Arash Akbarzadeh

Climate change is increasingly affecting Pacific salmon to combinations of thermal, hypoxic, and osmotic stressors that can impair survival. Although genomic tools have enabled detailed insight into stressor responses under controlled conditions, field applications remain limited by the lack of species-specific classifiers capable of resolving physiological condition across multiple stressor contexts. In this study, we developed and experimentally validated transcriptional classifiers for juvenile sockeye salmon ( Oncorhynchus nerka ) using controlled multi-stressor challenges. Fish exposed to a full factorial design of three temperatures (10 °C, 14 °C, 18 °C), three salinities (0, 20, 28 ppt), and two dissolved oxygen levels (> 8 mg L ^-1 , 3–3.5 mg L ^-1 ) over six days, across smolt and de-smolt stages; pre-smolts were additionally challenged with seawater (28 ppt). Gill tissue, a primary interface between fish and their environment, was analyzed using the “Salmon Fit-Chips” microfluidic qPCR tool to quantify gene expression responses. Random Forest models were then used to develop classifiers detecting transcriptional signatures of thermal stress, hypoxic stress, salinity acclimation, and smolt stage. Classifiers achieved high predictive accuracy (85.5%–100%) across all stressors and life stages. Recovery analysis showed that transcriptional stress signals diminished within three days for hypoxia, but lingered longer for thermal stress. By developing sockeye salmon–specific molecular classifiers within a multi-stressor experimental framework, this study advances the Fit-Chip approach and provides a foundation for future field applications, including potential non-lethal monitoring using gill biopsies.

Herbal bioactive compounds are effective in enhancing the antioxidant and immune status of fish, as they effectively neutralize oxidative stress. The present study examined the effectiveness of Ivy ( Hedera helix ) leaf extract (ILE) on growth performance, immune responses, antioxidant parameters, and the expression of immune‐related genes in rainbow trout ( Oncorhynchus mykiss ). Four test diets were prepared as follows: a control diet at 0 mg of ILE (ILE0) and diets of 100 mg (ILE100), 150 mg (ILE150), and 200 mg (ILE200) of ILE/kg. A total number of 240 rainbow trout, with an initial weight of 6.38 ± 0.30 g, were divided into 12 tanks and fed on the experimental diets for 8 weeks. Results indicated that ILE150 and ILE200 exhibited significantly higher final weight (FW), weight gain, and specific growth rate compared to the control and ILE100 treatments. Additionally, fish fed with ILE showed significantly higher activity of digestive enzymes, including trypsin, pepsin, proteases, lipase, and amylase compared to the control group. The experimental diets also significantly affected hematological indices, including erythrocytes, leukocytes, hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration, and mean corpuscular hemoglobin, and their highest levels were observed in ILE150 and ILE200. Furthermore, a notable reduction was observed in aspartate aminotransferase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) activities in all ILE‐supplemented diets. Serum antioxidant enzymes, including catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD), significantly increased in ILE150 and ILE200, but serum malondialdehyde (MDA) levels significantly declined in treatments fed with ILE compared to the control group. Alternative complement, total immunoglobulin, total protein, and lysozyme levels were significantly higher in ILE‐supplemented‐diet treatments. The results also showed a significant upregulation of immune‐related genes, such as interleukin 1β, interleukin‐6, interleukin‐8, and lysozyme, in ILE150 and ILE200 compared to the control and ILE100. In conclusion, administering ILE at doses of 150–200 mg/kg diet could improve the growth performance and immune‐antioxidant capacity of rainbow trout.

Citrus ( Citrus limon ) by‐products, particularly lemon peel, are rich in bioactive compounds with known antioxidant and anti‐inflammatory properties. This study evaluated the effects of lemon peel extract (LPE) supplementation on growth performance, immune responses, and antioxidant activity in Pacific white shrimp ( Litopenaeus vannamei ). Over a 60‐day period, shrimp were fed four diets: a control diet (0%) and three diets with varying levels of LPE (0.5%, 1%, and 2%). The results showed that shrimp fed the 1% LPE diet exhibited significantly higher weight gain (WG), specific growth rate (SGR), and lower feed conversion ratio (FCR) compared to the control group ( p < 0.05). Additionally, shrimp fed 0.5% and 1% LPE showed significantly enhanced activities of antioxidant enzymes (catalase [CAT], glutathione peroxidase [GPX], superoxide dismutase [SOD]), lysozyme, total protein, and hemocyte counts, including large granular cells (GCs), semi‐granulocytes, and hyaline cells (HCs) ( p < 0.05). Total hemocyte counts (THCs) were also significantly higher in all LPE‐treated groups compared to the control ( p < 0.05). Plasma glucose levels were reduced in the 0.5% and 1% LPE treatments, though the decrease in cholesterol was not statistically significant. Moreover, the expression of integrin β , PX , and α2-M mRNA was significantly upregulated in shrimp fed the 2% LPE diet, while LGBP and proPO1 mRNA expression were higher across all LPE treatments. In conclusion, dietary supplementation with LPE, particularly at 0.5% and 1%, enhances growth performance, immune response, and antioxidant activity in L. vannamei . Therefore, LPE presents a promising and cost‐effective feed additive for shrimp aquaculture, with the potential to enhance disease resistance, support immune function, and ultimately improve shrimp health and productivity under farming conditions.

