Trout Skin Research Articles

Skin microRNA transcriptomic and functional analysis revealed novel-m0065-3p regulating antiviral immune responses via targeting IRF7 in rainbow trout (Oncorhynchus mykiss) infected with IHNV

miRNAs are small non-coding RNA that instrumental in host immune response to pathogen infection. However, studies on the involvement of miRNAs in rainbow trout (Oncorhynchus mykiss) antiviral response are still lacking. In this study, miRNA profiles of 48 hpi (T48SKs) compared to control (C48SKs), novel-m0065-3p and interferon regulatory factor 7 (IRF7) expression, and novel-m0065-3p-IRF7 functions were examined in rainbow trout skin following infectious hematopoietic necrosis virus (IHNV) challenge through RNA-seq, qRT-PCR, and overexpression and inhibition assays. Transcriptome analysis identified 23 up-regulated and 25 down-regulated differentially expressed miRNAs (DEMs). Enrichment analysis revealed that target genes were enriched in MAPK, RIG-I-like receptor, and Toll-like receptor signaling pathways. The DEMs (miR-205-z, novel-m0065-3p, novel-m0215-5p, novel-m0384-5p, and novel-m0397‐3p) were identified, and targeted key immune-related genes. Expression patterns suggested that novel-m0065-3p and IRF7 were potential regulators in antiviral immune responses of rainbow trout. Functional analysis revealed that the overexpression of novel-m0065-3p reduced significantly IRF7 expression in liver cells, which was attenuated by the introduction of IRF7, whereas the opposite result was obtained by silencing novel-m0065-3p. Overexpressed novel-m0065-3p promoted liver cell proliferation and inhibited apoptosis, and co-transfection of IRF7 attenuated the effect of novel-m0065-3p. Furthermore, IRF7 overexpression inhibited significantly IHNV replication. In vivo, the injection of agomiR-m0065-3p and antagomiR-m0065-3p changed significantly the expression of IRF7 and downstream genes. This study provided valuable information for drug-targeted diseases research and directed breeding efforts.

Characterization of Bacterial Communities on Trout Skin and Eggs in Relation to Saprolegnia parasitica Infection Status.

We have investigated the changes in the microbial communities on the surface of trout eggs and the skin of adult trout in relation to the presence of Saprolegnia parasitica. This pathogen causes saprolegniosis, a disease responsible for significant losses in salmonid farms and hatcheries. It is known from other disease systems that the host-associated microbiome plays a crucial role in the defence against pathogens, but if the pathogen predominates, this can lead to dysbiosis. However, analyses of the effects of S. parasitica on the diversity, composition, and function of microbial communities on fish skin and eggs are scarce. Thus, we have collected skin swabs from injured and healthy trout (N = 12), which differed in S. parasitica load, from three different fish farms in Croatia (Kostanjevac, Radovan, and Solin), while trout egg samples (N = 12) were infected with S. parasitica in the laboratory. Illumina sequencing of the V4 region of the 16S rRNA marker gene showed that infection with S. parasitica reduced the microbial diversity on the surface of the eggs, as evidenced by decreased Pielou's evenness and Shannon's indices. We further determined whether the bacterial genera with a relative abundance of >5.0% in the egg/skin samples were present at significantly different abundances in relation to the presence of S. parasitica. The results have shown that some genera, such as Pseudomonas and Flavobacterium, decreased significantly in the presence of the pathogen on the egg surface. On the other hand, some bacterial taxa, such as Acinetobacter and Janthinobacterium, as well as Aeromonas, were more abundant on the diseased eggs and the injured trout skin, respectively. Finally, beta diversity analyses (weighted UniFrac, unweighted UniFrac, Bray-Curtis) have shown that the sampling location (i.e., fish farm), along with S. parasitica infection status, also has a significant influence on the microbial communities' composition on the trout skin and eggs, demonstrating the strong influence of the environment on the shaping of the host surface microbiome. Overall, we have shown that the presence of S. parasitica was associated with changes in the diversity and structure of the trout skin/egg microbiome. The results obtained could support the development of new strategies for the management of saprolegniosis in aquaculture.

