Non-model Fish Research Articles

Specific biomarkers and neurons distribution of different brain regions in largemouth bass (Micropterus salmoides).

The brain regulates multiple physiological processes in fish. Despite this, knowledge about the basic structure and function of distinct brain regions in non-model fish species remains limited due to their diversity and the scarcity of common biomarkers. In the present study, four major brain parts, the telencephalon, diencephalon, mesencephalon and rhombencephalon, were isolated in largemouth bass, Micropterus salmoides. Within these parts, nine brain regions and 74 nuclei were further identified through morphological and cytoarchitectonic analysis. Transcriptome analysis revealed a total of 7153 region-highly expressed genes and 176 region-specifically expressed genes. Genes related to growth, reproduction, emotion, learning, and memory were significantly overexpressed in the olfactory bulb and telencephalon (OBT). Feeding and stress-related genes were in the hypothalamus (Hy). Visual system-related genes were predominantly enriched in the optic tectum (OT), while vision and hearing-related genes were widely expressed in the cerebellum (Ce) region. Sensory input and motor output-related genes were in the medulla oblongata (Mo). Osmoregulation, stress response, sleep/wake cycles, and reproduction-related genes were highly expressed in the remaining brain (RB). Three candidate marker genes were further identified for each brain regions, such as neuropeptide FF (npff) for OBT, pro-melanin-concentrating hormone (pmch) for Hy, vesicular inhibitory amino acid transporter (viaat) for OT, excitatory amino acid transporter 1 (eaat1) for Ce, peripherin (prph) for Mo, and isotocin neurophysin (itnp) for RB. Additionally, the distribution of seven neurotransmitter-type neurons and five types of non-neuronal cells across different brain regions were analyzed by examining the expression of their marker genes. Notably, marker genes for glutamatergic and GABAergic neurons showed the highest expression levels across all brain regions. Similarly, the marker gene for radial astrocytes exhibited high expression compared to other markers, while those for microglia were the least expressed. Overall, our results provide a comprehensive overview of the structural and functional characteristics of distinct brain regions in the largemouth bass, which offers a valuable resource for understanding the role of central nervous system in regulating physiological processes in teleost.

Mapping the cellular landscape of Atlantic salmon head kidney by single cell and single nucleus transcriptomics

Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.

Identification of key regulatory pathways and genes involved in the dynamic progression of pigmentation in Sinibrama taeniatus

IntorductionColoration is a prominent trait in fish that is closely linked to their market value. Fish exhibit a diverse range of body colors, making them an excellent model for studying molecular mechanisms underlying pigmentation. Although regulatory pathways involved in pigmentation have been extensively studied in model fish such as zebrafish and medaka, the presence of varying chromatophore types across different fish species suggests that fish pigmentation mechanisms are not fixed. Therefore, more studies should be conducted on non-model fish. Sinibrama taeniatus is an endemic fish in the upper Yangtze River that is highly valued for its ornamental and edible properties.MethodsIn this study, we identified three chromatophore types in S. taeniatus and investigated changes in body color, chromatophores, and pigments at different developmental stages. Subsequently, RNA-seq analysis revealed that retinol metabolism, thyroid hormone synthesis, purine metabolism, and pyrimidine metabolism pathway are closely associated with pigmentation.ResultsWeighted gene co-expression network analysis (WGCNA) identified several hub genes, including mitf, wnt 9a, wnt10b, wnt11, adcy5, edn1, adcy8, and rdh8, that may play an important role in pigmentation.DiscussionOverall, our findings provide valuable insights into the role of genes and pathways in fish pigmentation and offer useful information for body color-based molecular breeding programs in aquaculture.

DNA repair genes play a variety of roles in the development of fish embryos.

