Epigenetic changes in pyloric caeca of Atlantic salmon fed diets containing increasing levels of lipids and choline.

An earlier study of ours investigating the effect of dietary lipid levels on the choline requirement of Atlantic salmon showed increasing severity of intestinal steatosis with increasing lipid levels. As choline is involved in epigenetic regulation by being the key methyl donor, pyloric caeca samples from the study were analysed for epigenetic effects of dietary lipid and choline levels. The diets varied in lipid levels between 16% and 28%, and choline levels between 1.9 and 2.3 g/kg. The diets were fed for 8 weeks to Atlantic salmon of 25 g of initial weight. Using reduced representation bisulfite sequencing (RRBS), this study revealed that increasing dietary lipid levels induced methylation differences in genes involved in membrane transport and signalling pathways, and in microRNAs important for the regulation of lipid homoeostasis. Increasing choline levels also affected genes involved in fatty acid biosynthesis and transport, lipolysis, and lipogenesis, as well as important immune genes. Our observations confirmed that choline is involved in epigenetic regulation in Atlantic salmon, as has been reported for higher vertebrates. This study showed the need for the inclusion of biomarkers of epigenetic processes in studies that must be conducted to define optimal choline levels in diets for Atlantic salmon.

Unmasking microsatellite deceptiveness and debunking hybridization with SNPs in four marine copepod species of Calanus.

Interspecific hybridization events are on the rise in natural systems due to climate change disrupting species barriers. Across taxa, microsatellites have long been the molecular markers of choice to identify admixed individuals. However, with the advent of high-throughput sequencing easing the generation of genome-wide datasets, incorrect reports of hybridization resulting from microsatellite technical artefacts have been uncovered in a growing number of taxa. In the marine zooplankton genus Calanus (Copepoda), whose species are used as climate change indicators, microsatellite markers have suggested hybridization between C. finmarchicus and C. glacialis, while other nuclear markers (InDels) never detected any admixed individuals, leaving the scientific community divided. Here, for the first time, we investigated the potential for hybridization among C. finmarchicus, C. glacialis, C. helgolandicus and C. hyperboreus using two large and independent SNP datasets. These were derived firstly from a protocol of target-capture applied to 179 individuals collected from 17 sites across the North Atlantic and Arctic Oceans, including sympatric areas, and second from published RNA sequences. All SNP-based analyses were congruent in showing that Calanus species are distinct and do not appear to hybridize. We then thoroughly re-assessed the microsatellites showing hybrids, with the support of published transcriptomes, and identified technical issues plaguing eight out of 10 microsatellites, including size homoplasy, paralogy, potential for null alleles and even two primer pairs targeting the same locus. Our study illustrates how deceptive microsatellites can be when applied to the investigation of hybridization.

Effects of dietary supplementation with prebiotics and Pediococcus acidilactici on gut health, transcriptome, microbiota, and metabolome in Atlantic salmon (Salmo salar L.) after seawater transfer.

BACKGROUND: Given the importance of gut microbiota for health, growth and performance of the host, the aquaculture industry has taken measures to develop functional fish feeds aiming at modulating gut microbiota and inducing the anticipated beneficial effects. However, present understanding of the impact of such functional feeds on the fish is limited. The study reported herein was conducted to gain knowledge on performance and gut health characteristics in post-smolt Atlantic salmon fed diets varying in content of functional ingredients. Three experimental diets, a diet containing fructo-oligosaccharides (FOS), a diet with a combination of FOS and Pediococcus acidilactici (BC) and a diet containing galacto-oligosaccharides (GOS) and BC, were used in a 10-weeks feeding trial. A commercial diet without functional ingredients was also included as a control/reference. Samples of blood plasma, mucosa and digesta were subjected to microbiota, transcriptome and metabolome profiling for evaluation of the diet effects. RESULTS: No significant growth differences were observed between fish fed the supplemented diets, but FOS-BC fed fish showed significantly faster growth than the control fed fish. The microbiota results showed that the BC was present in both the digesta, and the mucosa samples of fish fed the FOS-BC and GOS-BC diets. Digesta-associated microbiota was altered, while mucosa-associated microbiota was relatively unaffected by diet. Replacing FOS with GOS increased the level of metabolites linked to phospholipid, fatty acid, carnitine and sphingolipid metabolism. Variation in metabolite levels between the treatments closely correlated with genera mainly belonging to Firmicutes and Actinobacteria phyla. The transcriptome analyses indicated diet effects of exchanging FOS with GOS on immune functions, oxidative defense and stress responses. No significant diet effect was observed on intestinal inflammation in the pyloric caeca or in the distal intestine, or on lipid accumulation in the pyloric caeca. CONCLUSIONS: Dietary supplementation with BC induced moderate effects on the microbiota of the digesta, while the effects of replacing FOS with GOS were more marked and was observed also for nutrient metabolism. Our data indicates therefore that the quality of a prebiotic may be of great importance for the effects of a probiotic on gut microbiota, function, and health.

