The generation of the first chromosome-level de novo genome assembly and the development and validation of a 50K SNP array for the St. John River aquaculture strain of North American Atlantic salmon.

Atlantic salmon (Salmo salar) in Northeastern US and Eastern Canada has high economic value for the sport fishing and aquaculture industries. Large differences exist between the genomes of Atlantic salmon of European origin and North American (N.A.) origin. Given the genetic and genomic differences between the 2 lineages, it is crucial to develop unique genomic resources for N.A. Atlantic salmon. Here, we describe the resources that we recently developed for genomic and genetic research in N.A. Atlantic salmon aquaculture. Firstly, a new single nucleotide polymorphism (SNP) database for N.A. Atlantic salmon consisting of 3.1 million putative SNPs was generated using data from whole-genome resequencing of 80 N.A. Atlantic salmon individuals. Secondly, a high-density 50K SNP array enriched for the genic regions of the genome and containing 3 sex determination and 61 putative continent of origin markers was developed and validated. Thirdly, a genetic map composed of 27 linkage groups with 36K SNP markers was generated from 2,512 individuals in 141 full-sib families. Finally, a chromosome-level de novo genome assembly from a male N.A. Atlantic salmon from the St. John River aquaculture strain was generated using PacBio long reads. Information from Hi-C proximity ligation sequences and Bionano optical mapping was used to concatenate the contigs into scaffolds. The assembly contains 1,755 scaffolds and only 1,253 gaps, with a total length of 2.83 Gb and N50 of 17.2 Mb. A BUSCO analysis detected 96.2% of the conserved Actinopterygii genes in the assembly, and the genetic linkage information was used to guide the formation of 27 chromosome sequences. Comparative analysis with the reference genome assembly of the European Atlantic salmon confirmed that the karyotype differences between the 2 lineages are caused by a fission in chromosome Ssa01 and 3 chromosome fusions including the p arm of chromosome Ssa01 with Ssa23, Ssa08 with Ssa29, and Ssa26 with Ssa28. The genomic resources we have generated for Atlantic salmon provide a crucial boost for genetic research and for management of farmed and wild populations in this highly valued species.

Functional feeds marginally alter immune expression and microbiota of Atlantic salmon (Salmo salar) gut, gill, and skin mucosa though evidence of tissue-specific signatures and host-microbe coadaptation remain.

BACKGROUND: Mucosal surfaces of fish provide cardinal defense against environmental pathogens and toxins, yet these external mucosae are also responsible for maintaining and regulating beneficial microbiota. To better our understanding of interactions between host, diet, and microbiota in finfish and how those interactions may vary across mucosal tissue, we used an integrative approach to characterize and compare immune biomarkers and microbiota across three mucosal tissues (skin, gill, and gut) in Atlantic salmon receiving a control diet or diets supplemented with mannan-oligosaccharides, coconut oil, or both. Dietary impacts on mucosal immunity were further evaluated by experimental ectoparasitic sea lice (Lepeophtheirus salmonis) challenge. RESULTS: Fish grew to a final size of 646.5 g ± 35.8 during the 12-week trial, with no dietary effects on growth or sea lice resistance. Bacterial richness differed among the three tissues with the highest richness detected in the gill, followed by skin, then gut, although dietary effects on richness were only detected within skin and gill. Shannon diversity was reduced in the gut compared to skin and gill but was not influenced by diet. Microbiota communities clustered separately by tissue, with dietary impacts on phylogenetic composition only detected in the skin, although skin and gill communities showed greater overlap compared to the gut according to overall composition, differential abundance, and covariance networks. Inferred metagenomic functions revealed preliminary evidence for tissue-specific host-microbiota coadaptation, as putative microbiota functions showed ties to the physiology of each tissue. Immune gene expression profiles displayed tissue-specific signatures, yet dietary effects were also detected within each tissue and peripheral blood leukocytes. Procrustes analysis comparing sample-matched multivariate variation in microbiota composition to that of immune expression profiles indicated a highly significant correlation between datasets. CONCLUSIONS: Diets supplemented with functional ingredients, namely mannan-oligosaccharide, coconut oil, or a both, resulted in no difference in Atlantic salmon growth or resistance to sea lice infection. However, at the molecular level, functional ingredients caused physiologically relevant changes to mucosal microbiota and host immune expression. Putative tissue-specific metagenomic functions and the high correlation between expression profiles and microbiota composition suggest host and microbiota are interdependent and coadapted in a tissue-specific manner.