Identification and characterization of circular RNAs in the skin of rainbow trout (Oncorhynchus mykiss) infected with infectious hematopoietic necrosis virus

Rainbow trout (Oncorhynchus mykiss) is an economically significant freshwater-farmed fish worldwide, and the frequent outbreaks of infectious hematopoietic necrosis (IHN) in recent years have gravely compromised the healthy growth of the rainbow trout aquaculture industry. Fish skin is an essential immune barrier against the invasion of external pathogens, but it is poorly known about the role of circRNAs in rainbow trout skin. Therefore, we examined the expression profiles of circRNAs in rainbow trout skin following IHNV infection using RNA-seq. A total of 6607 circRNAs were identified, of which 34 circRNAs were differentially expressed (DE) and these DE circRNA source genes were related to immune-related pathways such as Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, Cytokine-cytokine receptor interaction, ubiquitin mediated proteolysis, and ferroptosis. We used qRT-PCR, Sanger sequencing, and subcellular localization to validate the chosen DE circRNAs, confirming their localization and expression patterns in rainbow trout skin. Further, 12 DE circRNAs were selected to construct the circRNA-miRNA-mRNA regulatory network, finding one miRNA could connect one or more circRNAs and mRNAs, and some miRNAs were reported to be associated with antiviral immunity. The functional prediction findings revealed that novel_circ_002779 and novel_circ_004118 may act as sponges for miR-205-z and miR-155-y to regulate the expression of target genes TLR8 and PIK3R1, respectively, and participated in the antiviral immune responses in rainbow trout. These results shed light on the immunological mechanism of circRNAs in rainbow trout skin and offer fundamental information for further research on the innate immune system and breeding rainbow trout resistant to disease.

Dedicated Protocol for Ultrastructural Analysis of Farmed Rainbow Trout (Oncorhynchus mykiss) Tissues with Red Mark Syndrome: The Skin-Part One.

The present study aims to provide a specific protocol for transmission electron microscopy of a sample of skin of rainbow trout affected by red mark syndrome (RMS). The red mark syndrome is a skin disease that affects the rainbow trout (Oncorhynchus mykiss). The disease, probably due to the Midichloria-like organism infection, is not lethal, but morbidity can reach up to 60%, leading to significant economic impact associated with the downgrading of the commercial product, increased labor, and susceptibility to secondary infections. The ultrastructure analyses allowed an earlier study to identify the presence of scattered microorganisms characterized by an oval shape, mainly in the cytoplasm of the cells. The protocol developed in this study will be instrumental in visualizing the ultrastructure of the microorganism, which is probably responsible for red mark syndrome infection.

Evaluation of potential antiviral activities of antimicrobial peptides in fish mucus.

Fish skin mucus contains innate immune factors and acts as the first line of physical or chemical defense against pathogens. The primary aim of this study was to determine the antiviral activity of sea bream (SBr), rainbow trout (RT), and sea bass (SBa) fish skin mucus against herpes simplex virus (HSV)-1. In addition, it was aimed to associate possible antiviral activity with antimicrobial peptides (AMPs) such as cathelicidin, hepcidin, galectin 2, and C10ORF99, whose levels were determined in the mucus. The antiviral activity and oxidative/antioxidant status of mucus against HSV-1 virus was evaluated. In addition, AMPs, SOD, and CAT activities, and immunoglobulin M levels were also analyzed in mucus of fish. Antiviral activity mucus of SBr, RT, and SBa against HSV-1 were determined as 2-4, 2-5, and 2-2, respectively. The higher antiviral activity of SBr and RT mucus compared to the mucus of SBa can be associated with higher AMP levels in them. The skin mucus of SBr and RT may be nutritional supplement, adjuvant, and a new agent that can potentiate the effects of antimicrobial/antiviral agents.

