs with critical regulatory roles in cells. Tens of thousands of circRNAs have been unveiled due to the recent advances in high throughput RNA sequencing technologies and bioinformatic tools development. At the same time, polymerase chain reaction (PCR) cross-validation for circRNAs predicted by bioinformatic tools remains an essential part of any circRNA study before publication., Results: Here, we present the CircPrime web-based platform, providing a user-friendly solution for DNA primer design and thermocycling conditions for circRNA identification with routine PCR methods., Conclusions: User-friendly CircPrime web platform ( http://circprime.elgene.net/ ) works with outputs of the most popular bioinformatic predictors of circRNAs to design specific circular RNA primers. CircPrime works with circRNA coordinates and any reference genome from the National Center for Biotechnology Information database)., (© 2023. The Author(s).)

Muscle growth in teleosts is a complex biological process orchestrated by numerous protein-coding genes and non-coding RNAs. A few recent studies suggest that circRNAs are involved in teleost myogenesis, but the molecular networks involved remain poorly understood. In this study, an integrative omics approach was used to determine myogenic circRNAs in Nile tilapia by quantifying and comparing the expression profile of mRNAs, miRNAs, and circRNAs in fast muscle from full-sib fish with distinct growth rates. There were 1947 mRNAs, 9 miRNAs, and 4 circRNAs differentially expressed between fast- and slow-growing individuals. These miRNAs can regulate myogenic genes and have binding sites for the novel circRNA circMef2c. Our data indicate that circMef2c may interact with three miRNAs and 65 differentially expressed mRNAs to form multiple competing endogenous RNA networks that regulate growth, thus providing novel insights into the role of circRNAs in the regulation of muscle growth in teleosts., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The complete mitogenome of the Atlantic spiny lumpsucker ( Eumicrotremus spinosus) was generated using the PacBio Sequel II HiFi sequencing platform. The mitogenome assembly has a length of 19,281 bp and contains 13 protein-coding sequences, 22 tRNA genes, 2 rRNA genes, one control region containing the D-loop (2383 bp) and a duplicate control region (1133 bp) Phylogenetic analysis using maximum likelihood revealed that E. spinosus is closely related to the Siberian lumpsucker ( E. asperrimus ). The mitogenome of the spiny lumpsucker will be useful in population genomics and systematic studies of Cyclopteridae, Liparidae, and Cottidae., Competing Interests: No conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The mechanisms underlying the fast genome evolution that occurs during animal domestication are poorly understood. Here, we present a genome-wide epigenetic dataset that quantifies DNA hydroxymethylation at single nucleotide resolution among full-sib Nile tilapia (Oreochromis niloticus) with distinct growth performance. In total, we obtained 355 million, 75 bp reads from 5 large- and 5 small-sized fish on an Illumina NextSeq500 platform. We identified several growth-related genes to be differentially hydroxymethylated, especially within gene bodies and promoters. Previously, we proposed that DNA hydroxymethylation greatly affects the earliest responses to adaptation and potentially drives genome evolution through its targeted enrichment and elevated nucleotide transversion rates. This dataset can be analysed in various contexts (e.g., epigenetics, evolution and growth) and compared to other epigenomic datasets in the future, namely DNA methylation and histone modifications. With forthcoming advancements in genome research, this hydroxymethylation dataset will also contribute to better understand the epigenetic regulation of key genomic features, such as cis-regulatory and transposable elements., (© 2023. The Author(s).)

Acetylcholine (Ach) represents the old neurotransmitter in central and peripheral nervous system. Its muscarinic and nicotinic receptors (mAChRs and nAChRs) constitute an independent cholinergic system that is found in immune cells and play a key role in the regulation of the immune function and cytokine production. Gas exchanging surfaces of the gills and air-breathing organs (ABOs) of the sharptooth catfish Clarias gariepinus were investigated using ultrastructural and confocal immunofluorescence techniques. This study was predominantly focused on the structure of the immune cell types, the expression of their neurotransmitters, including the antimicrobial peptide piscidin 1, and the functional significance of respiratory gas exchange epithelia. A network of immune cells (monocytes, eosinophils, and mast cells) was observed in the gill and the ABO epithelia. Eosinophils containing 5-hydroxytryptamine (5-HT) immunoreactivity were seen in close association with mast cells expressing acetylcholine (Ach), 5-HT, neuronal nitric oxide synthase, and piscidin 1. A rich and dense cholinergic innervation dispersing across the islet capillaries of the gas exchange barrier and the localization of Ach in the squamous pavement cells covering the capillaries were evidenced byVAChT antibodies. We report for the first time that piscidin 1 (Pis 1)-positive mast cells interact with Pis 1-positive nerves found in the epithelia of the respiratory organs. Pis 1 immunoreactivity was also observed in the covering respiratory epithelium of the ABOs and associated with a role in local mucosal immune defense. The above results anticipate future studies on the neuro-immune interactions at mucosal barrier surfaces, like the gill and the skin of fish, areas densely populated by different immune cells and sensory nerves that constantly sense and adapt to tissue-specific environmental challenges., (© 2022 American Association for Anatomy.)