Embryogenesis is one of the most important life stages because it determines an organism's healthy growth. However, embryos of externally fertilizing species, such as most fish, are directly exposed to the environment during development and may be threatened by DNA damaging factors (pollutants, UV, reactive oxygen species). To counteract the negative effects of DNA fragmentation, fish embryos evolved complex damage response pathways. DNA repair pathways have been extensively studied in some fish species, such as zebrafish (Danio rerio). Our literature review, on the other hand, revealed a paucity of knowledge about DNA damage response and repair in non-model aquaculture fish species. Further, several pieces of evidence underlie the additional role of DNA repair genes and proteins in organogenesis, spatiotemporal localization in different tissue, and its indispensability for normal embryo development. In this review, we will summarize features of different DNA repair pathways in course of fish embryo development. We describe how the expression of DNA repair genes and proteins is regulated during development, their organogenetic roles, and how the expression of DNA repair genes changes in response to genotoxic stress. This will aid in addressing the link between genotoxic stress and embryo phenotype. Furthermore, available data indicate that embryos can repair damaged DNA, but the effects of early-life stress may manifest later in life as behavioral changes, neoplasia, or neurodegeneration. Overall, we conclude that more research on DNA repair in fish embryos is needed.

Influence of RNA-Seq library construction, sampling methods, and tissue harvesting time on gene expression estimation.

RNA sequencing (RNA-Seq) is popular for measuring gene expression in non-model organisms, including wild populations. While RNA-Seq can detect gene expression variation among wild-caught individuals and yield important insights into biological function, sampling methods can also affect gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq library types: 3' RNA-Seq (QuantSeq) and whole mRNA-Seq (NEB). We evaluated effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 min after euthanasia on estimated gene expression in blood, gill, and muscle. We found no significant differences in gene expression between sampling methods or tissue collection times with either library type. When library types were compared using the same blood samples, 58% of genes detected by both NEB and QuantSeq showed significantly different expression between library types, and NEB detected 31% more genes than QuantSeq. Although the two library types recovered different numbers of genes and expression levels, results with NEB and QuantSeq were consistent in that neither library type showed differences in gene expression between sampling methods and tissue harvesting times. Our study suggests that researchers can safely rely on different fish sampling strategies in the field. In addition, while QuantSeq is more cost effective, NEB detects more expressed genes. Therefore, when it is crucial to detect as many genes as possible (especially low expressed genes), when alternative splicing is of interest, or when working with an organism lacking good genomic resources, whole mRNA-Seq is more powerful.

Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments.

Biomonitoring surveys make use of metabarcoding tools to describe the community composition. These studies match their sequencing results against public genomic databases to identify the species. However, mitochondrial genomic reference data are yet incomplete, only a few genes may be available, or the suitability of existing sequence data is suboptimal for species level resolution. Here, we present a dedicated and cost-effective workflow with no DNA amplification for generating complete fish mitogenomes for the purpose of strengthening fish mitochondrial databases. Two different strategies using long fragment sequencing with Oxford Nanopore technology coupled with mitochondrial DNA enrichment were used. One where the enrichment is achieved by preferential isolation of mitochondria followed by DNA extraction and nuclear DNA depletion ("mitoenrichment"). A second enrichment approach takes advantage of the CRISPR Cas9 targeted scission on previously dephosphorylated DNA ("targeted mitosequencing"). The sequencing results varied between tissue, species, and integrity of the DNA. The mitoenrichment method yielded 0.17%-12.33% of sequences on target and a mean coverage ranging from 74.9 to 805-fold. The targeted mitosequencing experiment from native genomic DNA yielded 1.83%-55% of sequences on target and a 38 to 2123-fold mean coverage. These produced complete mitogenomes of species with homopolymeric regions, tandem repeats, and gene rearrangements. We demonstrate that deep sequencing of long fragments of native fish DNA can be achieved with low computational resources in a cost-effective manner, opening the discovery of mitogenomes of nonmodel or understudied fish taxa to a broad range of laboratories worldwide.

Gut microbiota of Anabas testudineus (Bloch, 1792) in the e-waste dismantling region: In situ status and relationship with internal metal burden.