Use of rainbow trout skin treated with glutaraldehyde as a mesh for abdominal hernioplasty in rats.

To test the use of rainbow trout skin as a surgical mesh in abdominal hernioplasties in rats. The experiment involved 20 Wistar rats receiving implants of trout skin processed for disinfection in 0.5% glutaraldehyde and preserved in 100% glycerin. The animals were divided into four groups, divided at 7, 15, 30, and 90 days postoperatively. Clinical and infrared thermography evaluations were performed, and after euthanasia, assessments of adhesion formations and sample collection for histological evaluation were conducted. The implant was observed to be intact, ensuring the integrity of the abdominal wall, support for the viscera, and normal mobility for the rats for up to 90 days. Low rates of clinical alterations were observed, with an intense inflammatory reaction up to day 7, chronic inflammation and the onset of angiogenesis at day 15, and a low inflammatory reaction with collagenous infiltrate and fibrosis at day 30. At day 90, the implants showed a collagenous and fibrotic infiltrate with a minimal inflammatory infiltrate. The surgical mesh of trout skin performed well, making it a potential alternative for surgical procedures in muscle aponeurotic corrections in the abdominal wall.

Application of 4 × 44 Oligo Microarray to Transcriptomic Analysis of Immune Response in Rainbow Trout Infected with Aeromonas salmonicida

Rainbow trout, one of the most economically important aquaculture fish species worldwide, is affected by the pathogenic bacteria A. salmonicida, which causes furunculosis outbreaks, leading to huge economic losses. In this study, an oligonucleotide microarray was applied to identify transcriptional changes in the skin of rainbow trout individuals in response to a bacterial infection. Overall, 656 and 434 differentially expressed genes (DEGs) were identified at 2 and 6 days after a bacterial challenge (dpi), respectively. A comparison of moribund (2 dpi) and survivor fish (6 dpi) revealed 169 DEGs. Between these were many genes involved in immune response, including lysozymes, pattern recognition receptors (c-type lectins), antimicrobial peptides (cathelicidin and hepcidin), acute-phase proteins (serum amyloids and haptoglobin), complement cascade proteins (c3, c4, c6 and c7), interleukins (il11 and il1b) and chemokines (ccl19 and cxcl8). Alterations of leptin, eicosanoids and prostaglandins have been found, which suggest metabolic remodeling in conjunction with immune response. Further, the regulation of programmed cell death genes (caspase 8, bcl2 apoptosis regulator, nfkb inhibitor alpha and heme oxygenase) and structural proteins (collagens, myosins, keratins and metalloproteinases) was observed. This study provides, for the first time, a gene expression analysis of rainbow trout skin in response to A. salmonicida infection, revealing the complexity of defense strategies in response to furunculosis.

Involvement of miR-495 in the skin pigmentation of rainbow trout (Oncorhynchus mykiss) through the regulation of mc1r

MicroRNAs (miRNAs) play crucial roles in skin pigmentation in animals. Rainbow trout (Oncorhynchus mykiss) is a key economic fish species worldwide, and skin color directly affects its economic value. However, the functions of miRNAs in rainbow trout skin pigmentation remain largely unknown. Herein, we overexpressed and silenced miR-495 in vitro and in vivo to investigate its functions. The analysis of spatial and temporal expression patterns suggested that miR-495 is a potential regulator during the process of skin pigmentation. In vitro, mc1r was validated as a direct target for miR-495 by dual-luciferase reporter assay, and overexpression of miR-495 significantly inhibited mc1r expression; in contrast, mc1r and its downstream gene mitf levels were markedly upregulated by decreased miR-495. In vivo, overexpressed miR-495 by injecting agomiR-495 led to a substantial decrease in the expression of mc1r and mitf in dorsal skin and liver, while the opposite results were obtained after miR-495 silencing by antagomiR-495. These findings suggested that miR-495 can target mc1r to regulate rainbow trout skin pigmentation, which provide a potential basis for using miRNAs as target drugs to treat pigmentation disorders and melanoma.