Significant palaeoecological and paleoclimatic changes that took place during Late Pleistocene-Early Holocene transition are considered important factors that led to megafauna extinctions. Unlike many other species, the brown bear ( Ursus arctos ) has survived this geological time. Despite the fact that several mitochondrial DNA clades of brown bears became extinct at the end of the Pleistocene, this species is still widely distributed in Northeast Eurasia. Here, using the ancient DNA analysis of a brown bear individual that inhabited Northeast Asia in the Middle Holocene (3460 ± 40 years BP) and comparative phylogenetic analysis, we show a significant mitochondrial DNA similarity of the studied specimen with modern brown bears inhabiting Yakutia and Chukotka. In this study, we clearly demonstrate the maternal philopatry of the Northeastern Eurasian U. arctos population during the several thousand years of the Holocene.

Soybean meal evokes diet-induced intestinal inflammation in certain fishes. Although the molecular aspects of soybean-induced intestinal inflammation in zebrafish are known, the impact of the inflammatory diet on fish behavior remain largely underexplored. We fed zebrafish larvae with three diets - control, soybean meal and soybean meal with β-glucan to gain deeper insight into the behavioral changes associated with the soybean meal-induced inflammation model. We assessed the effect of the diets on the locomotor behavior, morphological development, oxygen consumption and larval transcriptome. Our study revealed that dietary soybean meal can reduce the locomotor activity, induce developmental defects and increase the oxygen demand in zebrafish larvae. Transcriptomic analysis pointed to the suppression of genes linked to visual perception, organ development, phototransduction pathway and activation of genes linked to the steroid biosynthesis pathway. On the contrary, β-glucan, an anti-inflammatory feed additive, counteracted the behavioral and phenotypic changes linked to dietary soybean. Although we did not identify any differentially expressed genes from the soybean meal alone fed group vs soybean meal + β-glucan-fed group comparison, the unique genes from the comparisons of the two groups with the control likely indicate reduction in inflammatory cytokine signaling, inhibition of proteolysis and induction of epigenetic modifications by the dietary glucan. Furthermore, we found that feeding an inflammatory diet at the larval stage can lead to long-lasting developmental defects. In conclusion, our study reveals the extra-intestinal manifestations associated with soybean meal-induced inflammation model., Competing Interests: Author JD was employed by company SPAROS Lda. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rehman, Gora, Varshney, Dias, Olsvik, Fernandes, Brugman and Kiron.)

Lampriformes are circumglobally distributed and contain several families of strictly marine bony fishes that have a peculiar morphology. Lampriformes systematics is affected by limitations in biometric, meristic, and molecular data; for this reason, it underwent several rearrangements in the past. This review aimed to describe the biological and ecological characteristics of the order Lampriformes, summarizing the current taxonomy of the group. The main aim was to clarify what is known about the distribution of the order Lampriformes in the Mediterranean Sea, collecting all the scarce and fragmented reports and notes on their occurrence. Knowledge scarcity is due to their solitary nature, in addition to their low to absent economic value. Despite this, the order Lampriformes represents a taxon of high biological and ecological importance. The high depth range of distribution characterizes their lifestyle. In the Mediterranean Sea, four families are present-Lampridae, Lophotidae, Regalecidae, and Trachipteridae-with the following species respectively, Lampris guttatus (Brünnich, 1788), Lophotus lacepede (Giorna, 1809), Regalecus glesne (Ascanius, 1772), Trachipterus arcticus (Brünnich, 1788), T. trachypterus (Gmelin, 1789), and Zu cristatus (Bonelli, 1819). Data deficiencies affect information on this taxon; the present review, which collected all the reports of the Mediterranean Sea, creates a baseline for depicting the biogeography of these rare and important species.

Morphology and feature selection are key approaches to address several issues in fisheries science and stock management, such as the hypothesis of admixture of Caspian common carp ( Cyprinus carpio ) and farmed carp stocks in Iran. The present study was performed to investigate the population classification of common carp in the southern Caspian basin using data mining algorithms to find the most important characteristic(s) differing between Iranian and farmed common carp. A total of 74 individuals were collected from three locations within the southern Caspian basin and from one farm between November 2015 and April 2016. A dataset of 26 traditional morphometric (TMM) attributes and a dataset of 14 geometric landmark points were constructed and then subjected to various machine learning methods. In general, the machine learning methods had a higher prediction rate with TMM datasets. The highest decision tree accuracy of 77% was obtained by rule and decision tree parallel algorithms, and "head height on eye area" was selected as the best marker to distinguish between wild and farmed common carp. Various machine learning algorithms were evaluated, and we found that the linear discriminant was the best method, with 81.1% accuracy. The results obtained from this novel approach indicate that Darwin's domestication syndrome is observed in common carp. Moreover, they pave the way for automated detection of farmed fish, which will be most beneficial to detect escapees and improve restocking programs.