Effects of functional ingredients on gut inflammation in Atlantic salmon (Salmo salar L).

Functional feed ingredients are frequently used in feeds for Atlantic salmon, often claimed to improve immune functions in the intestine and reduce severity of gut inflammation. However, documentation of such effects is, in most cases, only indicative. In the present study effects of two packages of functional feed ingredients commonly used in salmon production, were evaluated employing two inflammation models. One model employed soybean meal (SBM) as inducer of a severe inflammation, the other a mixture of corn gluten and pea meal (CoPea) inducing mild inflammation. The first model was used to evaluate effects of two packages of functional ingredients: P1 containing butyrate and arginine, and P2 containing ß-glucan, butyrate, and nucleotides. In the second model only the P2 package was tested. A high marine diet was included in the study as a control (Contr). The six diets were fed to salmon (average weight of 177g) in saltwater tanks (57 fish per tank), in triplicate, for 69 days (754 ddg). Feed intake was recorded. The growth rate of the fish was high, highest for the Contr (TGC: 3.9), lowest for SBM fed fish (TGC: 3.4). Fish fed the SBM diet showed severe symptoms of inflammation in the distal intestine as indicated by histological, biochemical, molecular, and physiological biomarkers. The number of differently expressed genes (DEG) between the SBM and Contr fed fish was 849 and comprised genes indicating alteration in immune functions, cellular and oxidative stress, and nutrient digestion, and transport functions. Neither P1 nor P2 altered the histological and functional symptoms of inflammation in the SBM fed fish importantly. Inclusion of P1 altered expression of 81 genes, inclusion of P2 altered 121 genes. Fish fed the CoPea diet showed minor signs of inflammation. Supplementation with P2 did not change these signs. Regarding composition of the microbiota in digesta from the distal intestine, clear differences regarding beta-diversity and taxonomy between Contr, SBM, and CoPea fed fish were observed. In the mucosa the microbiota differences were less clear. The two packages of functional ingredients altered microbiota composition of fish fed the SBM and the CoPea diet towards that of fish fed the Contr diet.

Bile components affect the functions and transcriptome of the rainbow trout intestinal epithelial cell line RTgutGC.

The concomitant increase in cultivation of fish and decrease in supply of marine ingredients, have greatly increased the demand for new nutrient sources. This also regards so-called functional ingredients which may benefit health and welfare of the fish. In vitro cell line-based intestinal epithelial barrier models may serve as tools for narrowing down the broad range of ingredient options, to identify the most promising candidates before in vivo feeding trials are run. In vivo, differentiation of the various epithelial cells in the fish intestine, from the multipotent stem cells, takes place in the presence of a variety of substances from dietary and endogenous origin. Among these, bile salts have recently received attention as regulators of epithelial function in health and disease but have not, until now, been included in the medium when culturing fish gut epithelial cells in vitro. As bile salts are present at high levels in the chyme of the fish intestine, in particular in salmon and rainbow trout, mostly as taurocholate (>90%), their role for effects of diet ingredients on the in vitro gut cell model should be understood. With this study, we wanted to investigate whether inclusion of bile from rainbow trout or pure taurocholate in the culture media would modulate functions of the RTgutGC epithelial cells. Here, we demonstrated that the rainbow trout intestinal epithelial cell line RTgutGC responded significantly to the presence of bile components. Treatment with rainbow trout bile taken from the gall bladder (RTbile) or pure taurocholate (TC) at taurocholate concentrations of ≤0.5 mg/mL retained normal cell morphology, cell viability as in cell oxidation-reduction metabolic activity and membrane integrity, and barrier features, while high concentrations of bile salts (≥1 mg/mL) were cytotoxic to the cells. After long-term (4 days) bile treatment, transcriptome responses showed how bile salts play important roles in intestinal epithelial cell metabolism. qPCR data demonstrated that barrier function genes, brush border enzyme genes and immune genes were significantly affected. Although similar trends were seen, treatment with bile salt as a component of rainbow trout bile or pure taurocholate, induced somewhat different effects. In conclusion, this study clearly indicates that bile salts should be included in the cell medium when running in vitro studies of gut cell functions, not at least immune functions, preferably at the level of ∼0.5 mg/mL supplemented as pure taurocholate to ensure reproducibility.