Yersinia ruckeri infection activates local skin and gill B cell responses in rainbow trout.

Teleost fish lack organized structures in mucosal tissues such as those of mammals, but instead contain dispersed B and T cells with the capacity to respond to external stimuli. Nonetheless, there is still a great lack of knowledge regarding how B cells differentiate to plasmablasts/plasma cells in these mucosal surfaces. To contribute to a further understanding of the mechanisms through which fish mucosal B cells are activated, in the current study, we have studied the B cell responses in the skin and gills of rainbow trout (Oncorhynchus mykiss) exposed to Yersinia ruckeri. We have first analyzed the transcription levels of genes related to B cell function in both mucosal surfaces, and in spleen and kidney for comparative purposes. In a second experiment, we have evaluated how the infection affects the presence and size of B cells in both skin and gills, as well as the presence of plasmablasts secreting total or specific IgMs. The results obtained in both experiments support the local differentiation of B cells to plasmablasts/plasma cells in the skin and gills of rainbow trout in response to Y. ruckeri. Interestingly, these plasmablasts/plasma cells were shown to secrete specific IgMs as soon as 5 days after the exposure. These findings contribute to a further understanding of how B cells in the periphery respond to immune stimulation in teleost fish.

In vitro modelling of the influence of alternative feeds (Hermetia illucens, Arthrospira platensis) on the resistance of different rainbow trout populations (Oncorhynchus mykiss) against the viral haemorrhagic septicaemia virus and Yersinia ruckeri.

Replacing fishmeal, a finite resource with high market demand, in the diet of carnivorous rainbow trout with proteins from alternative sources may be a challenge for these fish. Therefore, this study investigated whether replacing fishmeal with protein derived from Hermetia illucens or Arthrospira platensis could promote disease susceptibility in local trout populations with different growth performance. This was assessed in vitro by measuring susceptibility to infection with the viral haemorrhagic septicaemia virus (VHSV) or the bacterium Yersinia ruckeri. Analysis of fin tissue explants and primary cell cultures from scales from the three trout populations infected in vitro with VHSV and gill explants infected with Y. ruckeri showed no significant differences in virus replication or bacterial counts. Evaluation of the virucidal or bactericidal effect of skin mucus showed a significant reduction in viral load and bacterial count for all samples with mucus addition, but no significant difference was observed between the experimental groups. This study documents no apparent impairment of innate immune mechanisms in the skin and gills of trout after feeding a diet replacing fishmeal with Arthrospira or Hermetia proteins. This underlines the potential of these alternative protein sources for the further development of sustainable trout aquaculture.

Teleost IgD+IgM− B cells in gills and skin have a plasmablast profile, but functionally and phenotypically differ from IgM+IgD− B cells in these sites

Although most B cells in teleost systemic compartments co-express IgM and IgD on the surface, cells exclusively expressing either of the two Igs are common in fish mucosal tissues, providing us with a unique opportunity to further characterize IgD+IgM- B cells, an intriguing B cell subset. Hence, we compared the phenotype of IgD+IgM- cells to that of IgM+IgD- B cells in rainbow trout gills and skin, also establishing the response of these subsets to immune stimulation. The transcriptional profile and secreting capacity of IgD+IgM- B cells corresponded to that of cells that have started a differentiation program toward plasmablasts, similarly to IgM+IgD- B cells. Yet, IgM+IgD- B cells retained high levels of surface MHC II and antigen-processing abilities, while these were much lower in IgD+IgM- cells, suggesting important differences in their antigen-presenting capacities. Our findings contribute to a deeper understanding of the enigmatic role of IgD in mucosal surfaces.

