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Plastic pollution, including micro- and nanoplastics, is a growing concern. Tyre-wear particles (TWPs) are the second largest source of microplastics in the ocean following abrasion of synthetic fibres. In addition to the particles themselves, TWPs contain many harmful chemicals, including 6PPD. This chemical reacts with atmospheric ozone and forms the toxic compound 6PPD-quinone (6PPDq), which poses a danger to aquatic life. There is a knowledge gap in understanding risks associated with the combined toxicity of nanoplastics (NPs) and 6PPDq. The present study aimed to investigate the toxicity of NPs and 6PPDq on adult zebrafish using phenotypic (behaviour, histology) and transcriptomic endpoints. Zebrafish were exposed to four treatments: control (contaminant-free), 50 μg/L 6PPDq, 3 mg/L polystyrene (PS)-NPs, and a combination of 50 μg/L 6PPDq and 3 mg/L PS-NPs. We did not observe locomotory dysregulation in zebrafish exposed to NPs. However, we found significant hyperlocomotion in zebrafish exposed to 6PPDq and this effect was even more substantial after co-exposure with PS-NPs. This study explores the molecular mechanisms behind these effects, identifying genes associated with neurotransmitters and fatty acid metabolism that were dysregulated by the co-exposure. Transcriptomic analysis further showed that both 6PPDq and PS-NPs impacted cellular processes associated with sterol biosynthesis, cholesterol metabolism, and muscle tissue development. The effects on these mechanisms were stronger in co-exposed zebrafish, indicating a heightened risk to cellular integrity and mitochondrial dysfunction. These results highlight the significance of mixture toxicity when studying the effects of NPs and associated chemicals like 6PPDq., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Anthropogenic releases of plastics, persistent organic pollutants (POPs), and heavy metals can impact the environment, including aquatic ecosystems. Nanoplastics (NPs) have recently emerged as pervasive environmental pollutants that have the ability to adsorb POPs and can cause stress in organisms. Among POPs, DDT and its metabolites are ubiquitous environmental pollutants due to their long persistence. Despite the discontinued use of DDT in Europe, DDT and its metabolites (primarily p,p'-DDE) are still found at detectable levels in fish feed used in salmon aquaculture. Our study aimed to look at the individual and combined toxicity of NPs (50 mg/L polystyrene) and DDE (100 μg/L) using zebrafish larvae as a model. We found no significant morphological, cardiac, respiratory, or behavioural changes in zebrafish larvae exposed to NPs alone. Conversely, morphological, cardiac and respiratory alterations were observed in zebrafish larvae exposed to DDE and NPs + DDE. Interestingly, behavioural changes were only observed in zebrafish larvae exposed to NPs + DDE. These findings were supported by RNA-seq results, which showed that some cardiac, vascular, and immunogenic pathways were downregulated only in zebrafish larvae exposed to NPs + DDE. In summary, we found an enhanced toxicological impact of DDE when combined with NPs., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Intestinal inflammation in Atlantic salmon was studied by profiling the intestine mucus proteome, employing iTRAQ and 2D LC-MS/MS approach. Two fish groups were fed soy saponin-containing (inflammation inducer) diets (SO and SP) and two control fish groups were fed diets devoid of soy saponin (CO and CP) for 36 days. The CP and SP diets contained a health additive. Inflammation characteristics in the intestine were milder in the SP-fed fish compared to the SO-fed fish. The SO group was characterised by alterations of many proteins. KEGG pathways such as phagosome and lipid binding were possibly affected in the SO group due to the higher abundant proteins like Integrin beta 2 precursor, Coronin 1A, Cathepsin S precursor, Vesicle-trafficking protein, and Neutrophil cytosol factors. On the other hand, the SP group had fewer altered proteins and inflammation characteristics; aminoacyl-tRNA biosynthesis and ribosome in the fish group were plausibly changed due to the higher abundance of many large and small subunit of ribosomes. Elevation of the abundance of ribosomal proteins, aminoacyl-tRNA ligases, and appropriate abundance of Glycogen phosphorylase and Glutamine synthetase could possibly alleviate intestinal inflammation. Data are available via ProteomeXchange with identifier PXD027922 and PXD029849. SIGNIFICANCE: Intestinal inflammation, caused by dietary factors, can be considered as a non-infectious disease. Hence, researchers are gathering clues to avert the associated health issues. The present study was conducted to infer the alterations in the intestine