A transdisciplinary approach to the initial validation of a single cell protein as an alternative protein source for use in aquafeeds.

The human population is growing and, globally, we must meet the challenge of increased protein needs required to feed this population. Single cell proteins (SCP), when coupled to aquaculture production, offer a means to ensure future protein needs can be met without direct competition with food for people. To demonstrate a given type of SCP has potential as a protein source for use in aquaculture feed, a number of steps need to be validated including demonstrating that the SCP is accepted by the species in question, leads to equivalent survival and growth, does not result in illness or other maladies, is palatable to the consumer, is cost effective to produce and can easily be incorporated into diets using existing technology. Here we examine white shrimp (Litopenaeus vannamei) growth and consumer taste preference, smallmouth grunt (Haemulon chrysargyreum) growth, survival, health and gut microbiota, and Atlantic salmon (Salmo salar) digestibility when fed diets that substitute the bacterium Methylobacterium extorquens at a level of 30% (grunts), 100% (shrimp), or 55% (salmon) of the fishmeal in a compound feed. In each of these tests, animals performed equivalently when fed diets containing M. extorquens as when fed a standard aquaculture diet. This transdisciplinary approach is a first validation of this bacterium as a potential SCP protein substitute in aquafeeds. Given the ease to produce this SCP through an aerobic fermentation process, the broad applicability for use in aquaculture indicates the promise of M. extorquens in leading toward greater food security in the future.

Dose-dependent consumption of farmed Atlantic salmon (Salmo salar) increases plasma phospholipid n-3 fatty acids differentially.

Enhanced n-3 fatty acid intake benefits cardiovascular disease (CVD) risk reduction. Increasing consumption at a population level may be better addressed by diet than through supplementation. However, limited data are available on the effect of the dose response to fish intake on plasma levels of n-3 fatty acids. To compare the effects of different doses of farmed Atlantic salmon on plasma phospholipid fatty acid proportions and CVD risk biomarkers (eg, glucose, insulin, homeostasis model of assessment-insulin resistance, high-sensitivity C-reactive protein, and interleukin-6) in healthy subjects we performed a randomized three-period crossover-designed trial (4-week treatment, 4- to 8-week washout) to compare the effects of twice per week consumption of farmed Atlantic salmon at doses of 90, 180, and 270 g in 19 apparently healthy men and women (mean age 40 to 65 years) and a body mass index between 25 and 34.9. All study visits were conducted at the US Department of Agriculture Agricultural Research Service Grand Forks Human Nutrition Research Center. Eicosapentaenoic acid and total n-3 concentrations were increased (P<0.05) by all treatments in a dose-response manner, with total n-3 of 8.03% ± 0.26% and 9.21% ± 0.26% for 180- and 270-g doses, respectively. Linoleic acid did not change in response to treatment, whereas arachidonic acid (P<0.05) and total n-6 fatty acids decreased dose dependently (<0.0001). The addition of farmed Atlantic salmon to the diet twice per week for 4 weeks at portions of 180 g and 270 g modifies phospholipid fatty acid proportions of n-3 and n-6 in a level associated with decreased risk for CVD.

Baking reduces prostaglandin, resolvin, and hydroxy-fatty acid content of farm-raised Atlantic salmon (Salmo salar).

The consumption of seafood enriched in n-3 polyunsaturated fatty acids (PUFA) is associated with a decreased risk of cardiovascular disease. Several n-3 oxidation products from eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (22:6n-3) have known protective effects in the vasculature. It is not known whether the consumption of cooked seafood enriched in n-3 PUFA causes appreciable consumption of lipid oxidation products. We tested the hypothesis that baking Atlantic salmon (Salmo salar) increases the level of n-3 and n-6 PUFA oxidation products over raw salmon. We measured the contents of several monohydroxy-fatty acids (MHFA), prostanoids, and resolvins. Our data demonstrate that baking did not change the overall total levels of MHFA. However, baking resulted in selective regioisomeric loss of hydroxy fatty acids from arachidonic acid (20:4n-6) and EPA, while significantly increasing hydroxyl-linoleic acid levels. The contents of prostanoids and resolvins were reduced several-fold with baking. The inclusion of a coating on the salmon prior to baking reduced the loss of some MHFA but had no effect on prostanoid losses incurred by baking. Baking did not decrease n-3 PUFA contents, indicating that baking of salmon is an acceptable means of preparation that does not alter the potential health benefits of high n-3 seafood consumption. The extent to which the levels of MHFA, prostanoids, and resolvins in the raw or baked fish have physiologic consequence for humans needs to be determined.