Skin Microbiota of Salmonids: A Procedure to Examine Active Bacterial Populations Using an RNA-Based Approach

Fish microbiota studies have mostly addressed intestinal bacterial communities because of their role in fish physiology; however, the skin microbiota has been studied less despite its role as the first pathogen barrier in the host. DNA-based molecular techniques have contributed to improving our understanding of the skin microbiota, but this approach faces challenges, such as the low count of bacteria in healthy fish skin. To overcome this limitation, an RNA-based approach was developed to study the skin microbiota in salmonids, including tissue sampling, RNA extraction, and downstream procedures to obtain PCR amplicons for next-generation sequencing. The protocol originated in this work overcomes the limitations of low bacterial counts and is useful for describing active microbiota in fish skin. The application of the protocol to salmonids reared in both an experimental setting and on a farm revealed that the trout skin microbiota was dominated by bacteria from the phylum Proteobacteria (>65%). At the genus level, Piscirickettsia (46%) was highlighted as the most abundant in the experimental unit samples; in contrast, Pseudoalteromonas (26%), followed by Escherichia_Shigella (~25%), was the most abundant in farmed trout.

Obtaining and Study of Peptide Compositions Based on Hydrolysates of Collagen-Containing Fish Raw Materials

Experimental studies of fish cutting waste—scales and skin were carried out, their general biochemical composition was studied, a high content of collagen was established, and elastin was noted, which accounted for 76–86% of the protein mass. Processes for the hydrolysis of secondary fish raw materials have been developed: fish scales and skin. Technological schemes have been developed and the influence of the conditions of thermal, enzymatic, enzymatic-thermal, electrochemical hydrolysis on the amino acid composition and molecular weight distribution (MWD) and antioxidant activity of peptides and proteins in the obtained hydrolysates has been studied. It has been established that the enzymatic and enzymatic-thermal method of hydrolysis of fish scales using the enzyme Alcalase 2.5 L and the electrochemical hydrolysis of the skin of cod, trout and herring made it possible to obtain protein hydrolysates with a protein content of 80–90%. At the same time, 91–98% of enzymatic hydrolysates from scales and 62%, 74% and 82.5% of electrochemically obtained hydrolysates from the skin of trout, herring, cod, respectively, account for the share of low-molecular peptides with a molecular weight of less than 10 kDa. The prospects of their use in functional foods and oil-containing products are noted.

Mucin O-glycosylation and pathogen binding ability differ between rainbow trout epithelial sites

Mucins are highly glycosylated proteins that make up the mucus covering internal and external surfaces of fish. Mucin O-glycans regulate pathogen quorum sensing, growth, virulence and attachment to the host. Knowledge on this mucosal defense system can enable alternative treatments to diseases posing a threat to productivity and welfare in aquaculture. Here, we characterize the rainbow trout (Oncorhynchus mykiss) gill, skin, pyloric ceca and distal intestinal mucin O-glycosylation and compare it to known teleost O-glycomes. We identified 54 O-glycans, consisting of up to nine monosaccharide residues. Skin glycans were most acidic, shortest on average and consisted mainly of NeuAcα2-6GalNAc. Glycans from the gills were less acidic with predominantly core 1 and 2 glycans, whereas glycans from pyloric ceca and distal intestine expressed an increased number of core 5 glycans, distinctly decorated with NeuAcα2-8NeuAc- like epitopes. When compared to Atlantic salmon and Arctic charr, trends on the core distribution, average size and overall acidity remained similar, although the epitopes varied. Rainbow trout mucins from gill and intestine bound A. salmonicida and A. hydrophila more efficiently than skin mucins. This is in line with a model where skin mucins with small glycans limit bacterial adhesion to the fish surface whereas the complex intestinal mucin glycans aid in trapping and removing pathogens from the epithelial surface.