During colonial times, Nile tilapia Oreochromis niloticus (Linnaeus, 1758) was introduced into non-native parts of the Congo Basin (Democratic Republic of the Congo, DRC) for the first time. Currently, it is the most farmed cichlid in the DRC, and is present throughout the Congo Basin. Although Nile tilapia has been reported as an invasive species, documentation of historical introductions into this basin and its consequences are scant. Here, we study the genetic consequences of these introductions by genotyping 213 Nile tilapia from native and introduced regions, focusing on the Congo Basin. Additionally, 48 specimens from 16 other tilapia species were included to test for hybridization. Using RAD sequencing (27,611 single nucleotide polymorphisms), we discovered genetic admixture with other tilapia species in several morphologically identified Nile tilapia from the Congo Basin, reflecting their ability to interbreed and the potential threat they pose to the genetic integrity of native tilapias. Nile tilapia populations from the Upper Congo and those from the Middle-Lower Congo are strongly differentiated. The former show genetic similarity to Nile tilapia from the White Nile, while specimens from the Benue Basin and Lake Kariba are similar to Nile tilapia from the Middle-Lower Congo, suggesting independent introductions using different sources. We conclude that the presence of Nile tilapia in the Congo Basin results from independent introductions, reflecting the dynamic aquaculture history, and that their introduction probably leads to genetic interactions with native tilapias, which could lower their fitness. We therefore urge avoiding further introductions of Nile tilapia in non-native regions and to use native tilapias in future aquaculture efforts., (© 2022 John Wiley & Sons Ltd.)

Resident microbial communities that can support various host functions play a key role in their development and health. In fishes, microbial symbionts are vertically transferred from the parents to their progeny. Such transfer of microbes in mouthbrooder fish species has not been reported yet. Here, we employed Nile tilapia ( Oreochromis niloticus ) to investigate the vertical transmission of microbes across generations using a 16S rRNA amplicon sequencing approach, based on the presence of bacteria in different generations. Our analysis revealed that the core microbiome in the buccal cavity and posterior intestine of parents shapes the gut microbiome of the progeny across generations. We speculate that the route of this transmission is via the buccal cavity. The identified core microbiome bacteria, namely Nocardioides, Propionibacterium , and Sphingomonas have been reported to play an essential role in the health and development of offspring. These core microbiome members could have specific functions in fish, similar to mammals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abdelhafiz, Fernandes, Donati, Pindo and Kiron.)

A moderate surplus of the one carbon (1C) nutrients methionine, folic acid, vitamin B 6 and B 12 above dietary recommendations for Atlantic salmon has shown to improve growth and reduce hepatosomatic index in the on-growing saltwater period when fed throughout smoltification. Metabolic properties and molecular mechanisms determining the improved growth are unexplored. Here, we investigate metabolic and transcriptional signatures in skeletal muscle taken before and after smoltification to acquire deeper insight into pathways and possible nutrient–gene interactions. A control feed (Ctrl) or 1C nutrient surplus feed (1C+) were fed to Atlantic salmon 6 weeks prior to smoltification until 3 months after saltwater transfer. Both metabolic and gene expression signatures revealed significant 1C nutrient-dependent changes already at pre-smolt, but differences intensified when analysing post-smolt muscle. Transcriptional differences revealed lower expression of genes related to translation, growth and amino acid metabolisation in post-smolt muscle when fed additional 1C nutrients. The 1C+ group showed less free amino acid and putrescine levels, and higher methionine and glutathione amounts in muscle. For Ctrl muscle, the overall metabolic profile suggests a lower amino acid utilisation for protein synthesis, and increased methionine metabolisation in polyamine and redox homoeostasis, whereas transcription changes are indicative of compensatory growth regulation at local tissue level. These findings point to fine-tuned nutrient–gene interactions fundamental for improved growth capacity through better amino acid utilisation for protein accretion when salmon was fed additional 1C nutrients throughout smoltification. It also highlights potential nutritional programming strategies on improved post-smolt growth through 1C+ supplementation before and throughout smoltification.

Intestinal inflammation in Atlantic salmon was studied by profiling the intestine mucus proteome, employing iTRAQ and 2D LC-MS/MS approach. Two fish groups were fed soy saponin-containing (inflammation inducer) diets (SO and SP) and two control fish groups were fed diets devoid of soy saponin (CO and CP) for 36 days. The CP and SP diets contained a health additive. Inflammation characteristics in the intestine were milder in the SP-fed fish compared to the SO-fed fish. The SO group was characterised by alterations of many proteins. KEGG pathways such as phagosome and lipid binding were possibly affected in the SO group due to the higher abundant proteins like Integrin beta 2 precursor, Coronin 1A, Cathepsin S precursor, Vesicle-trafficking protein, and Neutrophil cytosol factors. On the other hand, the SP group had fewer altered proteins and inflammation characteristics; aminoacyl-tRNA biosynthesis and ribosome in the fish group were plausibly changed due to the higher abundance of many large and small subunit of ribosomes. Elevation of the abundance of ribosomal proteins, aminoacyl-tRNA ligases, and appropriate abundance of Glycogen phosphorylase and Glutamine synthetase could possibly alleviate intestinal inflammation. Data are available via ProteomeXchange with identifier PXD027922 and PXD029849. SIGNIFICANCE: Intestinal inflammation, caused by dietary factors, can be considered as a non-infectious disease. Hence, researchers are gathering clues to avert the associated health issues. The present study was conducted to infer the alterations in the intestine mucus proteome induced by a dietary health additive to counter intestinal inflammation in farmed Atlantic salmon. The reduction in the number of affected proteins and their alterations point to mechanisms evoked by the premix. Our knowledge on inflammation associated proteome in fish is limited and the present study not only highlights the changes, but also opens the possibility to avert the dysfunction of the organ through a dietary approach., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.

