Characterising the Physiological Responses of Chinook Salmon (Oncorhynchus tshawytscha) Subjected to Heat and Oxygen Stress.

In New Zealand, during the hottest periods of the year, some salmon farms in the Marlborough Sounds reach water temperatures above the optimal range for Chinook salmon. High levels of mortality are recorded during these periods, emphasising the importance of understanding thermal stress in this species. In this study, the responses of Chinook salmon (Oncorhynchus tshawytscha) to chronic, long-term changes in temperature and dissolved oxygen were investigated. This is a unique investigation due to the duration of the stress events the fish were exposed to. Health and haematological parameters were analysed alongside gene expression results to determine the effects of thermal stress on Chinook salmon. Six copies of heat shock protein 90 (HSP90) were discovered and characterised: HSP90AA1.1a, HSP90AA1.2a, HSP90AA1.1b, HSP90AA1.2b, HSP90AB1a and HSP90AB1b, as well as two copies of SOD1, named SOD1a and SOD1b. The amino acid sequences contained features similar to those found in other vertebrate HSP90 and SOD1 sequences, and the phylogenetic tree and synteny analysis provided conclusive evidence of their relationship to other vertebrate HSP90 and SOD1 genes. Primers were designed for qPCR to enable the expression of all copies of HSP90 and SOD1 to be analysed. The expression studies showed that HSP90 and SOD1 were downregulated in the liver and spleen in response to longer term exposure to high temperatures and lower dissolved oxygen. HSP90 was also downregulated in the gill; however, the results for SOD1 expression in the gill were not conclusive. This study provides important insights into the physiological and genetic responses of Chinook salmon to temperature and oxygen stress, which are critical for developing sustainable fish aquaculture in an era of changing global climates.

Low copper levels measured in the aortic wall of New Zealand patients with non-syndromic ascending thoracic aortic aneurysm.

OBJECTIVES: Studies in animals have shown causal relationships between copper (Cu) deficiency and the development of thoracic aortic aneurysms (TAAs) [1, 2]. Cu deficiency is widespread in New Zealand (NZ) soils; the high soil pH from the use of lime fertilizers reduces the bioavailability of Cu for grazing animals and growing plants; this, in turn, reduces Cu availability in the NZ human food chain. Our study is a pilot study to explore associations between Cu and TAA. We measured Cu levels in aneurysmal aortic tissues in patients undergoing Bentall procedures and non-aneurysmal aortic tissue from coronary artery bypass graft patients. METHODS: Aortic samples were collected from 2 groups of patients during elective open-heart surgery over 4 months between November 2017 and February 2018. The groups were a TAA group, patients with non-syndromic aortic aneurysm and without the bicuspid aortic valve or known infectious or inflammatory condition (ANEURYSM; n = 13), and a control coronary artery bypass graft group (CONTROL; n = 44). Standardized digested dry tissue weighed samples were analysed from both groups. Tissue extraction of trace elements was carried out using HCl-H2O2 digestion and a highly sensitive analytical technique, inductively coupled plasma mass spectrometry-used to measure elemental concentrations. RESULTS: Cu concentration (mean ± SD) was significantly lower in ANEURYSM (3.34 ± 0.16 µg/g) when compared to the CONTROL group tissues (4.33 ± 0.20 µg/g) (dry weight; mean ± SD; Student's t-test, P < 0.05). Over 46% of the Aneurysm patients were Maori and live in a geographically Cu-deficient NZ territory. CONCLUSIONS: Cu deficiency may play a role in the development or progression of non-syndromic ascending aortic aneurysms in NZ. Maori patients are more at risk as they commonly live in rural NZ, dependent on locally grown nutritional sources. Further studies are required to confirm this exciting finding and to establish cause and effect relationship.

The repertoire of vertebrate STAT transcription factors: Origin and variations in fish.