Tracing the oomycete pathogen Saprolegnia parasitica in aquaculture and the environment

Saprolegnia parasitica causes saprolegniosis, a disease responsible for significant economic losses in aquaculture and declines of fish populations in the wild, but the knowledge of its distribution and prevalence in the environment is limited. We developed a fast, sensitive and specific S. parasitica droplet digital PCR (ddPCR) assay and demonstrated its applicability for the detection and quantification of the pathogen in environmental samples: swab DNA collected from the host (trout skin, surface of eggs) and environmental DNA extracted from water. The developed assay was used to assess how abiotic (i.e. physico-chemical parameters of the water) and biotic (health status of the host) factors influence the S. parasitica load in the environment. The pathogen load in water samples was positively correlated with some site-specific abiotic parameters such as electrical conductivity (EC) and calcium, while fluorides were negatively correlated, suggesting that physico-chemical parameters are important for determining S. parasitica load in natural waters. Furthermore, skin swabs of injured trout had significantly higher pathogen load than swabs collected from healthy fish, confirming that S. parasitica is a widespread opportunistic pathogen. Our results provide new insights into various environmental factors that influence the distribution and abundance of S. parasitica.

MiR-382 Functions on the Regulation of Melanogenesis via Targeting dct in Rainbow Trout (Oncorhynchus mykiss).

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that are involved in a diverse collection of biological processes as important post-transcriptional regulators. However, little is known about the molecular regulatory mechanism of miRNAs in fish skin pigmentation. In this study, we first confirmed that dopachrome tautomerase (dct), a key gene of melanogenesis, is a target of miR-382 in rainbow trout (Oncorhynchus mykiss) using luciferase reporter assay. The analysis of different developmental stages and tissue expression patterns between wild-type and yellow mutant rainbow trout suggested that miR-382 is a potential regulator during the process of skin pigmentation. In vitro, miR-382 mimics in rainbow trout primary liver cells significantly downregulated dct expression and resulted in decreased expression of key melanogenic genes including tyrosine-related protein 1 (tyrp1) and premelanosome protein (pmel), whereas the expression level of dct was markedly increased after transfected with miR-382 inhibitor. In vivo, overexpression of miR-382 by injection of miR-382 agomir significantly depressed the expression of dct in dorsal skin, tail fin, and liver and then reduced the expression levels of tyrp1 and pmel. Furthermore, transfection of miR-382 mimics inhibited cell proliferation and induced apoptosis. Taken together, our results identified a functional role of miR-382 in rainbow trout skin pigmentation through targeting dct, which facilitate understanding the regulatory mechanism of rainbow trout skin color at the post-transcriptional level and provide a theoretical basis for molecular breeding with skin color as the target trait.

Seasonal variations in the skin epidermal structure and mucosal immune parameters of rainbow trout skin (Oncorhynchus mykiss) at different stages of farming

The aim of this study was to investigate the seasonal changes in the epidermal structure and the innate immunity parameters of skin mucus in rainbow trout. The skin epidermis and mucus samples were collected over three consecutive seasons including winter, spring and late summer from three different weight groups i.e., 2–20 g (W1), 100–200 g (W2) and 400–600 g (W3) fish. The skin mucosal immunity analysis of rainbow trout showed that the haemagglutination activity increased significantly with increasing fish size from W1 to W3 in all three seasons, while no significant seasonal changes occurred in haemagglutination activity. Moreover, the bactericidal activity against fish pathogens increased significantly with increasing water bacterial load in late summer. The SDS-PAGE analysis of mucus showed a high amount of low molecular weight proteins (<35 kDa) in the late summer that was correlated with the increase in bactericidal activity. Histological analysis of the epidermis structure of rainbow trout skin showed that the density and size of goblet cells and consequently the mucus secretion significantly increased in W3 group in all seasons. In all three weight groups of fish, the density of goblet cells significantly increased from winter to spring and late summer along with increasing water temperature. Moreover, the goblet cell density showed a significant positive relationship with the soluble protein concentration and haemagglutination activity (p < 0.01). The results of this study demonstrated the more active immune role of the skin epidermal cells and mucus in rainbow trout during summer to protect fish against the pathogenic microorganisms. Given its potent bactericidal properties and the lack of haemolytic activity, the rainbow trout mucus might be used as a safe and inexpensive source for developing antimicrobial agents to prevent and treat some bacterial diseases in human and fish.

