Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish worldwide. As an important fish pathogen, F. psychrophilum is frequently exposed to multiple invading genetic elements in natural environments. Endonuclease Cas9 provides bacteria with adaptive interference against invading genetic elements. Previous studies revealed that several F. psychrophilum strains harbored a type II-C Cas9 called Fp1Cas9, but little is known about the potential role of this endonuclease against invading genetic elements. In this work, we identified a gene encoding a novel type II-C Cas9 called Fp2Cas9 from F. psychrophilum strain CN46. Through bacterial RNA sequencing, we demonstrated active transcription of both Fp2Cas9 and pre-crRNAs in strain CN46. Bioinformatics analysis further revealed that the transcription of Fp2Cas9 and pre-crRNAs was driven by a newly integrated promoter sequence and a promoter element embedded within each CRISPR repeat, respectively. To formally demonstrate that Fp2Cas9 and associated crRNAs yielded functional interference in strain CN46, a plasmid interference assay was performed, resulting in adaptive immunity to target DNA sequences in Flavobacterium bacteriophages. Phylogenetic analysis demonstrated that Fp2Cas9 was present only in several F. psychrophilum isolates. Phylogenetic analysis revealed that this novel endonuclease was probably acquired through horizontal gene transfer from the CRISPR-Cas9 system in an unidentified Flavobacterium species. Comparative genomics analysis further showed that the Fp2Cas9 was integrated into the type II-C CRISPR-Cas locus in strain CN38 instead of the original Fp1Cas9. Taken together, our results shed light on the origin and evolution of Fp2Cas9 gene and demonstrated that this novel endonuclease provided adaptive interference against bacteriophage infections.

Off-flavors can have significant impacts on the quality of salmonid products. This study investigated the possibility of comprehensive off-flavor profiling considering both olfactory and taste sensory perspectives by combining near-infrared hyperspectral imaging (NIR-HSI) and machine/deep learning. Four feature extraction algorithms were employed for the extraction and interpretation of spectral fingerprint information regarding off-flavor-related compounds. Classification models, including the partial least squares discriminant analysis, least-squares support vector machine, extreme learning machine, and one-dimensional convolutional neural network (1DCNN) were constructed using the full wavelengths and selected spectral features for the identification of off-flavor salmonids. The 1DCNN achieved the highest discrimination accuracy with full and selected wavelengths (i.e., 91.11 and 86.39 %, respectively). Furthermore, the prediction and visualization of off-flavor-related compounds were achieved with acceptable performances (R2 > 0.6) for practical applications. These results indicate the potential of NIR-HSI for the off-flavor profiling of salmonid muscle samples for producers and researchers.

Brachymystax tsinlingensis Li, 1966 is an endangered freshwater fish with economic, ecological, and scientific values. Study of the genome of B. tsinlingensis might be particularly insightful given that this is the only Brachymystax species with genome. We present a high-quality chromosome-level genome assembly and protein-coding gene annotation for B. tsinlingensis with Illumina short reads, Nanopore long reads, Hi-C sequencing reads, and RNA-seq reads from 5 tissues/organs. The final chromosome-level genome size is 2,031,709,341 bp with 40 chromosomes. We found that the salmonids have a unique GC content and codon usage, have a slower evolutionary rate, and possess specific positively selected genes. We also confirmed the salmonids have undergone a whole-genome duplication event and a burst of transposon-mediated repeat expansion, and lost HoxAbβ Hox cluster, highly expressed genes in muscle may partially explain the migratory habits of B. tsinlingensis. The high-quality B. tsinlingensis assembled genome could provide a valuable reference for the study of other salmonids as well as aid the conservation of this endangered species.

So far, very few sex-determining genes have been identified in vertebrates and most of them, the so-called \'usual suspects\', evolved from genes which fulfil essential functions during sexual development and are thus already tightly linked to the process that they now govern. The single exception to this \'usual suspects\' rule in vertebrates so far is the conserved salmonid sex-determining gene, sdY (sexually dimorphic on the Y chromosome), that evolved from a gene known to be involved in regulation of the immune response. It is contained in a jumping sex locus that has been transposed or translocated into different ancestral autosomes during the evolution of salmonids. This special feature of sdY, i.e. being inserted in a \'jumping sex locus\', could explain how salmonid sex chromosomes remain young and undifferentiated to escape degeneration. Recent knowledge on the mechanism of action of sdY demonstrates that it triggers its sex-determining action by deregulating oestrogen synthesis that is a conserved and crucial pathway for ovarian differentiation in vertebrates. This result suggests that sdY has evolved to cope with a pre-existing sex differentiation regulatory network. Therefore, \'limited options\' for the emergence of new master sex-determining genes could be more constrained by their need to tightly interact with a conserved sex differentiation regulatory network rather than by being themselves \'usual suspects\', already inside this sex regulatory network. This article is part of the theme issue \'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)\'.