The stat gene family diversified during early vertebrate evolution thanks to two rounds of whole genome duplication (WGD) to produce a typical repertoire composed of 6 STAT factors (named 1-6). In contrast, only one or two stat genes have been reported in C. elegans and in D. melanogaster. The main types of STAT found from bony fish to mammals are present in Agnathan genomes, but a typical STAT1-6 repertoire is only observed in jawed vertebrates. Comparative syntenies showed that STAT6 was the closest to the ancestor of the family. An extensive survey of stat genes across fish including polyploid species showed that whole genome duplications did not lead to a uniform expansion of stat genes. While 2 to 5 stat1 are present in salmonids, whose genome duplicated about 35My ago, only one copy of stat2 and stat6 is retained. In contrast, common carp, with a recent whole genome duplication (5-10My), possesses a doubled stat repertoire indicating that the elimination of stat2 and stat6 additional copies is not immediate. Altogether our data shed light on the multiplicity of evolutionary pathways followed by key components of the canonical cytokine receptor signalling pathway, and point to differential selective constraints exerted on these factors.

Insights Into the Evolution of the prdm1/Blimp1 Gene Family in Teleost Fish.

In mammals, Blimp1 (B lymphocyte-induced maturation protein 1) encoded by the prdm1 gene and its homolog Hobit (homolog of Blimp1 in T cells) encoded by znf683, represent key transcriptional factors that control the development and differentiation of both B and T cells. Despite their essential role in the regulation of acquired immunity, this gene family has been largely unexplored in teleosts to date. Until now, one prdm1 gene has been identified in most teleost species, whereas a znf683 homolog has not yet been reported in any of these species. Focusing our analysis on rainbow trout (Oncorhynchus mykiss), an in silico identification and characterization of prdm1-like genes has been undertaken, confirming that prdm1 and znf683 evolved from a common ancestor gene, acquiring three gene copies after the teleost-specific whole genome duplication event (WGD) and six genes after the salmonid-specific WGD. Additional transcriptional studies to study how each of these genes are regulated in homeostasis, in response to a viral infection or in B cells in different differentiation stages, provide novel insights as to how this gene family evolved and how their encoded products might be implicated in the lymphocyte differentiation process in teleosts.

Comparison of three oral selenium compounds in cancer patients: Evaluation of differential pharmacodynamic effects in normal and malignant cells.

BACKGROUND: Selenium (Se) compounds have demonstrated therapeutic synergism in combination with anticancer treatments whilst reducing normal tissue toxicities in a range of experimental models. While reduction in some toxicities of chemotherapy and radiation has been confirmed in randomised clinical trials, they have not been powered to evaluate improved anticancer efficacy. A lack of data on the clinical potencies of the main nutritionally-relevant forms of Se and the relationship between their pharmacokinetic (PK) profiles and pharmacodynamic (PD) effects in cancer patients has hampered progress to date. The primary objective of this study was to determine the dose and form of Se that can be most safely and effectively used in clinical trials in combination with anti-cancer therapies. STUDY METHODS: In a phase I randomised double-blinded study, the PD profile of sodium selenite (SS), Se-methylselenocysteine (MSC) and seleno-l-methionine (SLM) were compared in two cohorts of 12 patients, one cohort with chronic lymphocytic leukaemia (CLL) and the other with solid malignancies. All 24 patients were randomised to receive 400 µg of elemental Se as either SS, MSC or SLM, taken orally daily for 8 weeks. PD parameters were assessed before, during and 4 weeks after Se compound exposure in plasma and peripheral blood mononuclear cells (PBMCs). RESULTS: No significant sustained changes were observed in plasma concentrations of vascular endothelial growth factor-α (VEGF-α), expression of proteins associated with endoplasmic reticulum stress (the unfolded protein response) or in intracellular total glutathione in PBMCs, in either disease cohort or when grouped by Se compound. CONCLUSIONS: At the 400 µg dose level no substantial changes in PD parameters were noted. Extrapolating from pre-clinical data, the dose examined in this cohort was too low to achieve the Se plasma concentration (≥ 5 µM) expected to elicit significant PD effects. Recruitment of a subsequent cohort at higher doses to exceed this PK threshold is planned.

Transcriptional changes in response to ketamine ester-analogs SN 35210 and SN 35563 in the rat brain.