mucus proteome induced by a dietary health additive to counter intestinal inflammation in farmed Atlantic salmon. The reduction in the number of affected proteins and their alterations point to mechanisms evoked by the premix. Our knowledge on inflammation associated proteome in fish is limited and the present study not only highlights the changes, but also opens the possibility to avert the dysfunction of the organ through a dietary approach., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Intraosseous cannulation can be life-saving when intravenous access cannot be readily achieved. However, it has been shown that the procedure may cause fat emboli to the lungs and brain. Fat embolization may cause serious respiratory failure and fat embolism syndrome. We investigated whether intraosseous fluid resuscitation in pigs in hemorrhagic shock caused pulmonary or systemic embolization to the heart, brain, or kidneys and if this was enhanced by open chest conditions., Methods: We induced hemorrhagic shock in anesthetized pigs followed by fluid-resuscitation through bilaterally placed tibial (hind leg) intraosseous cannulas. The fluid-resuscitation was limited to intraosseous or i.v. fluid therapy, and did not involve cardiopulmonary resuscitation or other interventions. A subgroup underwent median sternotomy with pericardiectomy and pleurotomy before hemorrhagic shock was induced. We used invasive hemodynamic and respiratory monitoring including Swan Ganz pulmonary artery catheter and transesophageal echocardiography and obtained biopsies from the lungs, heart, brain, and left kidney postmortem., Results: All pigs exposed to intraosseous infusion had pulmonary fat emboli in postmortem biopsies. Additionally, seven of twenty-one pigs had coronary fat emboli. None of the pigs with open chest had fat emboli in postmortem lung, heart, or kidney biopsies. During intraosseous fluid-resuscitation, three pigs developed significant ST-elevations on ECG; all of these animals had coronary fat emboli on postmortem biopsies., Conclusions: Systemic fat embolism occurred in the form of coronary fat emboli in a third of the animals who underwent intraosseous fluid resuscitation. Open chest conditions did not increase the incidence of systemic fat embolization., (© 2021. The Author(s).)

Nutrient digestibility, growth, and mucosal barrier status of fish skin, gills, and distal intestine were studied in Atlantic salmon fed feeds based on marine or plant-derived ingredients. The barrier status was assessed by considering the expression of four mucin genes, five genes that encode antimicrobial proteins, distal intestine micromorphology, and design-based stereology of the midgut epithelium. In addition, the head kidney leukocytes were examined using flow cytometry; to understand the differences in their counts and function. Five experimental feeds containing the main components i) fishmeal and fish oil (BG1), ii) soybean meal (BG2; to induce enteritis), iii) fishmeal as the main protein source and rapeseed oil as the main lipid source (BG3), iv) a mix of plant protein concentrates as the protein sources and fish oil as the lipid source (BG4), and v) plant and marine ingredients in the ratio 70:30 (BG5) were produced for the study. Atlantic salmon with initial weight 72.7 ± 1.2 g was offered the experimental feeds for 65 days. The results revealed that the weights of all fish groups doubled, except for fish fed BG2. Fish fed the BG2 diet had lower blood cholesterol concentration, developed enteritis, had lower expression of muc2 in the distal intestine, and had a compromised barrier status in the intestine. Expression of both the mucin genes and genes that encode antimicrobial peptides were tissue-specific and some were significantly affected by diet. The fish fed BG1 and BG3 had more head kidney lymphocyte-like cells compared to BG5-fed fish, and the phagocytic activity of macrophage-like cells from the head kidney was the highest in fish fed BG1. The intestinal micromorphology and the mucosal mapping suggest two different ways by which plant-based diets can alter the gut barrier status; by either reducing the mucous cell sizes, volumetric densities and barrier status (as noted for BG2) or increasing volumetric density of mucous cells (as observed for BG4 and BG5). The results of the compromised intestinal barrier in fish fed plant ingredients should be further confirmed through transcriptomic and immunohistochemical studies to refine ingredient composition for sustainable and acceptable healthy diets., Competing Interests: KK is employed by BioVivo Technologies AS, Bodø, Norway. KP is employed by Quantidoc AS, Bergen, Norway. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sørensen, Park, Gong, Vasanth, Dahle, Korsnes, Phuong, Kiron, Øyen, Pittman and Sørensen.)