In the present study, we applied the HDR (homology-directed DNA repair) CRISPR-Cas9-mediated knock-in system to accurately insert an optimized foreign bacterial phytase gene at a specific site of the nitrate reductase (NR) gene (exon 2) to achieve homologous recombination with the stability of the transgene and reduce insertion site effects or gene silencing. To this end, we successfully knocked-in the targeted NR gene of Chlamydomonas reinhardtii using the bacterial phytase gene cassette through direct delivery of the CRISPR/Cas9 system as the ribonucleoprotein (RNP) complex consisting of Cas9 protein and the specific single guide RNAs (sgRNAs). The NR insertion site editing was confirmed by PCR and sequencing of the transgene positive clones. Moreover, 24 clones with correct editing were obtained, where the phytase gene cassette was located in exon 2 of the NR gene, and the editing efficiency was determined to be 14.81%. Additionally, site-specific gene expression was analyzed and confirmed using RT-qPCR. Cultivation of the positive knocked-in colonies on the selective media during 10 generations indicated the stability of the correct editing without gene silencing or negative insertion site effects. Our results demonstrated that CRISPR-Cas9-mediated knock-in could be applied for nuclear expression of the heterologous gene of interest, and also confirmed its efficacy as an effective tool for site-specific gene knock-in, avoiding nuclear positional effects and gene silencing in C. reinhardtii. These findings could also provide a new perspective on the advantageous application of RNP-CRISPR/Cas9 gene-editing to accelerate the commercial production of complex recombinant proteins in the food-grade organism “C. reinhardtii”.

A few species of mollusks display color variation in their soft tissues. In pearl oysters, the color polymorphism in mantle tissue is associated with the color and radiance of shell and pearl. The study of biomineralization related genes in mantle tissue of pearl oysters can be used as a suitable approach to better identify the molecular mechanisms that influence shell and pearl quality and color variations. In this study, we investigated the expression of biomineralization-related genes in black and orange mantle morphotypes of pearl oyster, Pinctada persicain both warm and cool seasons using quantitative real-time PCR. Our results showed that the genes involved in biomineralization of the prismatic and nacre layer, i.e.;ASP, KRMP, MRNP34, SHELL, SHEM1B, LINKINE, PIF, SHEM5, NACREIN, and in pigmentation (TYR2A) were significantly higher expressed in orange phenotype compared to those of black one. The higher expression ofASP,KRMP,SHEM5,LINKINEandNACREINin orange phenotype was only observed in warm season, butPIF,SHELL,SHEM1B, andTYR2Awere upregulated in both warm and cool seasons. These results suggest the existence of different genetic processes between the two color morphs ofP. persicaand the more active role of genes in orange morphotype, particularly in warmer season. This study provides better understanding of the molecular mechanisms underlying biomineralization in pearl oysters.

Identifying early gene expression responses to hypoxia (i.e., low dissolved oxygen) as a tool to assess the degree of exposure to this stressor is crucial for salmonids, because they are increasingly exposed to hypoxic stress due to anthropogenic habitat change, e.g., global warming, excessive nutrient loading, and persistent algal blooms. Our goal was to discover and validate gill gene expression biomarkers specific to the hypoxia response in salmonids across multi-stressor conditions. Gill tissue was collected from 24 freshwater juvenile Chinook salmon (Oncorhynchus tshawytscha), held in normoxia [dissolved oxygen (DO) > 8 mg L^-1] and hypoxia (DO = 4‒5 mg L^-1) in 10 and 18°C temperatures for up to six days. RNA-sequencing (RNA-seq) was then used to discover 240 differentially expressed genes between hypoxic and normoxic conditions, but not affected by temperature. The most significantly differentially expressed genes had functional roles in the cell cycle and suppression of cell proliferation associated with hypoxic conditions. The most significant genes (n = 30) were selected for real-time qPCR assay development. These assays demonstrated a strong correlation (r = 0.88; p < 0.001) between the expression values from RNA-seq and the fold changes from qPCR. Further, qPCR of the 30 candidate hypoxia biomarkers was applied to an additional 322 Chinook salmon exposed to hypoxic and normoxic conditions to reveal the top biomarkers to define hypoxic stress. Multivariate analyses revealed that smolt stage, water salinity, and morbidity status were relevant factors to consider with the expression of these genes in relation to hypoxic stress. These hypoxia candidate genes will be put into application screening Chinook salmon to determine the identity of stressors impacting the fish.

In this study, we explored the gill mRNA expression ofNa⁺/K⁺-ATPase α1 (NKAα1) and Na⁺/K⁺/2Cl⁻ cotransporter 1a(NKCC1a) in an amphibious mudskipper species, Periophthalmus waltoniat high salinities of mangrove tidal zones. Fish were exposed to four salinities including 35 (control), 45, 55, and 70 g kg⁻¹. The branchial transcripts of NKAα1 were elevated in fish kept at 55 and 70 g kg⁻¹ salinities with a strong elevation after medium-term (11 days) of exposure. No obvious changes were observed in NKCC1a expression among different salinity groups after short-term (3 days) and long-term (21 days) of exposure, except for a medium-term strong upregulation of NKCC1agene at 55 g kg⁻¹. The NKAα1 and NKCC1a expressions returned to baseline levels after the long term exposure salinity period, suggesting complete molecular and physiological acclimation.These results demonstrate the osmoregulatory role of ion transporter enzymes in acclimation of amphibious fish to high salinities.