BACKGROUND: Ketamine ester analogs, SN 35210 and SN 35563, demonstrate different pharmacological profiles to ketamine in animal models. Both confer hypnosis with predictably rapid offset yet, paradoxically, SN35563 induces a prolonged anti-nociceptive state. To explore underlying mechanisms, broad transcriptome changes were measured and compared across four relevant target regions of the rat brain. RESULTS: SN 35563 produced large-scale alteration of gene expression in the Basolateral Amygdala (BLA) and Paraventricular Nucleus of the Thalamus (PVT), in excess of 10x that induced by ketamine and SN 35210. A smaller and quantitatively similar number of gene changes were observed in the Insula (INS) and Nucleus Accumbens (ACB) for all three agents. In the BLA and PVT, SN 35563 caused enrichment for gene pathways related to the function and structure of glutamatergic synapses in respect to: release of neurotransmitter, configuration of postsynaptic AMPA receptors, and the underlying cytoskeletal scaffolding and alignment. CONCLUSION: The analgesic ketamine ester analog SN 35563 induces profound large-scale changes in gene expression in key pain-related brain regions reflecting its unique prolonged pharmacodynamic profile.

Comparative Safety and Pharmacokinetic Evaluation of Three Oral Selenium Compounds in Cancer Patients.

Selenium (Se) compounds have demonstrated anticancer properties in both preclinical and clinical studies, with particular promise in combination therapy where the optimal form and dose of selenium has yet to be established. In a phase I randomised double-blinded study, the safety, tolerability and pharmacokinetic (PK) profiles of sodium selenite (SS), Se-methylselenocysteine (MSC) and seleno-l-methionine (SLM) were compared in patients with chronic lymphocytic leukaemia and a cohort of patients with solid malignancies. Twenty-four patients received 400 µg of elemental Se as either SS, MSC or SLM for 8 weeks. None of the Se compounds were associated with any significant toxicities, and the total plasma Se AUC of SLM was markedly raised in comparison to MSC and SS. DNA damage assessment revealed negligible genotoxicity, and some minor reductions in lymphocyte counts were observed. At the dose level used, all three Se compounds are well-tolerated and non-genotoxic. Further analyses of the pharmacodynamic effects of Se on healthy and malignant peripheral blood mononuclear cells will inform the future evaluation of higher doses of these Se compounds. The study is registered under the Australian and New Zealand Clinical Trials Registry No: ACTRN12613000118707.

Identification of the First Teleost CD5 Molecule: Additional Evidence on Phenotypical and Functional Similarities between Fish IgM+ B Cells and Mammalian B1 Cells.

Despite teleost fish being the first animal group in which all elements of adaptive immunity are present, the lack of follicular structures, as well as the fact that systemic Ab responses rely exclusively on unswitched low-affinity IgM responses, strongly suggests that fish B cell responses resemble mammalian B1 cell responses rather than those of B2 cells. In line with this hypothesis, in the current study, we have identified a homolog of CD5 in teleost fish. This pan-T marker belonging to the scavenger receptor cysteine-rich family of receptors is commonly used in mammals to distinguish a subset of B1 cells. Subsequently, we have demonstrated that a very high percentage of teleost IgM+ B cells express this marker, in contrast to the limited population of CD5-expressing B1 cells found in most mammals. Furthermore, we demonstrate that fish IgM+ B cells share classical phenotypic features of mammalian B1 cells such as large size, high complexity, high surface IgM, and low surface IgD expression, regardless of CD5 expression. Additionally, fish IgM+ B cells, unlike murine B2 cells, also displayed extended survival in cell culture and did not proliferate after BCR engagement. Altogether, our results demonstrate that although fish are evolutionarily the first group in which all the elements of acquired immunity are present, in the absence of follicular structures, most teleost IgM+ B cells have retained phenotypical and functional characteristics of mammalian B1 cells.

Sleep and stress hormone responses to training and competition in elite female athletes.