Due to the production of large quantities of electronic waste (e-waste), unsafe dismantling has caused serious pollution as well as toxicological impacts on both wildlife and humans. As an important aspect of physiology and health, the wildlife's gut microbiota and its changes induced by pollution have been recruiting increasing concerns. To reveal the gut microbiota-related ecotoxicology induced by e-waste dismantling, this study resolves the gut microbiota profile of Anabas testudineus, a native highly adapted nonmodel fish under the in situ exposure, and reveals whether and how the microbiota was altered. The comparisons are made by collecting samples from different e-waste polluted sites in Guiyu (a town in South China) and a nearby reference (nonpolluted) site. The overall gut microbiota landscape of A. testudineus is similar to that of other reported fishes, with an average of ∼300 OTUs, and constituted by Firmicutes (34.51%), Fusobacteria (29.16%) as the major phyla. Obviously different liver metal burdens/fingerprints were observed between the e-waste and reference sites. Accordingly, although the alpha-diversity (ACE, Simpson, and Shannon) of the gut microbiota did not significantly vary, a detailed exploration of the microbiota constitution indicated significant differences at various taxonomic levels, including a series of significantly different species and biomarkers, and showing site-specific beta-diversity clustering patterns. Interestingly, a few bacteria with greater abundance in the fish gut of e-waste polluted sites were also reported to present in other contaminated environments, have a role in wastewater treatment, be capable to transform metal, etc. Redundancy analysis (RDA) and Pearson association analyses indicated significant associations between Mn and Cetobacterium somerae (Pearson r=0.3612, p=0.0008) and between Pb and Clostridium colicanis (Pearson r=0.5151, p < 0.0001). In summary, pollution from e-waste dismantling may have a role in altering the fish gut microbiota, and this research provides insights for better understanding e-waste ecotoxicology and improving future conservation.

Transcriptome and Proteome Analyses Reveal Stage-Specific DNA Damage Response in Embryos of Sturgeon (Acipenser ruthenus).

DNA damage during early life stages may have a negative effect on embryo development, inducing mortality and malformations that have long-lasting effects during adult life. Therefore, in the current study, we analyzed the effect of DNA damage induced by genotoxicants (camptothecin (CPT) and olaparib) at different stages of embryo development. The survival, DNA fragmentation, transcriptome, and proteome of the endangered sturgeon Acipenser ruthenus were analyzed. Sturgeons are non-model fish species that can provide new insights into the DNA damage response and embryo development. The transcriptomic and proteomic patterns changed significantly after exposure to genotoxicants in a stage-dependent manner. The results of this study indicate a correlation between phenotype formation and changes in transcriptomic and proteomic profiles. CPT and olaparib downregulated oxidative phosphorylation and metabolic pathways, and upregulated pathways involved in nucleotide excision repair, base excision repair, and homologous recombination. We observed the upregulated expression of zona pellucida sperm-binding proteins in all treatment groups, as well as the upregulation of several glycolytic enzymes. The analysis of gene expression revealed several markers of DNA damage response and adaptive stress response, which could be applied in toxicological studies on fish embryos. This study is the first complex analysis of the DNA damage response in endangered sturgeons.

De novo assembly, transcriptome characterization and marker discovery in Indian major carp, Labeo rohita through pyrosequencing.

Labeo rohita, one of the Indian major carps, is the most popular culture species in Indian subcontinent due to its consumer preference and delicacy. A selective breeding program for harvest body weight has resulted in an average genetic gain of 17% per generation. Transcriptome resource for this species is scanty. Here, we have characterized the liver and muscle transcriptomes of rohu using Roche 454 GS-FLX next generation sequencing platform. In total, 1.2 million reads were generated, de novo assembly and clustering resulted in 4171 transcripts. Out of these, 4171 had significant blast hit against NCBI nr database, and 2130 transcripts were successfully annotated. In total, 289 SSRs were identified with an identification rate of 5.8%, and dinucleotide repeat motifs were observed to be the most abundant SSRs. Further, 2231 putative SNPs were identified with high confidence. Validation of eight putative SNPs using Sanger sequencing resulted in 100% true SNPs. Significant allelic imbalance of M1, M4 and M5 loci between growth selected and control individual were observed. Furthermore, 13 transcription factors were identified in the present study belonging to six different transcription factor families. The present study demonstrated the utility of RNAseq to develop genomics resources in non-model fish species, and the marker resources developed would support the genetic improvement program of this species.