In vitro and in vivo evaluation of the prebiotics GroBiotic-A, inulin, mannanoligosaccharide, and galactooligosaccharide on the digestive microbiota and performance of hybrid striped bass (Morone chrysops x Morone saxatilis).

Two separate experiments were conducted with hybrid striped bass to evaluate four potential prebiotics: GroBiotic-A (partially autolyzed brewer's yeast, dairy ingredient components, and fermentation products), mannanoligosaccharide (MOS), galactooligosaccharide (GOS), and inulin. In the in vitro experiment, intestinal contents were incubated with the individual prebiotics (0.5% by weight) at 25 degrees C for 24 and 48 h. Analysis of volatile fatty acids in the supernatant showed that GroBiotic-A, MOS, and GOS tended to produce lower acetate levels but higher butyrate levels at 48 h compared to diet alone. However, denaturing gradient gel electrophoresis (DGGE) analysis failed to detect any differences in the composition of the microbial community among treatments. DNA sequencing of a common band for all inoculated samples revealed close similarity to the anaerobic Fusobacteria bacterium. An 8-week feeding trial also was conducted to evaluate the four prebiotics looking at growth performance; weight gain, feed efficiency ratio, protein efficiency ratio, whole-body ash, moisture, and lipid did not vary among fish fed the various diets. However, DGGE analysis revealed that all prebiotics produced a different type of microbial community in the intestinal tract of hybrid striped bass compared to fish fed the basal diet. Thus, GroBiotic-A, FOS, GOS, and MOS exhibited prebiotic effects in hybrid striped bass.

A preliminary in vitro assessment of GroBiotic-A, brewer's yeast and fructooligosaccharide as prebiotics for the red drum Sciaenops ocellatus.

This study examined the effects of brewers yeast, fructooligosaccharide (FOS), and GroBiotic-A, a mixture of partially autolyzed brewers yeast, dairy components and dried fermentation products, on the intestinal microbial community of red drum, Sciaenops ocellatus. Gastrointestinal (GI) tracts were aseptically removed from three sub-adult red drum previously maintained on a commercial diet and placed in an anaerobic chamber. Intestinal contents were removed, diluted and incubated in vitro in one of four liquid media: normal diet alone, diet + 2% (w/w) GroBiotic-A, diet + 2% brewers yeast, and diet + 2% FOS. After 24 and 48 h of incubation at 25 degrees C, supernatants were removed for volatile fatty acid (VFA) analysis and DNA was extracted for denaturing gradient gel electrophoresis (DGGE) analysis. Polymerase chain reaction (PCR) was performed on a highly conserved region of M 16S rDNA and the amplicons were subjected to DGGE. The microbial community (MC) fingerprint was used to distinguish microbial populations. The intestinal contents incubated with GroBiotic-A had significantly (P<0.05) higher acetate and total VFA concentrations at 48 h compared to the other treatments. DGGE analysis demonstrated that the microbial community was significantly altered by Grobiotic-A and brewers yeast.

Dietary supplementation of short-chain fructooligosaccharides influences gastrointestinal microbiota composition and immunity characteristics of Pacific white shrimp, Litopenaeus vannamei, cultured in a recirculating system.

Supplementation of prebiotic compounds, including short-chain fructooligosaccharides (scFOS) has been shown to confer benefits on nutrient utilization, growth, and disease resistance of various animal species through improved gastrointestinal (GI) microbiota. However, potential uses of prebiotics for shrimp have not been defined. A 6-wk feeding trial was conducted in a recirculating system to determine the effects of scFOS supplementation on growth performance, immune functions, and GI microbiota composition of Pacific white shrimp (Litopenaeus vannamei). scFOS was supplemented in a nutritionally complete diet (35% crude protein) at 0.025, 0.0500, 0.075, 0.100, 0.200, 0.400, and 0.800% by weight. After 6 wk of feeding, shrimp fed 0, 0.1, and 0.8% scFOS were sampled for assays of immune function and GI microbiota. Dietary supplementation of scFOS did not improve weight gain, feed conversion ratio, or survival of shrimp. Denaturing gradient gel electrophoresis analysis suggested the intestinal tract microbial community from shrimp fed the basal diet was different from that of shrimp fed the scFOS diets [similarity coefficient (SC) = 74.9%)], although the intestinal tract microbial community from shrimp fed the scFOS-supplemented diets was very similar (SC = 92.3%). All the bacterial species contributing to the GI microbial differences were identified, although most of them are uncultured species. Both total hemocyte count and hemocyte respiratory burst increased (P < 0.05) by incremental dietary supplementation of scFOS (0-0.8%). This study is the first to our knowledge to show that dietary scFOS can selectively support growth of certain bacterial species in the GI tract of shrimp and enhance immunity, which may facilitate development of alternative strategies, including novel probiotics and synbiotics, for shrimp growth and health management.