Affordable aesthetic management for a patient from low income strata to improve social well-being: A case report.

Background: Aesthetic correction involves a holistic approach which considers the oral cavity as a whole by the norms of the treatment. Aesthetics enhance the facial profile and also the smile line of the patient which improves the quality of life of a patient enabling them to a better socioeconomic status. Smile correction of a patient paves the path for a systematic approach to the treatment and also imparts an improved level of confidence which leads to new opportunities both in the personal and professional field. AeEsthetic correction provides challenging aspects concerning patient satisfaction and functional demands. This is the most affordable treatment for patients' who are not financially sound in developing countries like India. The other treatment modalities such as Veneers or All-ceramic crowns are aesthetically pleasing but are quite expensive for people in the lower income strata. Due to this, direct composite treatment is very economical and it also improves the patient's overall confidence and it helps them to socialise well and carry out their daily routine activities without any hindrance and difficulty. Case Report: This case report deals with the esthetic correction of midline diastema and spacing in the maxillary and mandibular anterior region. Conclusion: Aesthetic correction involves meticulous treatment planning to revive the essence of lost beauty as well as improve the functional aspect for the patient. Conclusion: Aesthetic correction involves meticulous treatment planning to revive the essence of lost beauty as well as improve the functional aspect for the patient.

Plant-Based Diets Induce Transcriptomic Changes in Muscle of Zebrafish and Atlantic Salmon.

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.

The Seagrass Methylome Is Associated With Variation in Photosynthetic Performance Among Clonal Shoots.

Evolutionary theory predicts that clonal organisms are more susceptible to extinction than sexually reproducing organisms, due to low genetic variation and slow rates of evolution. In agreement, conservation management considers genetic variation as the ultimate measure of a population's ability to survive over time. However, clonal plants are among the oldest living organisms on our planet. Here, we test the hypothesis that clonal seagrass meadows display epigenetic variation that complements genetic variation as a source of phenotypic variation. In a clonal meadow of the seagrass Zostera marina, we characterized DNA methylation among 42 shoots. We also sequenced the whole genome of 10 shoots to correlate methylation patterns with photosynthetic performance under exposure to and recovery from 27°C, while controlling for somatic mutations. Here, we show for the first time that clonal seagrass shoots display DNA methylation variation that is independent from underlying genetic variation, and associated with variation in photosynthetic performance under experimental conditions. It remains unknown to what degree this association could be influenced by epigenetic responses to transplantation-related stress, given that the methylomes showed a strong shift under acclimation to laboratory conditions. The lack of untreated control samples in the heat stress experiment did not allow us to distinguish methylome shifts induced by acclimation from such induced by heat stress. Notwithstanding, the co-variation in DNA methylation and photosynthetic performance may be linked via gene expression because methylation patterns varied in functionally relevant genes involved in photosynthesis, and in the repair and prevention of heat-induced protein damage. While genotypic diversity has been shown to enhance stress resilience in seagrass meadows, we suggest that epigenetic variation plays a similar role in meadows dominated by a single genotype. Consequently, conservation management of clonal plants should consider epigenetic variation as indicator of resilience and stability.

Pseudozyma Priming Influences Expression of Genes Involved in Metabolic Pathways and Immunity in Zebrafish Larvae.

Fungi, particularly yeasts, are known essential components of the host microbiota but their functional relevance in development of immunity and physiological processes of fish remains to be elucidated. In this study, we used a transcriptomic approach and a germ-free (GF) fish model to determine the response of newly hatched zebrafish larvae after 24 h exposure to Pseudozyma sp. when compared to conventionally-raised (CR) larvae. We observed 59 differentially expressed genes in Pseudozyma-exposed GF zebrafish larvae compared to their naïve control siblings. Surprisingly, in CR larvae, there was not a clear transcriptome difference between Pseudozyma-exposed and control larvae. Differentially expressed genes in GF larvae were involved in host metabolic pathways, mainly peroxisome proliferator-activated receptors, steroid hormone biosynthesis, drug metabolism and bile acid biosynthesis. We also observed a significant change in the transcript levels of immune-related genes, namely complement component 3a, galectin 2b, ubiquitin specific peptidase 21, and aquaporins. Nevertheless, we did not observe any significant response at the cellular level, since there were no differences between neutrophil migration or proliferation between control and yeast-exposed GF larvae. Our findings reveal that exposure to Pseudozyma sp. may affect metabolic pathways and immune-related processes in germ-free zebrafish, suggesting that commensal yeast likely play a significant part in the early development of fish larvae.