The δ2H and δ18O of 105 salmonids cultured in freshwater and seawater and from different regions were combined with linear discriminant analysis (LDA), k-nearest neighbor (KNN), and random forest (RF) to create discrimination models. To assess the stability of the discrimination models, seasonal variation in δ2H and δ18O in salmonids cultured in different systems was studied. δ2H and δ18O were significantly different between salmonids cultured in freshwater and seawater and from different geographical origins. δ2H and δ18O of salmonids cultured in an open system were vulnerable to seasonal effects. The KNN model had 100% accuracy for identifying the production methods of salmonids and was less affected by seasonal variation. The RF model had the highest accuracy for identifying the geographical origins of salmonids with an accuracy of over 80%. Thus, δ2H and δ18O were more effective for identifying the production methods of salmonids than their geographical origins.

The flavor of salmonids is affected by species and origin. Sources of salmonid fish fillets are complex and difficult to identify and label fraud occasionally occurs in the market. In this study, headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS), electronic nose, electronic tongue and amino acid detection technologies were used to analyze flavor compounds in two salmonid species from different geographical origins. Fingerprints of volatile compounds of salmonid were constructed using HS-GC-IMS technology. Free amino acid (FAA) content differed in salmonids from different geographical origins. Regarding salmonid odor, HS-GC-IMS analysis results were basically consistent with those of the electronic nose. Regarding taste, the conclusions drawn from the electronic tongue were consistent with the amino acid test results. Therefore, our results demonstrate that flavor compounds can be used to distinguish salmonids from different geographical origins, providing a new dimension to food safety and authenticity. Furthermore, HS-GC-IMS, electronic nose and tongue can be used as tools in the market to identify food fraud.

Knowledge of how temperature influences animal behavior is critical to understanding and predicting impacts of changing climate on individual species and biotic interactions. However, the effects of climate change, especially winter warming in freshwater systems, on fish behaviors and the use of chemical information have been largely unexplored. Qinling lenok Brachymystax lenok tsinlingensis, an endangered salmonid species endemic to the Qinling Mountain Range, China, is currently experiencing population decline and is a potential biological indicator of warming winter climate effects on freshwater fishes due to its temperature sensitivity and required habitat of small, cold-water streams. Our objective was to determine if transient winter warming (increases of ~4 °C) consistent with seasonal maxima in line with near-future climate projections will affect antipredator responses to damage-released chemical alarm cues in B. lenok tsinlingensis. Wild fish were collected during winter and held in captivity under food deprivation for four days, during which half were acclimated to a warmer temperature (6 °C) while the other half were maintained at ambient levels (2 °C). Individual acclimated fish were then exposed to injections of either conspecific alarm cues to simulate elevated predation risk or stream water as a control treatment. Focal fish demonstrated responses consistent with antipredator behaviors to alarm cues at ambient temperature, but no significant behavioral responses to alarm cues were found relative to controls at the warmer temperature. These results support our hypothesis that winter warming will negatively influence antipredator responses and indicate that projected warmer temperature patterns in winter may have significant impacts on chemically mediated predator-prey interactions in cold-water streams.