Connexin 35/36 is phosphorylated at regulatory sites in the retina.

Connexin 35/36 is the most widespread neuronal gap junction protein in the retina and central nervous system. Electrical and/or tracer coupling in a number of neuronal circuits that express this connexin are regulated by light adaptation. In many cases, the regulation of coupling depends on signaling pathways that activate protein kinases such as PKA, and Cx35 has been shown to be regulated by PKA phosphorylation in cell culture systems. To examine whether phosphorylation might regulate Cx35/36 in the retina we developed phospho-specific polyclonal antibodies against the two regulatory phosphorylation sites of Cx35 and examined the phosphorylation state of this connexin in the retina. Western blot analysis with hybrid bass retinal membrane preparations showed Cx35 to be phosphorylated at both the Ser110 and Ser276 sites, and this labeling was eliminated by alkaline phosphatase digestion. The homologous sites of mouse and rabbit Cx36 were also phosphorylated in retinal membrane preparations. Quantitative confocal immunofluorescence analysis showed gap junctions identified with a monoclonal anti-Cx35 antibody to have variable levels of phosphorylation at both the Ser110 and Ser276 sites. Unusual gap junctions that could be identified by their large size (up to 32 microm2) and location in the IPL showed a prominent shift in phosphorylation state from heavily phosphorylated in nighttime, dark-adapted retina to weakly phosphorylated in daytime, light-adapted retina. Both Ser110 and Ser276 sites showed significant changes in this manner. Under both lighting conditions, other gap junctions varied from non-phosphorylated to heavily phosphorylated. We predict that changes in the phosphorylation states of these sites correlate with changes in the degree of coupling through Cx35/36 gap junctions. This leads to the conclusion that connexin phosphorylation mediates changes in coupling in some retinal networks. However, these changes are not global and likely occur in a cell type-specific or possibly a gap junction-specific manner.

Calcium-dependent binding of calmodulin to neuronal gap junction proteins.

We examined the interactions of calmodulin with neuronal gap junction proteins connexin35 (Cx35) from perch, its mouse homologue Cx36, and the related perch Cx34.7 using surface plasmon resonance. Calmodulin bound to the C-terminal domains of all three connexins with rapid kinetics in a concentration- and Ca2+-dependent manner. Dissociation was also very rapid. K(d)'s for calmodulin binding at a high-affinity site ranged from 11 to 72 nM, and K(1/2)'s for Ca2+ were between 3 and 5 microM. No binding to the intracellular loops was observed. Binding competition experiments with synthetic peptides mapped the calmodulin binding site to a 10-30 amino acid segment at the beginning of the C-terminal domain of Cx36. The micromolar K(1/2)'s and rapid on and off rates suggest that this interaction may change dynamically in neurons, and may occur transiently when Ca2+ is elevated to a level that would occur in the near vicinity of an activated synapse.

Protein kinase A mediates regulation of gap junctions containing connexin35 through a complex pathway.

Connexin 35 (Cx35) is a major component of electrical synapses in the central nervous system. Many gap junctions containing Cx35 are regulated by dopamine receptor pathways that involve protein kinase A (PKA). To study the mechanism of PKA regulation, we analyzed direct phosphorylation of Cx35 by PKA in vitro and studied the regulation of neurobiotin tracer coupling in HeLa cells expressing Cx35 or Cx35 mutants that lack phosphorylation sites. In Cx35-transfected cells, application of the PKA activator Sp-8-cpt-cAMPS caused a significant decline in coupling, while a PKA inhibitor, Rp-8-cpt-cAMPS, significantly increased tracer coupling. In vitro phosphorylation and mutagenic analysis showed that PKA phosphorylates Cx35 directly at two major sites, Ser110 in the intracellular loop and Ser276 in the carboxyl terminus. In addition, a minor phosphorylation site in the C-terminus was identified by truncation of the last 7 amino acids at Ser298. The mutations Ser110Ala or Ser276Ala significantly reduced regulation of coupling by the PKA activator while a combination of the two eliminated regulation. Truncation at Ser298 reversed the regulation such that the PKA activator significantly increased and the PKA inhibitor significantly decreased coupling. The activation was eliminated in the S110A, S276A, S298ter triple mutant. We conclude that PKA regulates Cx35 coupling in a complex manner that requires both major phosphorylation sites. Furthermore, the tip of the C-terminus acts as a "switch" that determines whether phosphorylation will inhibit or enhance coupling. Reliance on the combined states of three sites provides fine control over the degree of coupling through Cx35 gap junctions.