Susceptibility of polar cod (Boreogadus saida) to a model carcinogen

Studies that aim to characterise the susceptibility of the ecologically relevant and non-model fish polar cod (Boreogadus saida) to model carcinogens are required. Polar cod were exposed under laboratory conditions for six months to control, 0.03 μg BaP/g fish/week and 0.3 μg BaP/g fish/week dietary benzo(a)pyrene (BaP), a reference carcinogen. The concentrations of the 3-OH-BaP bile metabolite and transcriptional responses of genes involved in DNA adduct recognition (xpc), helicase activity (xpd), DNA repair (xpf, rad51) and tumour suppression (tp53) were assessed after 0, 1, 3 and 6 months of exposure, alongside body condition indexes (gonadosomatic index, hepatosomatic index and condition factor). Micronuclei and nuclear abnormalities in blood and spleen, and liver histopathological endpoints were assessed at the end of the experiment.Fish grew steadily over the whole experiment and no mortality was recorded. The concentrations of 3-OH-BaP increased significantly after 1 month of exposure to the highest BaP concentration and after 6 months of exposure to all BaP concentrations showing the biotransformation of the mother compound. Nevertheless, no significant induction of gene transcripts involved in DNA damage repair or tumour suppression were observed at the selected sampling times. These results together with the absence of chromosomal damage in blood and spleen cells, the subtle increase in nuclear abnormalities observed in spleen cells and the low occurrence of foci of cellular alteration suggested that the exposure was below the threshold of observable effects. Taken together, the results showed that polar cod was not susceptible to carcinogenesis using the BaP exposure regime employed herein.

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Studies that aim to characterise the susceptibility of the ecologically relevant and non-model fish polar cod (Boreogadus saida) to model carcinogens are required. Polar cod were exposed under laboratory conditions for six months to control, 0.03 μg BaP/g fish/week and 0.3 μg BaP/g fish/week dietary benzo(a)pyrene (BaP), a reference carcinogen. The concentrations of the 3-OH-BaP bile metabolite and transcriptional responses of genes involved in DNA adduct recognition (xpc), helicase activity (xpd), DNA repair (xpf, rad51) and tumour suppression (tp53) were assessed after 0, 1, 3 and 6 months of exposure, alongside body condition indexes (gonadosomatic index, hepatosomatic index and condition factor). Micronuclei and nuclear abnormalities in blood and spleen, and liver histopathological endpoints were assessed at the end of the experiment.Fish grew steadily over the whole experiment and no mortality was recorded. The concentrations of 3-OH-BaP increased significantly after 1 month of exposure to the highest BaP concentration and after 6 months of exposure to all BaP concentrations showing the biotransformation of the mother compound. Nevertheless, no significant induction of gene transcripts involved in DNA damage repair or tumour suppression were observed at the selected sampling times. These results together with the absence of chromosomal damage in blood and spleen cells, the subtle increase in nuclear abnormalities observed in spleen cells and the low occurrence of foci of cellular alteration suggested that the exposure was below the threshold of observable effects. Taken together, the results showed that polar cod was not susceptible to carcinogenesis using the BaP exposure regime employed herein.

RNA sequencing describes both population structure and plasticity-selection dynamics in a non-model fish

BackgroundMessenger RNA sequencing is becoming more common in studies of non-model species and is most often used for gene expression-based investigations. However, the method holds potential for numerous other applications as well—including analyses of alternative splicing, population structure, and signatures of selection. To maximize the utility of mRNA data sets, distinct analyses may be combined such as by exploring dynamics between gene expression with signatures of selection in the context of population structure. Here, we compare two published data sets describing two populations of a minnow species endemic to the San Francisco Estuary (Sacramento splittail, Pogonichthys macrolepidotus): a microsatellite data set showing population structure, and an mRNA whole transcriptome data set obtained after the two populations were exposed to a salinity challenge. We compared measures of population structure and genetic variation using single nucleotide polymorphisms (SNPs) called from mRNA from the whole transcriptome sequencing study with those patterns determined from microsatellites. For investigating plasticity and evolution, intra- and inter-population transcriptome plasticity was investigated with differential gene expression, differential exon usage, and gene expression variation. Outlier SNP analysis was also performed on the mRNA data set and signatures of selection and phenotypic plasticity were investigated on an individual-gene basis.ResultsWe found that mRNA sequencing revealed patterns of population structure consistent with those found with microsatellites, but with lower magnitudes of genetic variation and population differentiation consistent with widespread purifying selection expected when using mRNA. In addition, within individual genes, phenotypic plasticity or signatures of selection were found in almost mutual exclusion (except heatr6, nfu1, slc22a6, sya, and mmp13).ConclusionsThese results show that an mRNA sequencing data set may have multiple uses, including describing population structure and for investigating the mechanistic interplay of evolution and plasticity in adaptation. MRNA sequencing thus complements traditional sequencing methods used for population genetics, in addition to its utility for describing phenotypic plasticity.