Postovulatory maternal transcriptome in Atlantic salmon and its relation to developmental potential of embryos.

BACKGROUND: Early development of an oviparous organism is based on maternally stocked structural, nutritional and regulatory components. These components influence the future developmental potential of an embryo, which is referred to as egg quality. Until zygotic genome activation, translational activity in a fish early embryo is limited to parentally inherited transcripts only. In this study, we asked whether egg transcriptome is associated with egg quality in Atlantic salmon (Salmo salar), which is capable of storing ovulated eggs in its abdominal cavity for a long time before spawning. RESULTS: We analyzed messenger RNA (mRNA) and micro RNA (miRNA) transcriptomes throughout the post-ovulatory egg retention period in batches of eggs from two quality groups, good and poor, classified based on the future developmental performance. We identified 28,551 protein-coding genes and 125 microRNA families, with 200 mRNAs and 5 miRNAs showing differential abundance between egg quality groups and/or among postovulatory ages. Transcriptome dynamics during the egg retention period was different in the two egg quality groups. We identified only a single gene, hepcidin-1, as a potential marker for Atlantic salmon egg quality evaluation. CONCLUSION: The overlapping effect of post-ovulatory age on intrinsic egg developmental competence makes the quantification of egg quality difficult when based on transcripts abundance only.

Towards population genomics in non-model species with large genomes: a case study of the marine zooplankton Calanus finmarchicus.

Advances in next-generation sequencing technologies and the development of genome-reduced representation protocols have opened the way to genome-wide population studies in non-model species. However, species with large genomes remain challenging, hampering the development of genomic resources for a number of taxa including marine arthropods. Here, we developed a genome-reduced representation method for the ecologically important marine copepod Calanus finmarchicus (haploid genome size of 6.34 Gbp). We optimized a capture enrichment-based protocol based on 2656 single-copy genes, yielding a total of 154 087 high-quality SNPs in C. finmarchicus including 62 372 in common among the three locations tested. The set of capture probes was also successfully applied to the congeneric C. glacialis. Preliminary analyses of these markers revealed similar levels of genetic diversity between the two Calanus species, while populations of C. glacialis showed stronger genetic structure compared to C. finmarchicus. Using this powerful set of markers, we did not detect any evidence of hybridization between C. finmarchicus and C. glacialis. Finally, we propose a shortened version of our protocol, offering a promising solution for population genomics studies in non-model species with large genomes.

Genetics redraws pelagic biogeography of Calanus.

Planktonic copepods of the genus Calanus play a central role in North Atlantic/Arctic marine food webs. Here, using molecular markers, we redrew the distributional ranges of Calanus species inhabiting the North Atlantic and Arctic Oceans and revealed much wider and more broadly overlapping distributions than previously described. The Arctic shelf species, C. glacialis, dominated the zooplankton assemblage of many Norwegian fjords, where only C. finmarchicus has been reported previously. In these fjords, high occurrences of the Arctic species C. hyperboreus were also found. Molecular markers revealed that the most common method of species identification, prosome length, cannot reliably discriminate the species in Norwegian fjords. Differences in degree of genetic differentiation among fjord populations of the two species suggested that C. glacialis is a more permanent resident of the fjords than C. finmarchicus We found no evidence of hybridization between the species. Our results indicate a critical need for the wider use of molecular markers to reliably identify and discriminate these morphologically similar copepod species, which serve as important indicators of climate responses.

Liver transcriptome changes in zebrafish during acclimation to transport-associated stress.