Background: DNA methylation has an important role in intergenerational inheritance. An increasing number of studies have reported evidence of germline inheritance of DNA methylation induced by nutritional signals in mammals. Vitamins and minerals as micronutrients contribute to growth performance in vertebrates, including Atlantic salmon (Salmo salar), and also have a role in epigenetics as environmental factors that alter DNA methylation status. It is important to understand whether micronutrients in the paternal diet can influence the offspring through alterations of DNA methylation signatures in male germ cells., Results: Here, we show the effect of micronutrient supplementation on DNA methylation profiles in the male gonad through a whole life cycle feeding trial of Atlantic salmon fed three graded levels of micronutrient components. Our results strongly indicate that micronutrient supplementation affects the DNA methylation status of genes associated with cell signalling, synaptic signalling, and embryonic development. In particular, it substantially affects DNA methylation status in the promoter region of a glutamate receptor gene, glutamate receptor ionotropic, NMDA 3A-like (grin3a-like), when the fish are fed both medium and high doses of micronutrients. Furthermore, two transcription factors, histone deacetylase 2 (hdac2) and a zinc finger protein, bind to the hyper-methylated site in the grin3a-like promoter. An estimated function of hdac2 together with a zinc finger indicates that grin3a-like has a potential role in intergenerational epigenetic inheritance and the regulation of embryonic development affected by paternal diet., Conclusions: The present study demonstrates alterations of gene expression patterns and DNA methylation signatures in the male gonad when Atlantic salmon are fed different levels of micronutrients. Alterations of gene expression patterns are of great interest because the gonads are supposed to have limited metabolic activities compared to other organs, whereas alterations of DNA methylation signatures are of great importance in the field of nutritional epigenetics because the signatures affected by nutrition could be transferred to the next generation. We provide extensive data resources for future work in the context of potential intergenerational inheritance through the male germline., (© 2022. The Author(s).)

Consumption of lipid-rich foods can increase the blood cholesterol content. β-glucans have hypocholesterolemic effect. However, subtle changes in their molecular branching can influence bioactivity. Therefore, a comparative investigation of the cholesterol-lowering potential of two β-glucans with different branching patterns and a cholesterol-lowering drug, namely simvastatin was undertaken employing the zebrafish ( Danio rerio ) model of diet-induced hypercholesterolemia. Fish were allocated to 5 dietary treatments; a control group, a high cholesterol group, two β-glucan groups, and a simvastatin group. We investigated plasma total cholesterol, LDL and HDL cholesterol levels, histological changes in the tissues, and explored intestinal transcriptomic changes induced by the experimental diets. Dietary cholesterol likely caused the suppression of endogenous cholesterol biosynthesis, induced dysfunction of endoplasmic reticulum and mitochondria, and altered the histomorphology of the intestine. The two β-glucans and simvastatin significantly abated the rise in plasma cholesterol levels and restored the expression of specific genes to alleviate the endoplasmic reticulum-related effects induced by the dietary cholesterol. Furthermore, the distinct patterns of transcriptomic changes in the intestine elicited by the oat and microalga β-glucans impacted processes such as fatty acid metabolism, protein catabolic processes, and nuclear division. Oat and microalgal β-glucans also altered the pattern of lipid deposition in the liver. Our study provides insights into the effectiveness of different β-glucans to alleviate dysfunctions in lipid metabolism caused by dietary cholesterol., Competing Interests: JD was employed by company SPAROS Lda. MC was employed by company MadeBiotech and NatureXtracts S.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gora, Rehman, Kiron, Dias, Fernandes, Olsvik, Siriyappagouder, Vatsos, Schmid-Staiger, Frick and Cardoso.)

The intestine has many types of cells that are present mostly in the epithelium and lamina propria. The importance of the intestinal cells for the mammalian mucosal immune system is well-established. However, there is no in-depth information about many of the intestinal cells in teleosts. In our previous study, we reported that adherent intestinal cells (AIC) predominantly express macrophage-related genes. To gather further evidence that AIC include macrophage-like cells, we compared their phagocytic activity and morphology with those of adherent head kidney cells (AKC), previously characterized as macrophage-like cells. We also compared equally abundant as well as differentially expressed mRNAs and miRNAs between AIC and AKC. AIC had lower phagocytic activity and were larger and more circular than macrophage-like AKC. RNA-Seq data revealed that there were 18309 mRNAs, with 59 miRNAs that were equally abundant between AIC and AKC. Integrative analysis of the mRNA and miRNA transcriptomes revealed macrophage heterogeneity in both AIC and AKC. In addition, analysis of AIC and AKC transcriptomes revealed functional characteristics of mucosal and systemic macrophages. Five pairs with significant negative correlations between miRNA and mRNAs were linked to macrophages and epithelial cells and their interaction could be pointing to macrophage activation and differentiation. The potential macrophage markers suggested in this study should be investigated under different immune conditions to understand the exact macrophage phenotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Park, Zhang, Fernandes, Wiegertjes and Kiron.)