Stress hormone and sleep differences in a competition versus training setting are yet to be evaluated in elite female team-sport athletes. The aim of the current study was to evaluate salivary cortisol and perceptual stress markers during competition and training and to determine the subsequent effects on sleep indices in elite female athletes. Ten elite female netball athletes (mean ± SD; age: 23 ± 6 years) had their sleep monitored on three occasions; following one netball competition match (MATCH), one netball match simulation session (TRAIN), and one rest day (CONTROL). Perceived stress values and salivary cortisol were collected immediately pre- (17:15 pm) and post-session (19:30 pm), and at 22:00 pm. Sleep monitoring was performed using wrist actigraphy assessing total time in bed, total sleep time (TST), efficiency (SE%), latency, sleep onset time and wake time. Cortisol levels were significantly higher (p < .01) immediately post MATCH compared with TRAIN and CONTROL (mean ± SD; 0.700 ± 0.165, 0.178 ± 0.127 and 0.157 ± 0.178 µg/dL, respectively) and at 22:00 pm (0.155 ± 0.062, 0.077 ± 0.063, and 0.089 ± 0.083 µg/dL, respectively). There was a significant reduction in TST (-118 ± 112 min, p < .01) and SE (-7.7 ± 8.5%, p < .05) following MATCH vs. TRAIN. Salivary cortisol levels were significantly higher, and sleep quantity and quality were significantly reduced, following competition when compared to training and rest days.

Antimicrobial peptides within the Yellowtail Kingfish (Seriola lalandi).

A number of Seriola species are currently farmed or being investigated as future aquaculture species in countries around the world. However they face a number of issues and limitations which will need to be overcome to ensure future stability and growth, one of which are disease outbreaks. Despite this, very little has been done to understand the immune system of Seriola species and very few immune genes have been characterised. Antimicrobial peptides (AMP) are naturally occurring low molecular weight polypeptides that play a major role in an organism's immune system and act effectively as a first line of defence. This investigation isolates the full length cDNA sequences of two AMP's, piscidin and hepcidin from the yellowtail kingfish (Seriola lalandi). The full-length cDNA of the piscidin gene encodes a 65 amino acid prepropeptide, containing a 25-residue peptide, predicted to form an amphipathic helix-loop-helix structure. Phylogenetic analysis using fish piscidin sequences, showed that this AMP is only found in bony fish within the Acanthomorpha clade and that a possible three groups within the piscidin family exists, with S. lalandi belonging to a particular group. The full-length cDNA of the hepcidin gene encodes a 90 amino acid preprohepcidin, which contains a typical RX(R/K)R motif for cleavage of the mature peptide which comprises of eight conserved cysteine residues. Phylogenetic analysis of known vertebrate hepcidin antimicrobial peptide (HAMP) sequences, shows sequences from the Neoteleostei clade of bony fish form two very separate groups, HAMP1 and HAMP2, with the S. lalandi hepcidin gene grouped with the HAMP1 sequences. HAMP2 sequences are found to have multiple copies within fish and genome analysis showed very clearly that these two groups of genes are located on separate regions on the genome, with the multiple HAMP2 copies formed from tandem gene duplications. Lastly, using qPCR the expression of the S. lalandi piscidin gene within healthy fish was highest within, spleen and gills and lowest in liver, whereas hepcidin was highest in the liver with little or no expression in the spleen and gills.

Omics and cytokine discovery in fish: Presenting the Yellowtail kingfish (Seriola lalandi) as a case study.

A continued programme of research is essential to overcome production bottlenecks in any aquacultured fish species. Since the introduction of genetic and molecular techniques, the quality of immune research undertaken in fish has greatly improved. Thousands of species specific cytokine genes have been discovered, which can be used to conduct more sensitive studies to understand how fish physiology is affected by aquaculture environments or disease. Newly available transcriptomic technologies, make it increasingly easier to study the immunogenetics of farmed species for which little data exists. This paper reviews how the application of transcriptomic procedures such as RNA Sequencing (RNA-Seq) can advance fish research. As a case study, we present some preliminary findings using RNA-Seq to identify cytokine related genes in Seriola lalandi. These will allow in-depth investigations to understand the immune responses of these fish in response to environmental change or disease and help in the development of therapeutic approaches.

Correction: First Demonstration of Antigen Induced Cytokine Expression by CD4-1+ Lymphocytes in a Poikilotherm: Studies in Zebrafish (Danio rerio).

[This corrects the article DOI: 10.1371/journal.pone.0126378.].