Assessment on the adverse effects on different kinds of fish induced by methamphetamine during the natural attenuation process based on adverse outcome pathway.

The adverse effects on model fish induced by methamphetamine (METH) have been revealed. However, the toxicity of METH on different kinds of non-model fish during the natural attenuation remained unclear. Hence, in this study, we for the first time established a static lab-scale aquatic ecosystem spiked with METH (initial levels at 25μg/L) for 40days to estimate its metabolism and toxicity in Chinese medaka, rosy bitterling, loach, and mosquito fish. The concentrations of METH in water and fish's brain were detected termly. The physiological functions, histopathology of brain, neurotransmitters contents, and expressions of associated genes of the four kinds of fish were determined at day 0, 20, and 40, respectively. The results indicated METH could be remarkably accumulated in fish brains with the distribution factor vs water (DFw) at 232.5-folds, and attenuated both in water and fish body during the exposure. METH caused physiological functions (i.e., swimming trajectories, locomotion distances, and feeding rates) disorders of the four kinds of fish, and stimulated surfacing behavior of loach. Tissue and macro/micromolecular biomarkers including histopathology, neurotransmitters (i.e., dopamine, serotonin, and norepinephrine), and mRNA, were similarly affected by METH. Mitogen-activated protein kinase (MAPKs) signaling pathway, P53-regulated apoptosis signaling pathway, N-methyl-d-aspartate-dopamine system, and mTOR signaling pathway of different kinds of fish were regulated by METH. Additionally, the impairments of the physiological and macromolecular indicators of fish could be alleviated as the natural attenuation of METH occurred. All the biomarkers, as well as the recovery effects during the exposure were integrated onto an adverse outcome pathway (AOP) framework. The key event was the micromolecular indicators (genes). The adverse outcomes at individual and population levels would result in the ecological consequences, implying the imperative to consider the natural attenuation process while assessing the environmental risk of METH.

ReCodLiver0.9: Overcoming Challenges in Genome-Scale Metabolic Reconstruction of a Non-model Species.

The availability of genome sequences, annotations, and knowledge of the biochemistry underlying metabolic transformations has led to the generation of metabolic network reconstructions for a wide range of organisms in bacteria, archaea, and eukaryotes. When modeled using mathematical representations, a reconstruction can simulate underlying genotype-phenotype relationships. Accordingly, genome-scale metabolic models (GEMs) can be used to predict the response of organisms to genetic and environmental variations. A bottom-up reconstruction procedure typically starts by generating a draft model from existing annotation data on a target organism. For model species, this part of the process can be straightforward, due to the abundant organism-specific biochemical data. However, the process becomes complicated for non-model less-annotated species. In this paper, we present a draft liver reconstruction, ReCodLiver0.9, of Atlantic cod (Gadus morhua), a non-model teleost fish, as a practicable guide for cases with comparably few resources. Although the reconstruction is considered a draft version, we show that it already has utility in elucidating metabolic response mechanisms to environmental toxicants by mapping gene expression data of exposure experiments to the resulting model.

Polyunsaturated Fatty Acids Influence LPS-Induced Inflammation of Fish Macrophages Through Differential Modulation of Pathogen Recognition and p38 MAPK/NF-κB Signaling.

Polyunsaturated fatty acids (PUFAs) not only serve as essential nutrients but also function as modulators of the immune response in marine fish. However, their immunomodulatory mechanism is poorly understood given that the underlying regulation of the innate immune response in fish has not been fully elucidated. Hence, study of the innate immunity of fish could help elucidate the mechanism by which PUFAs affect the fish immune response. Here, we used combined transcriptome analysis and in vitro experimentation to study the mechanism of LPS-induced inflammation. Transcriptome profiling indicated that LPS elicited strong pro-inflammatory responses featuring high expression levels of pathogen recognition receptors (PRRs) and cytokines along with the activation of NF-κB and MAPK signaling pathways. The transcription factor p65 alone could increase the transcription of IL1β by binding to the promoter of IL1β, and this promoting effect disappeared after mutation or deletion of its binding sites. We then examined the effects of PUFAs on the levels of gene expression and the abundance of proteins of critical kinases associated with LPS-induced inflammation. We found that LA exerts pro-inflammatory response while ALA, EPA, and DHA induced anti-inflammatory effects by modulating the expression of PRRs, phosphorylation of IKK and p38, and the nuclear translocation of p65. Overall, this study advances our understanding of the regulatory mechanisms by which PUFAs regulate LPS-induced inflammation in a non-model fish species.