Fish are widely exposed to higher microbial loads compared to land and air animals. It is known that the microbiome plays an essential role in the health and development of the host. The oral microbiome is vital in females of different organisms, including the maternal mouthbrooding species such as Nile tilapia ( Oreochromis niloticus ). The present study reports for the first time the microbial composition in the buccal cavity of female and male Nile tilapia reared in a recirculating aquaculture system. Mucus samples were collected from the buccal cavity of 58 adult fish (∼1 kg), and 16S rRNA gene amplicon sequencing was used to profile the microbial communities in females and males. The analysis revealed that opportunistic pathogens such as Streptococcus sp. were less abundant in the female buccal cavity. The power play of certain bacteria such as Acinetobacter , Acidobacteria (GP4 and GP6), and Saccharibacteria that have known metabolic advantages was evident in females compared to males. Association networks inferred from relative abundances showed few microbe-microbe interactions of opportunistic pathogens in female fish. The findings of opportunistic bacteria and their interactions with other microbes will be valuable for improving Nile tilapia rearing practices. The presence of bacteria with specific functions in the buccal cavity of female fish points to their ability to create a protective microbial ecosystem for the offspring., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abdelhafiz, Fernandes, Stefani, Albanese, Donati and Kiron.)

Micronutrients (vitamins and minerals) have been less well studied compared to macronutrients (fats, proteins, and carbohydrates) although they play important roles in growth, metabolism, and maintenance of tissues. Hence, there is growing interest to understand the influence of micronutrients across various aspects in nutritional research. In the last two decades, aquaculture feeds have been shifted to containing more plant-based materials to meet the increasing demand and maintain the sustainability in the industry. A recent whole life cycle feeding trial of Atlantic salmon ( Salmo salar ) with graded levels of micronutrient packages has concluded that the levels of several B-vitamins and microminerals need to be increased from the current recommendation levels for optimal growth and fish welfare when plant-based diets are used. Here, we show the effect of micronutrient supplementation on hepatic transcriptional and epigenetic regulation in a dose dependent manner. . Specifically, our aim is to reveal the mechanisms of altered cell metabolism, which results in improved growth performance by micronutrient surpluses, at gene expression and DNA methylation levels. Our results strongly indicate that micronutrient supplementation suppresses gene expression in lipid metabolism in a dose-dependent manner and broadly affects DNA methylation in cell-adhesion and cell-signalling. In particular, it increases DNA methylation levels on the acetyl-CoA carboxylase alpha promoter in a concentration-dependent manner, which further suggests that acetyl-CoA carboxylase alpha is an upstream epigenetic regulator controlling its downstream lipid biosynthesis activities. This study demonstrates a comprehensive analysis to reveal an important role of micronutrients in lipid metabolism through epigenetic control of gene expression.

Anti-nutritional factors in dietary components can have a negative impact on the intestinal barrier. Here, we present soya bean-induced changes in the intestine of juvenile zebrafish and the effect of yeast β-glucan through a transcriptomic approach. The inclusion of soya bean meal affected the expression of several intestinal barrier function-related genes like arl4ca, rab25b, rhoub, muc5ac, muc5d, clcn2c and cltb in zebrafish. Several metabolic genes like cyp2x10.2, cyp2aa2, aldh3a2b, crata, elovl4, elovl6, slc51a, gpat2 and ATP-dependent peptidase activity (lonrf, clpxb) were altered in the intestinal tissue. The expression of immune-related genes like nlrc3, nlrp12, gimap8, prdm1 and tph1a, and genes related to cell cycle, DNA damage and DNA repair (e.g. spo11, rad21l1, nabp1b, spata22, tdrd9) were also affected in the soya bean fed group. Furthermore, our study suggests the plausible effect of yeast β-glucan through the modulation of several genes that regulate immune responses and barrier integrity. Our findings indicate a subdued inflammation in juvenile zebrafish fed soya bean meal and the efficacy of β-glucan to counter these subtle inflammatory responses., (© 2021 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Heteropneustes fossilis is an air-breathing teleost inhabiting environments with very poor O 2 conditions, and so it has evolved to cope with hypoxia. In the gills and respiratory air-sac, the sites for O 2 sensing and the response to hypoxia rely on the expression of acetylcholine (Ach) acting via its nicotinic receptor (nAChR). This study examined the expression patterns of neuronal markers and some compounds in the NECs of the gills and respiratory air sac having an immunomodulatory function in mammalian lungs. Mucous cells, epithelial cells and neuroepithelial cells (NECs) were immunopositive to a variety of both neuronal markers (VAChT, nAChR, GABA-B-R1 receptor, GAD679) and the antimicrobial peptide piscidin, an evolutionary conserved humoral component of the mucosal immune system in fish. We speculate that Ach release via nAChR from mucous cells may be modulated by GABA production in the NECs and it is required for the induction of mucus production in both normoxic and hypoxic conditions. The presence of piscidin in mucous cells may act in synergy with the autocrine/paracrine signals of Ach and GABA binding to GABA B R1B receptor that may play a local immunomodulatory function in the mucous epithelia of the gills and the respiratory air sac. The potential role of the NECs in the immunobiological behaviour of the gill/air-sac is at moment a matter of speculation. The extent to which the NECs as such may participate is elusive at this stage and waits investigation., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

In industrial animal production, breeding strategies are essential to produce offspring of better quality and vitality. It is also known that host microbiome has a bearing on its health. Here, we report for the first time the influence of crossbreeding strategy, inbreeding or outbreeding, on the buccal and intestinal bacterial communities in female Nile tilapia ( Oreochromis niloticus ). Crossbreeding was performed within a family and between different fish families to obtain the inbred and outbred study groups, respectively. The genetic relationship and structure analysis revealed significant genetic differentiation between the inbred and outbred groups. We also employed a 16S rRNA gene sequencing technique to understand the significant differences between the diversities of the bacterial communities of the inbred and outbred groups. The core microbiota composition in the mouth and the intestine was not affected by the crossbreeding strategy but their abundance varied between the two groups. Furthermore, opportunistic bacteria were abundant in the buccal cavity and intestine of the outbred group, whereas beneficial bacteria were abundant in the intestine of the inbred group. The present study indicates that crossbreeding can influence the abundance of beneficial bacteria, core microbiome and the inter-individual variation in the microbiome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abdelhafiz, Fernandes, Larger, Albanese, Donati, Jafari, Nedoluzhko and Kiron.)

