Plasma Proteome Responses in Salmonid Fish Following Immunization.

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout (Oncorhynchus mykiss, Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.

The AMPK system of salmonid fishes was expanded through genome duplication and is regulated by growth and immune status in muscle.

5'adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. This study identified expansions in the AMPK-α, -ß and -γ families of salmonid fishes due to a history of genome duplication events, including five novel salmonid-specific AMPK subunit gene paralogue pairs. We tested the hypothesis that the expanded AMPK gene system of salmonids is transcriptionally regulated by growth and immunological status. As a model, we studied immune-stimulated coho salmon (Oncorhynchus kisutch) from three experiment groups sharing the same genetic background, but showing highly-divergent growth rates and nutritional status. Specifically, we compared wild-type and GH-transgenic fish, the latter achieving either enhanced or wild-type growth rate via ration manipulation. Transcript levels for the fifteen unique salmonid AMPK subunit genes were quantified in skeletal muscle after stimulation with bacterial or viral mimics to alter immune status. These analyses revealed a constitutive up-regulation of several AMPK-α and -γ subunit-encoding genes in GH-transgenic fish achieving accelerated growth. Further, immune stimulation caused a decrease in the expression of several AMPK subunit-encoding genes in GH-transgenic fish specifically. The dynamic expression responses observed suggest a role for the AMPK system in balancing energetic investment into muscle growth according to immunological status in salmonid fishes.

Proteomic comparison of selective breeding and growth hormone transgenesis in fish: Unique pathways to enhanced growth.

In fish used for food production and scientific research, fast growth can be achieved via selective breeding or induced instantaneously via growth hormone (GH) transgenesis (GHT). The proteomic basis for these distinct routes towards a similar higher phenotype remains uncharacterized, as are associated implications for health parameters. We addressed this knowledge gap using skeletal muscle proteomics in coho salmon (Oncorhynchus kisutch), hypothesising that i) selective breeding and GHT are underpinned by both parallel and unique changes in growth systems, and ii) rapidly-growing fish strains have lowered scope to allocate resources towards immune function. Quantitative profiling of GHT and growth-selected strains was done in comparison to wild-type after injection with PBS (control) or Poly I:C (to mimic infection). We identified remodelling of the muscle proteome in each growth-enhanced strain that was strikingly non-overlapping. GHT was characterized by focal upregulation of systems driving protein synthesis, while the growth-selected fish presented a larger and more diverse set of changes, consistent with complex alterations to many metabolic and cellular pathways. Poly I:C had little detectable effect on the muscle proteome. This study demonstrates that distinct proteome profiles can explain outwardly similar enhanced growth phenotypes, improving our understanding of growth mechanisms in anthropogenic animal strains. SIGNIFICANCE: This work provides the first proteomic insights into mechanisms underpinning different anthropogenic routes to rapid growth in salmon. High-throughput proteomic profiling was used to reveal changes supporting enhanced growth, comparing skeletal muscle of growth hormone transgenic (GHT) and selectively-bred salmon strains with their wild-type counterparts. Contrasting past mRNA-level comparisons of the same fish strains, our data reveals a surprisingly substantial proteomic divergence between the GHT and selectively bred strains. The findings demonstrate that many unique molecular mechanisms underlie growth-enhanced phenotypes in different types of fish strain used for food production and scientific research.

High-throughput proteomic profiling of the fish liver following bacterial infection.

BACKGROUND: High-throughput proteomics was used to determine the role of the fish liver in defense responses to bacterial infection. This was done using a rainbow trout (Oncorhynchus mykiss) model following infection with Aeromonas salmonicida, the causative agent of furunculosis. The vertebrate liver has multifaceted functions in innate immunity, metabolism, and growth; we hypothesize this tissue serves a dual role in supporting host defense in parallel to metabolic adjustments that promote effective immune function. While past studies have reported mRNA responses to A. salmonicida in salmonids, the impact of bacterial infection on the liver proteome remains uncharacterized in fish. RESULTS: Rainbow trout were injected with A. salmonicida or PBS (control) and liver extracted 48 h later for analysis on a hybrid quadrupole-Orbitrap mass spectrometer. A label-free method was used for protein abundance profiling, which revealed a strong innate immune response along with evidence to support parallel rewiring of metabolic and growth systems. 3076 proteins were initially identified against all proteins (n = 71,293 RefSeq proteins) annotated in a single high-quality rainbow trout reference genome, of which 2433 were maintained for analysis post-quality filtering. Among the 2433 proteins, 109 showed significant differential abundance following A. salmonicida challenge, including many upregulated complement system and acute phase response proteins, in addition to molecules with putative functions that may support metabolic re-adjustments. We also identified novel expansions in the complement system due to gene and whole genome duplication events in salmonid evolutionary history, including eight C3 proteins showing differential changes in abundance. CONCLUSIONS: This study provides the first high-throughput proteomic examination of the fish liver in response to bacterial challenge, revealing novel markers for the host defense response, and evidence of metabolic remodeling in conjunction with activation of innate immunity.