An increase in urban and agricultural application of pyrethroid insecticides in the San Francisco Bay Estuary and Sacramento San Joaquin Delta has raised concern for the populations of several salmonids, including Chinook salmon (Oncorhynchus tshawytscha). Bifenthrin, a type I pyrethroid, is among the most frequently detected pyrethroids in the Bay-Delta watershed, with surface water concentrations often exceeding chronic toxicity thresholds for several invertebrate and fish species. To better understand the mechanisms of bifenthrin-induced neurotoxicity, juvenile Chinook salmon were exposed to concentrations of bifenthrin previously measured in the Delta. Non-targeted metabolomic profiles were used to identify transcriptomic changes in the brains of bifenthrin-exposed fish. Pathway analysis software predicted increased apoptotic, inflammatory, and reactive oxygen species (ROS) responses in Chinook following exposure to 0.15 and 1.50 μg/L bifenthrin for 96 h. These responses were largely driven by reduced levels of inosine, hypoxanthine, and guanosine. Subsequently, in the brain, the expression of caspase 3, a predominant effector for apoptosis, was significantly upregulated following exposure to 1.50 μg/L bifenthrin. This data suggests that metabolites involved in inflammatory and apoptotic responses, as well as those involved in maintaining proper neuronal function may be disrupted following sublethal exposure to bifenthrin and further suggests that additional population studies should focus on behavioral responses associated with impaired brain function.

BACKGROUND: Financial loss and health risk caused by the substitution of rainbow trout for other salmonid species have become a common issue around the world. The situation could be further exacerbated in China by the \'abused\' common name of San Wen Yu (the corresponding Chinese ideogram ) for salmonids, considering the absence of a standardized naming system for seafood species. To prevent such episodes, the present study aimed to develop novel loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays targeting the mitochondrial cytochrome b gene for rapid identification of rainbow trout in processed fish products.
RESULTS: Rainbow trout-specific primers (LAMP and PCR) were designed, and the specificity against 23 different fish species was confirmed. The minimum amount of detectable DNA for LAMP assay reached 500 pg, up to 10-fold less than for PCR assay. In addition to agarose gel electrophoresis, naked-eye inspection of the LAMP-positive samples using SYBR Green I under daylight or ultraviolet light was also validated. Finally, commercial San Wen Yu products made from rainbow trout could be accurately identified using the newly developed LAMP and PCR assays, further cross-confirmed by mini DNA barcoding and neighbor-joining dendrograms.
CONCLUSIONS: The LAMP and PCR assays established in the study allow a fast and accurate identification of rainbow trout in processed fish products. © 2020 Society of Chemical Industry.

Mammalian interleukin (IL)-2 is a cytokine centrally involved in the differentiation and survival of CD4+ T helper subsets and CD4+ T regulatory cells and in activation of cytotoxic effector lymphocytes. In bony fish, IL2 orthologs have been identified with an additional divergent IL2-Like gene on the same locus present in several fish species. We report here two divergent IL2 paralogs, IL2A and IL2B, in salmonids that originated from the whole genome duplication event in this fish lineage. The salmonid IL2 paralogs differ not only in sequence but also in exon sizes. The IL-2 isoforms that are encoded have disparate pI values and may have evolved to preferentially bind specific IL-2 receptors. Rainbow trout IL2 paralogs are highly expressed in thymus, spleen, gills, kidney and intestine, important tissues/organs in fish T cell development and function. Their expression in peripheral blood leukocytes (PBL) is low constitutively but can be upregulated by the mixed leukocyte reaction, by the T cell mitogen phytohemagglutinin and by signal mimics of T cell activation (phorbol 12-myristate 13-acetate and calcium ionophore). Both trout IL-2 isoforms promoted PBL proliferation and sustained high-level expression of CD4 and CD8, suggesting that trout IL-2 isoforms are T cell growth/survival factors mainly expressed by activated T cells. The recombinant proteins for these two trout IL2 paralogs have been produced in E. coli and possess shared but also distinct bioactivities. IL-2A, but not IL-2B, induced IL12P35A1 and CXCR1 expression in PBL. IL-2B had a stronger effect on upregulation of the T helper 1 (Th1) cytokine interferon-γ (IFNγ) and could sustain CD8α and CD8β expression levels. Nevertheless, both cytokines upregulated key Th1 (IFNγ1, IFNγ2, TNFα2 and IL12) and T helper 2 (Th2) cytokines (IL4/13B1 and IL4/13B2), cytokine and chemokine receptors and the antimicrobial peptide cathelicidin-1 but had limited effects on T helper 17 cytokines and TGFβ1 in PBL. They could also enhance PBL phagocytosis. These results suggest, for the first time in fish, that IL-2 isoforms may have an important role in regulating Th1 and Th2 cell development, and innate and adaptive host defenses in fish, and shed light on lineage-specific expansion, evolution, and functional diversification of IL2 in vertebrates.