DNA methylation is one of the main epigenetic mechanisms that regulate gene expression in a manner that depends on the genomic context and varies considerably across taxa. This DNA modification was first found in nuclear genomes of eukaryote several decades ago and it has also been described in mitochondrial DNA. It has recently been shown that mitochondrial DNA is extensively methylated in mammals and other vertebrates. Our current knowledge of mitochondrial DNA methylation in fish is very limited, especially in non-model teleosts. In this study, using whole-genome bisulfite sequencing, we determined methylation patterns within non-CpG (CH) and CpG (CG) contexts in the mitochondrial genome of Nile tilapia, a non-model teleost of high economic importance. Our results demonstrate the presence of mitochondrial DNA methylation in this species predominantly within a non-CpG context, similarly to mammals. We found a strand-specific distribution of methylation, in which highly methylated cytosines were located on the minus strand. The D-loop region had the highest mean methylation level among all mitochondrial loci. Our data provide new insights into the potential role of epigenetic mechanisms in regulating metabolic flexibility of mitochondria in fish, with implications in various biological processes, such as growth and development., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In vertebrates, the somatotropic axis comprising the pituitary gland, liver and muscle plays a major role in myogenesis. Its output in terms of muscle growth is highly affected by nutritional and environmental cues, and thus likely epigenetically regulated. Hydroxymethylation is emerging as a DNA modification that modulates gene expression but a holistic characterization of the hydroxymethylome of the somatotropic axis has not been investigated to date. Using reduced representation 5-hydroxymethylcytosine profiling we demonstrate tissue-specific localization of 5-hydroxymethylcytosines at single nucleotide resolution. Their abundance within gene bodies and promoters of several growth-related genes supports their pertinent role in gene regulation. We propose that cytosine hydroxymethylation may contribute to the phenotypic plasticity of growth through epigenetic regulation of the somatotropic axis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod ( Gadus morhua ), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.

Circular RNAs (circRNAs) are an emerging class of regulatory RNAs with a covalently closed-loop structure formed during pre-mRNA splicing. Recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of novel approaches to their identification and functional characterization. CircRNAs are stable, developmentally regulated, and show tissue- and cell-type-specific expression across different taxonomic groups. They play a crucial role in regulating various biological processes at post-transcriptional and translational levels. However, the involvement of circRNAs in fish immunity has only recently been recognized. There is also broad evidence in mammals that the timely expression of circRNAs in muscle plays an essential role in growth regulation but our understanding of their expression and function in teleosts is still very limited. Here, we discuss the available knowledge about circRNAs and their role in growth and immunity in vertebrates from a comparative perspective, with emphasis on cultured teleost fish. We expect that the interest in teleost circRNAs will increase substantially soon, and we propose that they may be used as biomarkers for selective breeding of farmed fish, thus contributing to the sustainability of the aquaculture sector.

Acetylcholine (Ach) is the main neurotransmitter in the neuronal cholinergic system and also works as a signaling molecule in non-neuronal cells and tissues. The diversity of signaling pathways mediated by Ach provides a basis for understanding the biology of the cholinergic epithelial cells and immune cells in the gill of the species studied. NECs in the gill were not found surprisingly, but specialized cells showing the morphological, histochemical and ultrastructural characteristics of eosinophils were located in the gill filaments and respiratory lamellae. Much remains unknown about the interaction between the nerves and eosinophils that modulate both the release of acetylcholine and its nicotinic and muscarinic receptors including the role of acetylcholine in the mechanisms of O 2 chemosensing. In this study we report for the first time the expression of Ach in the pavement cells of the gill lamellae in fish, the mast cells associated with eosinophils and nerve interaction for both immune cell types, in the gill of the extant butterfly fish Pantodon buchholzi. Multiple roles have been hypothesized for Ach and alpha nAChR in the gills. Among these there are the possible involvement of the pavement cells of the gill lamellae as O 2 chemosensitive cells, the interaction of Ach positive mast cells with eosinophils and interaction of eosinophils with nerve terminals. This could be related to the use of the vesicular acetylcholine transporter (VAChT) and the alpha 2 subunit of the acetylcholine nicotinic receptor (alpha 2 nAChR). These data demonstrate the presence of Ach multiple sites of neuronal and non-neuronal release and reception within the gill and its ancestral signaling that arose during the evolutionary history of this conservative fish species., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Triploid, sterile Atlantic salmon (Salmo salar) could make a contribution to the development of the farming industry, but uncertainties about the performance and welfare of triploids have limited their adoption by farmers. In this study, we compared the ontogeny of digestive tract morphology and enzyme activities (pepsin, trypsin, chymotrypsin, alkaline phosphatase and aminopeptidase) of diploid and triploid Atlantic salmon. Fish were fed diets based on fishmeal (STD) or a mix of fishmeal and hydrolysed fish proteins (HFM) whilst being reared at low temperature from start-feeding to completion of the parr-smolt transformation. Fish weights for each ploidy and feed combination were used to calculate thermal growth coefficients (TGCs) that spanned this developmental period, and the data were used to examine possible relationships between enzyme activities and growth. At the end of the experiment, faeces were collected and analyzed to determine the apparent digestibility coefficients (ADCs) of the dietary amino acids (AAs). Digestive tract histo-morphology did not differ substantially between ploidies and generally reflected organ maturation and functionality. There were no consistent differences in proteolytic enzyme activities resulting from the inclusion of HFM in the diet, nor was there improved digestibility and AA bioavailability of the HFM feed in either diploid or triploid fish. The triploid salmon had lower ADCs than diploids for most essential and non-essential AAs in both diets (STD and HFM), but without there being any indication of lower intestinal protease activity in triploid fish. When trypsin-to-chymotrypsin activity and trypsin and alkaline phosphatase (ALP) ratios (T:C and T:ALP, respectively) were considered in combination with growth data (TGC) low T:C and T:ALP values coincided with times of reduced fish growth, and vice versa, suggesting that T:C and T:ALP may be used to predict recent growth history and possible growth potential., Competing Interests: The authors have declared that no competing interests exist. The commercial affiliation of one author [VP] does not alter our adherence to PLOS ONE policies on sharing data and materials.