Transport and Barrier Functions in Rainbow Trout Trunk Skin Are Regulated by Environmental Salinity.

The mechanisms underpinning ionic transport and barrier function have been relatively well characterised in amphibians and fish. In teleost fish, these processes have mostly been characterised in the gill and intestine. In contrast, these processes remain much less clear for the trunk skin of fish. In this study, we measured barrier function and active transport in the trunk skin of the rainbow trout, using the Ussing chamber technique. The effects of epithelial damage, skin region, salinity, and pharmacological inhibition were tested. Skin barrier function decreased significantly after the infliction of a superficial wound through the removal of scales. Wound healing was already underway after 3 h and, after 24 h, there was no significant difference in barrier function towards ions between the wounded and control skin. In relation to salinity, skin permeability decreased drastically following exposure to freshwater, and increased following exposure to seawater. Changes in epithelial permeability were accompanied by salinity-dependent changes in transepithelial potential and short-circuit current. The results of this study support the idea that barrier function in rainbow trout trunk skin is regulated by tight junctions that rapidly respond to changes in salinity. The changes in transepithelial permeability and short circuit current also suggest the presence of an active transport component. Immunostaining and selective inhibition suggest that one active transport component is an apical V-ATPase. However, further research is required to determine the exact role of this transporter in the context of the trunk skin.

Isolation of Chromatophores from Brown Trout (Salmo trutta) Skin

As new techniques for single-cell analysis are evolving, there is a growing need for streamlined and flexible protocols for single-cell suspension preparation and single-cell isolation. Based on previous research on the model species zebrafish (Danio rerio), a novel protocol for the isolation of chromatophores from brown trout (Salmo trutta) skin was developed and specific gene expression in chromatophore types evaluated with qPCR. This is the first report on a protocol for the isolation of chromatophores from the skin of salmonids. The method is simple and fast (taking about 90 min), requires no special equipment and is in line with the high ethical standards required in animal research, as no animals need to be sacrificed. In addition, skin biopsies can be taken from the same fish at multiple time points, allowing monitoring of chromatophore differentiation and their involvement in the formation of pigmentation patterns. The protocol should be effective for the vast majority of salmonid species due to similarities in skin morphology.

Genetic and correlative light and electron microscopy evidence for the unique differentiation pathway of erythrophores in brown trout skin

Based on their cell ultrastructure, two types of erythrophores in the spotted skin regions of brown trout (Salmo trutta) were previously described. To test the hypothesis regarding the origin of a new cell type following genome duplication, we analysed the gene and paralogue gene expression patterns of erythrophores in brown trout skin. In addition, the ultrastructure of both erythrophore types was precisely examined using transmission electron microscopy (TEM) and correlative light microscopy and electron microscopy (CLEM). Ultrastructural differences between the sizes of erythrophore inclusions were confirmed; however, the overlapping inclusion sizes blur the distinction between erythrophore types, which we have instead defined as cell subtypes. Nevertheless, the red spots of brown trout skin with subtype 2 erythrophores, exhibited unique gene expression patterns. Many of the upregulated genes are involved in melanogenesis or xanthophore differentiation. In addition, sox10, related to progenitor cells, was also upregulated in the red spots. The expressions of paralogues derived from two genome duplication events were also analysed. Multiple paralogues were overexpressed in the red spots compared with other skin regions, suggesting that the duplicated gene copies adopted new functions and contributed to the origin of a new cell subtype that is characteristic for red spot. Possible mechanisms regarding erythrophore origin are proposed and discussed. To the best of our knowledge, this is the first study to evaluate pigment cell types in the black and red spots of brown trout skin using the advanced CLEM approach together with gene expression profiling.