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ABSTRACT Fluctuating salinity is symptomatic of climate change challenging aquatic species. The melting of polar ice, rising sea levels, coastal surface and groundwater salinization, and increased evaporation in arid habitats alter salinity worldwide. Moreover, the frequency and intensity of extreme weather events such as rainstorms and floods increase, causing rapid shifts in brackish and coastal habitat salinity. Such salinity alterations disrupt homeostasis and ultimately diminish the fitness, of aquatic organisms by interfering with metabolism, reproduction, immunity, and other critical aspects of physiology. Proteins are central to these physiological mechanisms. They represent the molecular building blocks of phenotypes that govern organismal responses to environmental challenges. Environmental cues regulate proteins in a concerted fashion, necessitating holistic analyses of proteomes for comprehending salinity stress responses. Proteomics approaches reveal molecular causes of population declines and enable holistic bioindication geared toward timely interventions to prevent local extinctions. Proteomics analyses of salinity effects on aquatic organisms have been performed since the mid‐1990s, propelled by the invention of two‐dimensional protein gels, soft ionization techniques for mass spectrometry (MS), and nano‐liquid chromatography in the 1970s and 1980s. This review summarizes the current knowledge on salinity regulation of proteomes from aquatic organisms, including key methodological advances over the past decades. 

Abstract BackgroundHistone post-translational modifications (PTMs) are epigenetic marks that can be induced by environmental stress and elicit heritable patterns of gene expression. To investigate this process in an ecological context, we characterized the influence of salinity stress on histone PTMs within the gills, kidney, and testes of Mozambique tilapia (Oreochromis mossambicus). A total of 221 histone PTMs were quantified in each tissue sample and compared between freshwater-adapted fish exposed to salinity treatments that varied in intensity and duration. ResultsFour salinity-responsive histone PTMs were identified in this study. When freshwater-adapted fish were exposed to seawater for two hours, the relative abundance of H1K16ub significantly increased in the gills. Long-term salinity stress elicited changes in both the gills and testes. When freshwater-adapted fish were exposed to a pulse of severe salinity stress, where salinity gradually increased from freshwater to a maximum of 82.5 g/kg, the relative abundance of H1S1ac significantly decreased in the gills. Under the same conditions, the relative abundance of both H3K14ac and H3K18ub decreased significantly in the testes of Mozambique tilapia. ConclusionsThis study demonstrates that salinity stress can alter histone PTMs in the gills and gonads of Mozambique tilapia, which, respectively, signify a potential for histone PTMs to be involved in salinity acclimation and adaptation in euryhaline fishes. These results thereby add to a growing body of evidence that epigenetic mechanisms may be involved in such processes. 

Abstract Species living in changing environments require the acclimatization of individual organisms, which may be significantly influenced by allele specific expression (ASE). Data from RNA-seq experiments can be used to identify and quantify the expressed alleles. However, conventional allele matching to the reference genome creates a mapping bias towards the reference allele that prevents a reliable estimation of the allele counts. We developed a pipeline that allows identification and unbiased quantification of the alleles corresponding to an RNA-seq dataset, without any previous knowledge of the haplotype. To achieve the unbiased mapping, we generate two pseudogenomes by substituting the alternative alleles on the reference genome. The SNPs are further called against each pseudogenome, providing two SNP data-sets that are averaged for calculation of the allele depth to be merged in a final SNP calling file. The pipeline presented here can calculate ASE in non-model organisms and can be applied to previous RNA-seq data-sets for expanding studies in gene expression regulation. 

Abstract MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize anO. mossambicusbrain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonalmyca(a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced amycako cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonalmycako cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linkingmycato cellular osmoregulation and other cell functions. 