Intestinal inflammation in farmed fish is a non-infectious disease that deserves attention because it is a major issue linked to carnivorous fishes. The current norm is to formulate feeds based on plant-derived substances, and the ingredients that have antinutritional factors are known to cause intestinal inflammation in fishes such as Atlantic salmon. Hence, we studied inflammatory responses in the distal intestine of Atlantic salmon that received a feed rich in soybean derivatives, employing histology, transcriptomic and flow cytometry techniques. The fish fed on soy products had altered intestinal morphology as well as upregulated inflammation-associated genes and aberrated ion transport-linked genes. The enriched pathways for the upregulated genes were among others taurine and hypotaurine metabolism, drug metabolism-cytochrome P450 and steroid biosynthesis. The enriched gene ontology terms belonged to transmembrane transporter- and channel-activities. Furthermore, soybean products altered the immune cell counts; lymphocyte-like cell populations were significantly higher in the whole blood of fish fed soy products than those of control fish. Interestingly, the transcriptome of the head kidney did not reveal any differential gene expression, unlike the observations in the distal intestine. The present study demonstrated that soybean derivatives could evoke marked changes in intestinal transport mechanisms and metabolic pathways, and these responses are likely to have a significant impact on the intestine of Atlantic salmon. Hence, soybean-induced enteritis in Atlantic salmon is an ideal model to investigate the inflammatory responses at the cellular and molecular levels., Competing Interests: JD is employed by the company SPAROS Lda. Olhão, Portugal. VVT was employed by the company DSM Nutritional Products, Global Innovations, Kaiseraugst, Switzerland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from DSM Nutritional Products. The funder had the following involvement in the study: research design and feed formulation., (Copyright © 2020 Kiron, Park, Siriyappagouder, Dahle, Vasanth, Dias, Fernandes, Sørensen and Trichet.)

Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain-pituitary-gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis.

In the aquaculture industry, fish are stunned using a wide range of methods, but all of them trigger stress responses and affect the fish flesh quality. Chilled water is considered one of the most efficient methods, but even this is not a stress-free experience for the fish. Anesthetics included in the ice slurry or in water could decrease this stress and delay the loss of flesh quality. In this work, we analyze the effect of clove oil (CO) nanoencapsulated in β-cyclodextrins (β-CD) (CO + β-CD), incorporated in the stunning bath, on the stress response and the organoleptic attributes of fresh marine and freshwater fish from four economically important fish species: Atlantic salmon , European seabass , Nile tilapia , and Rainbow trout . CO + β-CD reduces the time required to induce anesthesia, independently of water salinity, habitat or water temperature. The plasmatic glucose and cortisol levels decreased in all four species, although the concentrations of CO varied between species. Moreover, plasmatic lactate level differed between the marine and freshwater fish. The use of CO + β-CD extended the shelf life of fish from all the species studied (by 3-7 days). In conclusion, using CO encapsulated in β-CD for anesthetizing fish can be regarded as an improved fish-stunning technique that reduces the anesthesia-induction time, decreases the stress response, and extends the shelf life of fresh fish.

