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Microalgae are emerging as functional feed ingredients in aquaculture due to their immune-stimulating and stress-modulating properties. We investigated the potential of the microalgae Chlorella vulgaris as a feed supplement to improve the health and modulate microbiota and stress responses of Atlantic salmon. Triplicate groups of Atlantic salmon (~ 126 g) were reared in a recirculating aquaculture system (RAS) at 15 °C and received diets supplemented with 2% (CV2) or 14% (CV14) spray-dried C. vulgaris daily, 14% once weekly (CV14w), or a control diet (CD) for 8 weeks. Subsequently, all groups were exposed to an acute one-hour peracetic acid (CH 3 CO 3 H; PAA) treatment, a commonly used disinfectant in RAS. While CV14 increased feed conversion (FCR) significantly, feeding the diets CV2 and CV14w improved protein retention efficiency. CV14 significantly modulated beta-diversity in the intestinal digesta and mucosa, but this effect was already visible in fish fed CV2. Feeding CV14 and, to a lesser degree, CV2 increased the relative abundances of Paenarthrobacter and Trichococcus in the digesta and mucosa, which are able to metabolize complex carbohydrates. However, the same diets reduced the abundance of the lactic acid bacteria Lactobacillus and Weissella in the digesta and Floricoccus in the mucosa. Peracetic acid exposure induced systemic stress (increase in plasma glucose and cortisol) and a local immune response in the gill, with the most prominent upregulation of several immune- and stress-regulated genes ( clra , cebpb , marco , tnfrsf14 , ikba , c1ql2 , drtp1 ) 18 h after exposure in fish fed the control diet. Fish receiving CV14 once a week showed a reduced transcriptional response to PAA exposure. Catalase protein abundance in the liver increased following exposure to PAA, while superoxide dismutase abundance in the gill and liver was increased in response to C. vulgaris inclusion before stress. Overall, the results highlight that a high (14%) inclusion rate of C. vulgaris in feed for Atlantic salmon impairs feed conversion and shifts the intestinal microbiota composition in digesta and mucosa. Weekly feeding of C. vulgaris proves a viable approach in improving protein retention and improving transcriptional resilience towards oxidative stress in increasingly intensive production systems. Thereby this study may motivate future studies on optimizing temporal feeding schedules for health-promoting aquafeeds.

Microalgae are increasingly being investigated as functional feed additives in a variety of fish species, but our knowledge on how microalgae supplementation affects Atlantic salmon remains limited. We hypothesized that microalgae inclusion of 8% in the feed would improve performance, fatty acid and pigment deposition as well as health and immunity of Atlantic salmon reared in recirculating aquaculture systems (RAS). We fed Atlantic salmon smolts with five different microalgae enriched diets containing Tetraselmis chuii (TC), Arthrospira platensis (AP), Schizochytrium limacinum (SL) or Chlorella vulgaris , either intact (CVI) or as broken cell wall derivative (CVB) or a control diet (CD). After eight weeks of feeding in brackish water (13 psu), all groups were transferred to seawater (32 psu) for additional two weeks. Our results indicate that CVB improved feed conversion and protein retention, but reduced condition factor (p < 0.05) compared to fish fed with a control diet. Voluntary feed intake decreased in seawater, but was similar among diet groups. The amount of docosahexaenoic acid was particularly high in SL-fed fish and alpha-linolenic acid was enriched in fish fed CVI, CVB and TC (p < 0.05). Following seawater transfer, fat content and monounsaturated fatty acids decreased in the muscle, while polyunsaturated fatty acids increased. Lutein was present in all muscle samples, but highest concentrations were found in CVB-, CVI- and TC-fed fish. In the anterior intestine, microalgae supplementation induced differentially regulated trout protein 1 ( drtp1 ) expression in CVI- and CVB-fed fish, but reduced the expression of interleukin 1 and 10 receptor ( il1r2 & il10rb ) in CVI-fed fish. In the liver, feeding CVI and SL induced complement C1q like 2 ( c1ql2 ) expression, while reducing serum amyloid A5 ( saa5 ) expression. Superoxide-dismutase protein concentration was induced in the liver of fish fed SL, while myeloperoxidase was reduced in most microalgae-fed groups. In conclusion, we show that commercially relevant microalgae can be used as functional feed additives for Atlantic salmon promoting different health aspects without negatively affecting their growth performance when cultivated in RAS.