Liver plays a key role during the stress acclimation, and liver transcriptome analysis of shipped zebrafish could reveal the molecular adjustments that occur in the organ. Transcriptional changes in liver were analyzed with a 44 K oligo array using total RNA from fish prior to transport and during a mock transport process--immediately after packing (0 h), at 48 and 72 h. Large numbers of genes related to a variety of biological processes and pathways were regulated, mainly during transport (at 48/72 h). Immediately after packing, transcripts of genes related to both gluconeogenesis and glycolysis were induced. During transport, induction of gluconeogenesis-linked genes and reduction of glycolysis-related genes may be supporting the increase in blood glucose levels. Inhibition of genes involved in fatty acid beta-oxidation may be pointing to the poor ability of fish to utilize energy from fatty acids, under transport conditions. Genes involved in some of the mechanisms that regulate body ammonia were also affected. Even though genes associated with certain transaminases were inhibited in liver, sustained glutamate deamination may have led to high ammonia accumulation in liver/body. Enhanced levels of gene transcripts in ubiquitination and MAPK signalling cascade and reduced levels of gene transcripts related to ROS generation via peroxisomal enzymes as well as xenobiotic metabolism may be signifying the importance of such cellular and tissue responses to maintain homeostasis. Furthermore, transcripts connected with stress and thyroid hormones were also regulated. Moreover, suppression of genes related to specific immune components may be denoting the deleterious impact of transport on fish health. Thus, this study has revealed the complex molecular adjustments that occur in zebrafish when they are transported.

Acclimation of zebrafish to transport stress.

Welfare of fish is commonly neglected when they are transported. This study examines the effect of a 72-h mock transport on certain aspects of the stress physiology of two groups of zebrafish-the first transported in water enriched with a nitrifying bacterial consortium and the second in water without the enrichment. Zebrafish were examined at different time points-before packing (BP), immediately after packing them in transport bags (AP), at the end of transport (AT), and 72 h thereafter (PT)-to assess the primary (cortisol) and secondary (glucose) stress responses. In addition, the relevant genes in hypothalamic-pituitary-interrenal (HPI) axis (crf in brain, mc2r, star, cyp11c1, and hsd11b2 in kidney), including that of mineralocorticoid receptor (mr in kidney), were studied. Procedures during packing caused an increase in whole body cortisol levels of both fish groups. Only in the fish transported without the bacterial consortium, an increase in the levels of whole body cortisol as well as blood glucose was observed at the end of the transport. At the same time point and in the same fish group, the transcripts of mr and hsd11b2 were enhanced, probably to cope with the stress and to maintain homeostasis. The mRNA levels of the other genes in the HPI stress axis (crf, mc2r, star, and cyp11c1) were not significantly altered. Zebrafish transported in water enriched with the bacterial consortium exhibited a speedier stress acclimation. Nevertheless, only through in-depth studies the beneficial effect of the consortium can be confirmed.

Glutamine synthetase activity and the expression of three glul paralogues in zebrafish during transport.

The enzyme glutamine synthetase (GS; glutamate-ammonia ligase, EC 6.3.1.2) plays an important role in the nitrogen metabolism of fish. In this study the GS activity and the corresponding genes were examined to understand how they are regulated in zebrafish in response to hyperammonemic stress during a 72 h simulated transport. Whole body ammonia levels, the activity of the enzyme GS and the mRNA expression of the splice variants of three paralogues of glul, glutamine synthetase gene (glula, glulb and glulc) were examined in brain, liver and kidney of zebrafish. Whole body ammonia reached significantly higher levels by 48 h, while brain showed higher levels as early as 24 h, compared to the values at the start of the transport. The GS activities in brain, liver and kidney were significantly higher at the end of 72 h transport than those at the start. However, only the expression of mRNA of glulb-002 and glulb-003 were significantly upregulated during the simulated transport. In silico analysis of the putative promoter regions of glul paralogues revealed glucocorticoid receptor binding sites. However, glucocorticoid response elements of glulb were not different. The up-regulation of GS enzyme activity and hitherto unreported mRNA expression of glul paralogues during zebrafish transport indicate a physiological response of fish to ammonia.

Changes in the intestinal microbiota of wild Atlantic cod Gadus morhua L. upon captive rearing.

The commensal microbiota plays an important role in the well-being of the host organism, and it would be worthwhile to know the tenacious communities among them. Therefore, a study was undertaken to examine the changes in constitution of the intestinal microbiota of wild fish consequential to captivity. At first, the composition of intestinal microorganisms of Atlantic cod caught from the coastal area off Bodø, Norway, was examined. Thereafter, the changes in the bacterial community of the captive fish after offering them artificial feed or subjecting them to starvation were studied. The microbiota from the intestinal contents and wall segments were analyzed quantitatively by spread plate technique and DAPI staining and qualitatively by denaturing gradient gel electrophoresis. The study revealed that the counts of intestinal microbes in wild-caught Atlantic cod were not affected by captive rearing for 6 weeks, either when fed or when starved. However, the diversity of intestinal bacterial community was reduced in response to artificial feeding, whereas the change was restricted upon starvation.