Development of a qPCR Method to Measure Mitochondrial and Genomic DNA Damage with Application to Chemotherapy-Induced DNA Damage and Cryopreserved Cells.

DNA damage quantitation assays such as the comet assay have focused on the measurement of total nuclear damage per cell. The adoption of PCR-based techniques to quantify DNA damage has enabled sequence- and organelle-specific assessment of DNA lesions. Here we report on an adaptation of a qPCR technique to assess DNA damage in nuclear and mitochondrial targets relative to control. Novel aspects of this assay include application of the assay to the Rotor-Gene platform with optimized DNA polymerase/fluorophore/primer set combination in a touchdown PCR protocol. Assay validation was performed using ultraviolet C radiation in A549 and THP1 cancer cell lines. A comparison was made to the comet assay applied to peripheral blood mononuclear cells, and an estimation of the effects of cryopreservation on ultraviolet C-induced DNA damage was carried out. Finally, dose responses for DNA damage were measured in peripheral blood mononuclear cells following exposure to the cytotoxic agents bleomycin and cisplatin. We show reproducible experimental outputs across the tested conditions and concordance with published findings with respect to mitochondrial and nuclear genotoxic susceptibilities. The application of this DNA damage assay to a wide range of clinical and laboratory-derived samples is both feasible and resource-efficient.

Analysis of interferon gamma protein expression in zebrafish (Danio rerio).

IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo. We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels.

Teleost Chemokines and Their Receptors.

Chemokines are a superfamily of cytokines that appeared about 650 million years ago, at the emergence of vertebrates, and are responsible for regulating cell migration under both inflammatory and physiological conditions. The first teleost chemokine gene was reported in rainbow trout in 1998. Since then, numerous chemokine genes have been identified in diverse fish species evidencing the great differences that exist among fish and mammalian chemokines, and within the different fish species, as a consequence of extensive intrachromosomal gene duplications and different infectious experiences. Subsequently, it has only been possible to establish clear homologies with mammalian chemokines in the case of some chemokines with well-conserved homeostatic roles, whereas the functionality of other chemokine genes will have to be independently addressed in each species. Despite this, functional studies have only been undertaken for a few of these chemokine genes. In this review, we describe the current state of knowledge of chemokine biology in teleost fish. We have mainly focused on those species for which more research efforts have been made in this subject, specially zebrafish (Danio rerio), rainbow trout (Oncorhynchus mykiss) and catfish (Ictalurus punctatus), outlining which genes have been identified thus far, highlighting the most important aspects of their expression regulation and addressing any known aspects of their biological role in immunity. Finally, we summarise what is known about the chemokine receptors in teleosts and provide some analysis using recently available data to help characterise them more clearly.

Characterisation of the TNF superfamily members CD40L and BAFF in the small-spotted catshark (Scyliorhinus canicula).

The tumour necrosis factor superfamily (TNFSF) members CD40L and BAFF play critical roles in mammalian B cell survival, proliferation and maturation, however little is known about these key cytokines in the oldest jawed vertebrates, the cartilaginous fishes. Here we report the cloning of CD40L and BAFF orthologues (designated ScCD40L and ScBAFF) in the small-spotted catshark (Scyliorhinus canicula). As predicted both proteins are type II membrane-bound proteins with a TNF homology domain in their extracellular region and both are highly expressed in shark immune tissues. ScCD40L transcript levels correlate with those of TCRα and transcription of both genes is modulated in peripheral blood leukocytes following in vitro stimulation. Although a putative CD40L orthologue was identified in the elephant shark genome the work herein is the first molecular characterisation and transcriptional analysis of CD40L in a cartilaginous fish. ScBAFF was also cloned and its transcription characterised in an attempt to resolve the discrepancies observed between spiny dogfish BAFF and bamboo shark BAFF in previously published studies.

First Demonstration of Antigen Induced Cytokine Expression by CD4-1+ Lymphocytes in a Poikilotherm: Studies in Zebrafish (Danio rerio).