Intestinal inflammation in farmed fish is a non-infectious disease that deserves attention because it is a major issue linked to carnivorous fishes. The current norm is to formulate feeds based on plant-derived substances, and the ingredients that have antinutritional factors are known to cause intestinal inflammation in fishes such as Atlantic salmon. Hence, we studied inflammatory responses in the distal intestine of Atlantic salmon that received a feed rich in soybean derivatives, employing histology, transcriptomic and flow cytometry techniques. The fish fed on soy products had altered intestinal morphology as well as upregulated inflammation-associated genes and aberrated ion transport-linked genes. The enriched pathways for the upregulated genes were among others taurine and hypotaurine metabolism, drug metabolism-cytochrome P450 and steroid biosynthesis. The enriched gene ontology terms belonged to transmembrane transporter- and channel-activities. Furthermore, soybean products altered the immune cell counts; lymphocyte-like cell populations were significantly higher in the whole blood of fish fed soy products than those of control fish. Interestingly, the transcriptome of the head kidney did not reveal any differential gene expression, unlike the observations in the distal intestine. The present study demonstrated that soybean derivatives could evoke marked changes in intestinal transport mechanisms and metabolic pathways, and these responses are likely to have a significant impact on the intestine of Atlantic salmon. Hence, soybean-induced enteritis in Atlantic salmon is an ideal model to investigate the inflammatory responses at the cellular and molecular levels., Competing Interests: JD is employed by the company SPAROS Lda. Olhão, Portugal. VVT was employed by the company DSM Nutritional Products, Global Innovations, Kaiseraugst, Switzerland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from DSM Nutritional Products. The funder had the following involvement in the study: research design and feed formulation., (Copyright © 2020 Kiron, Park, Siriyappagouder, Dahle, Vasanth, Dias, Fernandes, Sørensen and Trichet.)

Epigenetic modifications, such as DNA methylation, can be regulated by nutrition and dietary factors. There has been a large increase in the use of sustainable plant-based protein sources in fish feed due to limitations of fishmeal resources, which are needed to sustain a rapidly growing aquaculture industry. With this major transition from marine ingredients to plant-based diets, fish are abruptly introduced to changes in dietary composition and exposed to a variety of phytochemicals, some of which known to cause epigenetic changes in mammals. However, the effect of plant ingredients on the epigenome of fish is barely understood. In the present study, the nutriepigenomic effects of the addition of pea, soy, and wheat gluten protein concentrate to aquafeeds were investigated using zebrafish as a model. A genome-wide analysis of DNA methylation patterns was performed by reduced representation bisulphite sequencing to examine global epigenetic alterations in the mid intestine after a 42-day feeding trial. We found that inclusion of 30% of wheat gluten, pea and soy protein concentrate in the diet induced epigenetic changes in the mid intestine of zebrafish. A large number of genes and intergenic regions were differentially methylated with plant-based diets. The genes concerned were related to immunity, NF-κB system, ubiquitin-proteasome pathway, MAPK pathway, and the antioxidant defence system. Epigenetic regulation of several biological processes, including neurogenesis, cell adhesion, response to stress and immunity was also observed. Ultimately, the observed epigenetic changes may enable zebrafish to rapidly regulate inflammation and maintain intestinal homoeostasis when fed plant protein-based diets.