Exploring the Expression of Cardiac Regulators in a Vertebrate Extremophile: The Cichlid Fish Oreochromis (Alcolapia) alcalica.

Although it is widely accepted that the cellular and molecular mechanisms of vertebrate cardiac development are evolutionarily conserved, this is on the basis of data from only a few model organisms suited to laboratory studies. Here, we investigate gene expression during cardiac development in the extremophile, non-model fish species, Oreochromis (Alcolapia) alcalica. We first characterise the early development of O. alcalica and observe extensive vascularisation across the yolk prior to hatching. We further investigate heart development by identifying and cloning O. alcalica orthologues of conserved cardiac transcription factors gata4, tbx5, and mef2c for analysis by in situ hybridisation. Expression of these three key cardiac developmental regulators also reveals other aspects of O. alcalica development, as these genes are expressed in developing blood, limb, eyes, and muscle, as well as the heart. Our data support the notion that O. alcalica is a direct-developing vertebrate that shares the highly conserved molecular regulation of the vertebrate body plan. However, the expression of gata4 in O. alcalica reveals interesting differences in the development of the circulatory system distinct from that of the well-studied zebrafish. Understanding the development of O. alcalica embryos is an important step towards providing a model for future research into the adaptation to extreme conditions; this is particularly relevant given that anthropogenic-driven climate change will likely result in more freshwater organisms being exposed to less favourable conditions.

Probing SWATH-MS as a tool for proteome level quantification in a nonmodel fish.

Quantitative proteomics via mass spectrometry can provide valuable insight into molecular and phenotypic characteristics of a living system. Recent mass spectrometry developments include data‐independent acquisition (SWATH/DIA‐MS), an accurate, sensitive and reproducible method for analysing the whole proteome. The main requirement for this method is the creation of a comprehensive spectral library. New technologies have emerged producing larger and more accurate species‐specific libraries leading to a progressive collection of proteome references for multiple molecular model species. Here, for the first time, we set out to compare different spectral library constructions using multiple tissues from a coral reef fish to demonstrate its value and feasibility for nonmodel organisms. We created a large spectral library composed of 12,553 protein groups from liver and brain tissues. Via identification of differentially expressed proteins under fish exposure to elevated pCO2 and temperature, we validated the application and usefulness of these different spectral libraries. Successful identification of significant differentially expressed proteins from different environmental exposures occurred using the library with a combination of data‐independent and data‐dependent acquisition methods as well as both tissue types. Further analysis revealed expected patterns of significantly up‐regulated heat shock proteins in a dual condition of ocean warming and acidification indicating the biological accuracy and relevance of the method. This study provides the first reference spectral library for a nonmodel organism. It represents a useful guide for future building of accurate spectral library references in nonmodel organisms allowing the discovery of ecologically relevant changes in the proteome.

Characterization of antiviral immune response induced by poly(I:C) in macrophages of farmed large yellow croaker (Larimichthys crocea)

Fish tend to rely more on their innate immunity to executing defense against viral infection by inducing antiviral gene production. However, the expression pattern and underlying mechanism of fish antiviral responses have yet to be fully defined. In the present study, an in vitro viral infection model was established by exposing head kidney-derived macrophages of large yellow croaker to virus analog, poly(I:C). Transcriptome analysis indicated that poly(I:C) appeared to induce potent antiviral activity featuring dominant interferon a3 (IFNa3) expression through activation of toll-like receptors (TLRs)/TIR-domain-containing adapter-inducing interferon-β (TRIF) and retinoic acid-inducible gene I-like receptors (RLRs)/mitochondrial antiviral signaling protein (MAVS) pathways. Inhibition of nuclear factor κB (NF-κB) and stimulator of interferon genes (STING)/interferon regulatory factor 3 (IRF3) pathways diminished the expression of IFNa3. Mechanistically, transcription factors including p65 and IRF3 could promote expression of IRF3, and activated IRF3 alone further increased the transcriptional activity of IFNa3. We also characterized the promoter of IFNa3 with direct IRF3 binding site which was sufficient to render the transcription of IFNa3. This effect was attenuated after deletion or mutation of the IRF3 binding sites. Taken together, our findings illustrate the distinct transcriptional profiling of fish macrophages triggered by poly(I:C). Also, this work provides new insights into the molecular mechanism underpinning coordinated activation of pathogen recognition and signaling transduction in the antiviral responses of non-model fish species.