Broadly distributed chemical reactivity of natural antibodies expressed in coordination with specific antigen binding activity.

Antibody (Ab) nucleophilic reactivity was studied using hapten and polypeptide antigens containing biotinylated phosphonate diester groups (covalently reactive antigen analogs, CRAs). Polyclonal IgG from healthy donors formed covalent adducts with a positively charged hapten CRA at levels superior to trypsin. Each of the 16 single chain Fv clones studied expressed a similar reactivity, indicating the V domain location of the nucleophiles and their broad distribution in diverse Abs. The formation of hapten CRA-Fv adducts was correlated with Fv proteolytic activity determined by cleavage of a model peptide substrate. Despite excellent nucleophilicity, proteolysis by IgG proceeded at lower rates than trypsin, suggesting that events occurring after nucleophilic attack on the substrate limit the rate of Ab proteolysis. The extracellular domain of the epidermal growth factor receptor with phosphonate diester groups at Lys side chains and a synthetic peptide corresponding to residues 421- 431 of human immunodeficiency virus glycoprotein (gp) 120 with the phosphonate diester at the C terminus formed covalent adducts with specific polyclonal and monoclonal Abs raised by immunization with epidermal growth factor receptor and synthetic gp120-(421- 436) devoid of phosphonate diester groups, respectively. Adduct formation was inhibited by extracellular domain of the epidermal growth factor receptor (exEGFB) and synthetic gp120-(421- 436) devoid of phosphonate groups, suggesting that the nucleophiles are located within the antigen binding sites. These results suggest the innate character of the Ab nucleophilic reactivity, its functional coordination with non-covalent adaptive binding interactions developing over the course of B cell maturation, and novel routes toward permanent inhibition of Abs.

A mechanism-based probe for gp120-Hydrolyzing antibodies.

An antigenic peptide analogue consisting of HIV gp120 residues 421-431 (an antigen recognition site probe) with diphenyl amino(4-amidinophenyl)methanephosphonate located at the C-terminus (a catalytic site probe) was synthesized and its trypsin and antibody reactivity characteristics were studied. Antibodies to the peptide determinant recognized the peptidyl phosphonate probe. Trypsin was inhibited equipotently by the peptidyl phosphonate and its simple phosphonate counterpart devoid of the peptide determinant. The peptidyl phosphonate inhibited the gp120-hydrolyzing activity of a catalytic antibody light chain. It was bound covalently by the light chain and the binding was inhibited by the classical active-site directed inhibitor of serine proteinase, diisopropyl fluorophosphate. These results reveal that the peptidyl phosphonate ester can serve as a probe for the antigen recognition and catalytic subsites of proteolytic antibodies.

Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs.

Phosphonate monoesters have been assumed to serve as noncovalent transition state analogs for enzymes capable of catalyzing transacylation reactions. Here, we present evidence for the covalent reaction of certain serine proteinases and peptidase antibody fragments with monophenyl amino(4-amidinophenyl)methanephosphonate derivatives. Stable adducts of the N-biotinylated monophenyl ester with trypsin and antibody fragments were evident under conditions that disrupt noncovalent interactions. The reaction was inhibited by the active-site-directed reagent diisopropyl fluorophosphate. Mass spectrometry of the fragments from monoester-labeled trypsin indicated phosphonylation of the active site. Irreversible inhibition of trypsin- and thrombin-catalyzed hydrolysis of model substrates was observed. Kinetic analysis of inactivation of trypsin by the N-benzyloxycarbonylated monoester suggested that the first-order rate constant for formation of covalent monoester adducts is comparable to that of the diester adducts (0.47 vs 2.0 min(-1)). These observations suggest that the covalent reactivity of phosphonate monoesters contributes to their interactions with serine proteinases, including certain proteolytic antibodies.