Abstract Salinity tolerance in fish involves a suite of physiological changes, but a cohesive theory leading to a mechanistic understanding at the organismal level is lacking. To examine the potential of adapting energy homeostasis theory in the context of salinity stress in teleost fish,Oreochromis mossambicuswere acclimated to hypersalinity at multiple rates and durations to determine salinity ranges of tolerance and resistance. Over 3000 proteins were quantified simultaneously to analyze molecular phenotypes associated with hypersalinity. A species‐ and tissue‐specific data‐independent acquisition (DIA) assay library of MSMS spectra was created. Protein networks representing complex molecular phenotypes associated with salinity acclimation were generated.O. mossambicushas a wide “zone of resistance” from 75 g/kg salinity to 120 g/kg. Crossing into the zone of resistance resulted in marked phenotypic changes including blood osmolality over 400 mOsm/kg, reduced body condition, and cessation of feeding. Protein networks impacted by hypersalinity consist of electron transport chain (ETC) proteins and specific osmoregulatory proteins. Cytoskeletal, cell adhesion, and extracellular matrix proteins are enriched in networks that are sensitive to the critical salinity threshold. These network analyses identify specific proteome changes that are associated with distinct zones described by energy homeostasis theory and distinguish them from general hypersalinity‐induced proteome changes. 

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia ( Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyper-osmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (p < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (p<0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO induced activation of the MIB pathway. 

The California Grunion, Leuresthes tenuis, can experience a broad range of salinities during their early life stages, with their nursery grounds measuring salinities of 21 to 42ppt under normal conditions. Due to the unique subterrestrial incubation of their eggs within the sand of California beaches, it is not unlikely for this marine fish to hatch in non-seawater conditions, whereafter the larvae may transition to relatively diluted estuaries or concentrated harbors to develop as juveniles for several months. Consequently, they are an ideal system to study the impacts of ecologically relevant salinity stress on the alteration of proteins and histone post-translational modifications (PTMs) during early development. The study of the relationship between the critical window of development (CWD), in which sexual bipotential is lost, and the longevity of protein landscape and histone PTM changes has not been widely explored. The CWD is of interest as this is when the gametes of this species become set. Therefore, we hypothesize that histone PTM changes that occur during this time may thus be a form of heritable epigenetics. Thus, hypo-and-hyperosmotic exposures of L. tenuis hatchlings during this window, and past it, attempt to identify if the CWD is relevant to the presence and persistence of protein and histone PTM changes under salinity stress. We hypothesize that exposure to osmotic stress during the CWD will result in induced compensatory mechanisms in the protein landscape of the California grunion larvae, as well as persistent alterations in the histone PTMs in the larvae. To elucidate the relationship between these factors, California grunion larvae were exposed in replicate to one of three chronic salinity treatments (16ppt, 32ppt, or 40ppt) for the duration of their CWD (68dph), with 16ppt and 40ppt inducing hypoosmotic and hyperosmotic stress respectively. Chronic post-CWD exposures and recovery times until 98dph were used to determine whether the CWD or prolonged salinity exposures were key to alterations in the histone PTM and protein landscape of the larvae. After each timepoint, L. tenuis were culled and processed for histone PTM enrichment and proteomic analysis. At the time of culling, there were no significant differences in survivorship between replicates and experimental groups, with statistically significant different deaths between groups occurring only shortly after hatching. Skyline and MSstats were used in the analysis of the statistical significance of differential regulation of proteins and histone PTMs under stress conditions versus the controls. Of special interest is the osmoregulatory mechanisms involved in the myo-inositol biosynthesis pathway, with analysis focusing on the abundance of histone acetylation, amidation, ubiquitylation, and 4-hydroxynonelation in histone PTMs, as the accumulations of these have been found to be correlated with osmotic stress in some fish tissues. Funding provided by NSF IOS-2209383 and NSF GRFP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process. 

This study explored the feasibility of using fish skin bandages as a therapeutic option for third-degree skin burns. Following the California wildfires, clinical observations of animals with third-degree skin burns demonstrated increased comfort levels and reduced pain when treated with tilapia fish skin. Proteomic analysis of the fish skin revealed the presence of antimicrobial peptides. In combination with histological and other data, these results suggest that fish skin can serve as an innovative and cost-effective therapeutic alternative for burn victims to facilitate vascularization and reduce bacterial infection.