Adaptive immunity in homeotherms depends greatly on CD4+ Th cells which release cytokines in response to specific antigen stimulation. Whilst bony fish and poikilothermic tetrapods possess cells that express TcR and CD4-related genes (that exist in two forms in teleost fish; termed CD4-1 and CD4-2), to date there is no unequivocal demonstration that cells equivalent to Th exist. Thus, in this study we determined whether CD4-1+ lymphocytes can express cytokines typical of Th cells following antigen specific stimulation, using the zebrafish (Danio rerio). Initially, we analyzed the CD4 locus in zebrafish and found three CD4 homologues, a CD4-1 molecule and two CD4-2 molecules. The zfCD4-1 and zfCD4-2 transcripts were detected in immune organs and were most highly expressed in lymphocytes. A polyclonal antibody to zfCD4-1 was developed and used with an antibody to ZAP70 and revealed double positive cells by immunohistochemistry, and in the Mycobacterium marinum disease model CD4-1+ cells were apparent surrounding the granulomas typical of the infection. Next a prime-boost experiment, using human gamma globulin as antigen, was performed and revealed for the first time in fish that zfCD4-1+ lymphocytes increase the expression of cytokines and master transcription factors relevant to Th1/Th2-type responses as a consequence of boosting with specific antigen.

Identification and expression analysis of two interleukin-23α (p19) isoforms, in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar.

Interleukin (IL)-23 is a heterodimeric IL-12 family cytokine composed of a p19 α-chain, linked to a p40 ß-chain that is shared with IL-12. IL-23 is distinguished functionally from IL-12 by its ability to induce the production of IL-17, and differentiation of Th17 cells in mammals. Three isoforms of p40 (p40a, p40b and p40c) have been found in some 3R teleosts. Salmonids also possess three p40 isoforms (p40b1, p40b2 and p40c) although p40a is missing, and two copies (paralogues) of p40b are present that have presumably been retained following the 4R duplication in this fish lineage. Teleost p19 has been discovered recently in zebrafish, but to date there is limited information on expression and modulation of this molecule. In this report we have cloned two p19 paralogues (p19a and p19b) in salmonids, suggesting that a salmonid can possess six potential IL-23 isoforms. Whilst Atlantic salmon has two active p19 genes, the rainbow trout p19b gene may have been pseudogenized. The salmonid p19 translations share moderate identities (22.8-29.9%) to zebrafish and mammalian p19 molecules, but their identity was supported by structural features, a conserved 4 exon/3 intron gene organisation, and phylogenetic tree analysis. The active salmonid p19 genes are highly expressed in blood and gonad. Bacterial (Yersinia ruckeri) and viral infection in rainbow trout induces the expression of p19a, suggesting pathogen-specific induction of IL-23 isoforms. Trout p19a expression was also induced by PAMPs (poly IC and peptidoglycan) and the proinflammatory cytokine IL-1ß in primary head kidney macrophages. These data may indicate diverse functional roles of trout IL-23 isoforms in regulating the immune response in fish.

The longevity of the antimicrobial response in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) supplemented diet.

This study builds upon previous work studying antimicrobial peptide (AMP) gene expression in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) enriched diet. The aims here were 1) to evaluate how long AMP expression is elevated in skin with continuous feeding of fish with the PG enriched diet for 21 or 28 days, and 2) to assess the impact of stopping PG feeding at day 14 when sampled at day 21 or 28. The rainbow trout were divided into 6 groups, with two fed a control commercial diet for the duration of the experiment and the other four given the same diet enriched with 10 mg PG/Kg for 14 days (PG 1-14) or continuously (PG continuous), the former reverting back to the commercial diet at day 14. No mortalities occurred during the study and there were no significant differences in growth among the fish in the different diet groups. The expression of six AMP genes was studied by real-time PCR in the skin, since these genes were shown to be induced in response to the PG enriched diets in a previous experiment. We show that continuous PG treatment for 21 or 28 days maintained high levels of AMP expression, although in general the levels decreased with time on the diets. Withdrawal of the PG diets at day 14 resulted in a fall in expression level especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated levels (x10) in comparison to fish given a control diet. These results confirm that orally administered PG clearly enhances the trout innate immune system and could be used as a means to boost fish defences. Future studies should be conducted to verify the impact on survival after pathogen challenge in trout fed PG enriched diets under these regimes.