With the expansion of the aquaculture industry in the last two decades, there has been a large increase in the use of plant ingredients in aquafeeds, which has created new challenges in fish growth, health and welfare. Fish muscle growth is an important trait that is strongly affected by diet, but our knowledge on the effect of plant protein-based diets on global gene expression in muscle is still scant. The present study evaluated nutrigenomic effects of the inclusion of proteins from pea, soy and wheat into aquafeeds, compared to a control diet with fishmeal as the main protein source using the zebrafish model by RNA-seq; these results were extended to an important aquaculture species by analyzing selected differentially expressed genes identified in the zebrafish model on on-growing Atlantic salmon fed with equivalent plant protein-based diets. Expression of selected Atlantic salmon paralogues of the zebrafish homologs was analyzed using paralogue-specific qPCR assays. Global gene expression changes in muscle of zebrafish fed with plant-based diets were moderate, with the highest changes observed in the soy diet-fed fish, and no change for the pea diet-fed fish compared to the control diet. Among the differentially expressed genes were mylpfb , hsp90aa1.1 , col2a1a , and odc1 , which are important in regulating muscle growth, maintaining muscle structure and function, and muscle tissue homeostasis. Furthermore, those genes and their paralogues were differentially expressed in Atlantic salmon fed with the equivalent percentage of soy or wheat protein containing diets. Some of these genes were similarly regulated in both species while others showed species-specific regulation. The present study expands our understanding on the molecular effects of plant ingredients in fish muscle. Ultimately, the knowledge gained would be of importance for the improved formulation of sustainable plant-based diets for the aquaculture industry., (Copyright © 2020 Dhanasiri, Johny, Xue, Berge, Bogevik, Rise, Fæste and Fernandes.)

Our knowledge of the intestinal immune system of fish is rather limited compared to mammals. Very little is known about the immune cells including the phagocytic cells in fish intestine. Hence, employing imaging flow cytometry and RNA sequencing, we studied adherent cells isolated from healthy Atlantic salmon. Phagocytic activity and selected gene expression of adherent cells from the distal intestine (adherent intestinal cells, or AIC) were compared with those from head kidney (adherent kidney cells, or AKC). Phagocytic activity of the two cell types was assessed based on the uptake of Escherichia coli BioParticles TM . AIC showed phagocytic ability but the phagocytes were of different morphology compared to AKC. Transcriptomic analysis revealed that AIC expressed genes associated with macrophages, T cells, and endothelial cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related genes. We believe that the adherent intestinal cells have phagocytic characteristics and high expression of genes commonly associated with macrophages. We envisage the possibilities for future studies on enriched populations of adherent intestinal cells., (Copyright © 2020 Park, Zhang, Wiegertjes, Fernandes and Kiron.)

Triploid Atlantic salmon (Salmo salar L.) is seen as one of the best solutions to solve key issues in the salmon farming industry, such as the impact of escapees on wild stocks and pre-harvest sexual maturation. However, the effects of triploidy on salmon smoltification are poorly understood at the molecular level, even though smoltification is a very sensitive period that has a major influence on survival rate and performance of farmed salmon. In this study, we have compared the liver transcriptomes of diploid and triploid Atlantic salmon at three ontogeny stages: fry, parr and smolt. In diploid fish, a total of 2,655 genes were differentially expressed between fry and parr, whereas 506 genes had significantly different transcript levels between parr and smolts. In triploids, 1,507 and 974 genes were differentially expressed between fry and parr, and between parr and smolts, respectively. Most of these genes were down-regulated and 34 genes were differentially expressed between ploidies at the same stage. In both ploidy groups, the top differentially expressed genes with ontogeny stage belonged to common functional categories that can be related to smoltification. Nucleotide and energy metabolism were significantly down-regulated in fry when compared to parr, while immune system processes were significantly down-regulated in parr when compared to smolts. The close resemblance of enriched biological processes and pathways between ploidy groups suggests that triploidy is regulated by genome dosage compensation in Atlantic salmon. Histological analysis revealed that areas of vacuolization (steatosis) were present only in fry and parr stages, in contrast to a compact cellular histology with glycogen granules after smoltification. There was no significant difference in vacuolization between ploidy groups at the fry stage but the liver of diploid parr had a 33.5% higher vacuolization area compared to their triploid counterparts. Taken together, our data provide novel insights into the changes that occur at the molecular and histological level in the liver of both diploid and triploid Atlantic salmon prior to and during smoltification.

Epigenetic modifications, such as DNA methylation, can be regulated by nutrition and dietary factors. There has been a large increase in the use of sustainable plant-based protein sources in fish feed due to limitations of fishmeal resources, which are needed to sustain a rapidly growing aquaculture industry. With this major transition from marine ingredients to plant-based diets, fish are abruptly introduced to changes in dietary composition and exposed to a variety of phytochemicals, some of which known to cause epigenetic changes in mammals. However, the effect of plant ingredients on the epigenome of fish is barely understood. In the present study, the nutriepigenomic effects of the addition of pea, soy, and wheat gluten protein concentrate to aquafeeds were investigated using zebrafish as a model. A genome-wide analysis of DNA methylation patterns was performed by reduced representation bisulphite sequencing to examine global epigenetic alterations in the mid intestine after a 42-day feeding trial. We found that inclusion of 30% of wheat gluten, pea and soy protein concentrate in the diet induced epigenetic changes in the mid intestine of zebrafish. A large number of genes and intergenic regions were differentially methylated with plant-based diets. The genes concerned were related to immunity, NF-κB system, ubiquitin-proteasome pathway, MAPK pathway, and the antioxidant defence system. Epigenetic regulation of several biological processes, including neurogenesis, cell adhesion, response to stress and immunity was also observed. Ultimately, the observed epigenetic changes may enable zebrafish to rapidly regulate inflammation and maintain intestinal homoeostasis when fed plant protein-based diets.