Defatted microalgal biomass derived from biorefinery can be potential feed ingredients for carnivorous fish. The present study investigated the growth, feed intake:gain and health parameters in Atlantic salmon fed for 84 days with defatted Nannochloropsis oceania as a fishmeal replacer. Fish fed feeds containing the algal biomass (at 10 and 20% inclusion, alga groups) were compared with groups that consumed alga-devoid feeds (control group). The fish that received 20% alga tended to have reduced weight gain and specific growth rate. Condition factor, feed conversion ratio and feed intake of this fish group were significantly different when compared with the control group. Hepatosomatic and viscerosomatic indices, whole body and fillet proximate composition were not affected by the dietary treatments. Digestibility of dry matter, protein, lipid, ash and energy, as well as retention of lipid and energy of the fish that received feed with 20% alga meal were also significantly different from those of the control group. Serum superoxide dismutase activity of the 10% alga-fed fish was significantly higher compared with the control fish. Although alga feeding did not cause any distal intestinal inflammation, the intestinal proteins that were altered upon feeding 20% algal meal might be pointing to systemic physiological disturbances. In conclusion, feeds with 20% alga had a negative effect on feed intake, FCR, lipid and energy retention and health of the fish. The defatted Nannochloropsis oceania can be used at modest inclusion levels, around 10%, without negative effects on the performance of Atlantic salmon.

Atlantic salmon was orally intubated with a highly purified β-glucan product (MacroGard(®)) to study the recognition of the molecule by the receptor genes, the regulation of the downstream signalling genes and global proteins, and the micromorphological changes in the intestine. The β-glucan receptor genes of Atlantic salmon, sclra, sclrb, sclrc and cr3, seem to recognize the molecule, and initiate the downstream ITAM-motif signalling, as evident from the significantly high mRNA levels of ksyk, mapkin2, il1b and mip2a levels. Among the altered proteins, the Apoa4 (involved in carbohydrate and lipid metabolism); Tagln, Actb (uptake of β-glucan); Psma2 (associated with substrate recognition); and Ckt (energy metabolism-related) were the overexpressed ones. The underexpressed proteins included the Uk114, Rpl9, Ctsb and Lgal that are connected to proliferation, LPS-stimulation, Il1b and lactose recognition, respectively. Furthermore, the mRNA levels of igt and the number of immune cells in the distal intestine were found to increase upon β-glucan uptake by the fish. This study provides some clues on the mechanisms by which the β-glucan evokes response in Atlantic salmon, particularly at the intestinal level., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The efficacy of a microbial feed additive (Bactocell(®)) in countering intestinal inflammation in Atlantic salmon was examined in this study. Fish were fed either the additive-coated feed (probiotic) or feed without it (control). After an initial 3-week feeding, an inflammatory condition was induced by anally intubating all the fish with oxazolone. The fish were offered the feeds for 3 more weeks. Distal intestine from the groups was obtained at 4 h, 24 h, and 3 weeks, after oxazolone treatment. Inflammatory responses were prominent in both groups at 24 h, documented by changes in intestinal micromorphology, expression of inflammation-related genes, and intestinal proteome. The control group was characterized by edema, widening of intestinal villi and lamina propria, infiltration of granulocytes and lymphocytes, and higher expression of genes related to inflammatory responses, mul1b, il1b, tnfa, ifng, compared to the probiotic group or other time points of the control group. Further, the protein expression in the probiotic group at 24 h after inducing inflammation revealed five differentially regulated proteins - Calr, Psma5, Trp1, Ctsb, and Naga. At 3 weeks after intubation, the inflammatory responses subsided in the probiotic group. The findings provide evidence that the microbial additive contributes to intestinal homeostasis in Atlantic salmon.