Blast2Fish: a reference-based annotation web tool for transcriptome analysis of non-model teleost fish

BackgroundTranscriptome analysis by next-generation sequencing has become a popular technique in recent years. This approach is quite suitable for non-model organism study, as de novo assembly is independent of prior genomic sequences of organisms. De novo sequencing has benefited many studies on commercially important fish species. However, to understand the functions of these assembled sequences, they still need to be annotated with existing sequence databases. By combining Basic Local Alignment Search Tool (BLAST) and Gene Ontology analysis, we were able to identify homologous sequences of assembled sequences and describe their characteristics using pre-defined tags for each gene, though the above conventional annotation results obtained for non-model assembled sequences was still associated with a lack of pre-defined tags and poorly documented records in the database.ResultsWe introduced Blast2Fish, a novel approach for performing functional enrichment analysis on non-model teleost fish transcriptome data. The Blast2Fish pipeline was designed to be a reference-based enrichment method. Instead of annotating the BLAST single top hit by a pre-defined gene-to-tag database, we included 500 hits to search related PubMed articles and parse biological terms. These descriptive terms were then sorted and recorded as annotations for the query. The results showed that Blast2Fish was capable of providing meaningful annotations on immunology topics for non-model fish transcriptome analysis.ConclusionBlast2Fish provides a novel approach for annotating sequences of non-model fish. The reference-based strategy allows annotation to be performed without pre-defined tags for each gene. This method strongly benefits non-model teleost fish studies for gene functional enrichment analysis.

Population Genomics of an Anadromous Hilsa Shad Tenualosa ilisha Species across Its Diverse Migratory Habitats: Discrimination by Fine-Scale Local Adaptation

The migration of anadromous fish in heterogenic environments unceasingly imposes a selective pressure that results in genetic variation for local adaptation. However, discrimination of anadromous fish populations by fine-scale local adaptation is challenging because of their high rate of gene flow, highly connected divergent population, and large population size. Recent advances in next-generation sequencing (NGS) have expanded the prospects of defining the weakly structured population of anadromous fish. Therefore, we used NGS-based restriction site-associated DNA (NextRAD) techniques on 300 individuals of an anadromous Hilsa shad (Tenualosa ilisha) species, collected from nine strategic habitats, across their diverse migratory habitats, which include sea, estuary, and different freshwater rivers. The NextRAD technique successfully identified 15,453 single nucleotide polymorphism (SNP) loci. Outlier tests using the FST OutFLANK and pcadapt approaches identified 74 and 449 SNPs (49 SNPs being common), respectively, as putative adaptive loci under a divergent selection process. Our results, based on the different cluster analyses of these putatively adaptive loci, suggested that local adaptation has divided the Hilsa shad population into two genetically structured clusters, in which marine and estuarine collection sites were dominated by individuals of one genetic cluster and different riverine collection sites were dominated by individuals of another genetic cluster. The phylogenetic analysis revealed that all the riverine populations of Hilsa shad were further subdivided into the north-western riverine (turbid freshwater) and the north-eastern riverine (clear freshwater) ecotypes. Among all of the putatively adaptive loci, only 36 loci were observed to be in the coding region, and the encoded genes might be associated with important biological functions related to the local adaptation of Hilsa shad. In summary, our study provides both neutral and adaptive contexts for the observed genetic divergence of Hilsa shad and, consequently, resolves the previous inconclusive findings on their population genetic structure across their diverse migratory habitats. Moreover, the study has clearly demonstrated that NextRAD sequencing is an innovative approach to explore how dispersal and local adaptation can shape genetic divergence of non-model anadromous fish that intersect diverse migratory habitats during their life-history stages.