Airbreathing catfish are stenohaline freshwater fish capable of withstanding various environmental conditions and farming practices, including breathing atmospheric oxygen. This unique ability has enabled them to thrive in semi-terrestrial habitats. However, the genomic mechanisms underlying their adaptation to adverse ecological environments remain largely unexplored, primarily due to the limited availability of high-quality genomic resources. Here, we present a haplotype-resolved and near telomere-to-telomere (T2T) genome assembly of the African catfish ( Clarias gariepinus ), utilizing Oxford Nanopore, PacBio HiFi, Illumina and Hi-C sequencing technologies. The primary assembly spans 969.62 Mb with only 47 contigs, achieving a contig N50 of 33.71 Mb. Terminal telomeric signals were detected in 22 of 47 contigs, suggesting T2T assembled chromosomes. BUSCO analysis confirmed gene space completeness of 99% against the Actinopterygii dataset, highlighting the high quality of the assembly. Genome annotation identified 25,655 protein-coding genes and estimated 43.94% genome-wide repetitive elements. This data provides valuable genomic resources to advance aquaculture practices and to explore the genomic underpinnings of the ecological resilience of airbreathing catfish and related teleosts.

The interleukin-1-receptor-associated kinase 3 (IRAK3) is known in mammals as a negative feedback regulator of NF-κB-mediated innate-immune mechanisms. Our RNA-seq experiments revealed a prototypic 1920-nt sequence encoding from rainbow trout ( ), as well as 20 variants that vary in length and nucleotide composition. Based on the DNA-sequence information from two closely related genes from rainbow trout and an sequence fragment from Atlantic salmon retrieved from public databases, we elucidated the underlying genetic causes for this striking diversity. Infecting rainbow trout with a lethal dose of enhanced the expression of all variants in the liver, head kidney, and peripheral blood leucocytes. We analyzed the functional impact of the full-length factor and selected structural variants by overexpressing them in mammalian HEK-293 cells. The full-length factor enhanced the basal activity of NF-κB, but did not dampen the TLR2-signaling-induced levels of NF-κB activation. Increasing the basal NF-κB-activity through Irak3 apparently does not involve its C-terminal domain. However, more severely truncated factors had only a minor impact on the activity of NF-κB. The TLR2-mediated stimulation did not alter the spatial distribution of Irak3 inside the cells. In salmonid CHSE-214 cells, we observed that the Irak3-splice variant that prominently expresses the C-terminal domain significantly quenched the stimulation-dependent production of interleukin-1β and interleukin-8, but not the production of other immune regulators. We conclude that the different gene and splice variants of Irak3 from trout play distinct roles in the activation of immune-regulatory mechanisms.

The immediate stress response involves the activation of the monoaminergic neurotransmitter systems including serotonin, dopamine and noradrenaline in particular areas of the fish brain. We chose maraena whitefish as a stress-sensitive salmonid species to investigate the influence of acute and chronic handling on the neurochemistry of monoamines in the brain. Plasma cortisol was quantified to assess the activation of the stress axis. In addition, we analyzed the expression of 37 genes related to the monoamine system to identify genes that could be used as markers of neurophysiological stress effects. Brain neurochemistry responded to a single handling (1 min netting and chasing) with increased serotonergic activity 3 h post-challenge. This was accompanied by a modulated expression of monoaminergic receptor genes in the hindbrain and a significant increase of plasma cortisol. The initial response was compensated by an increased monoamine synthesis at 24 h post-challenge, combined with the modulated expression of serotonin-receptor genes and plasma cortisol concentrations returning to control levels. After 10 days of repeated handling (1 min per day), we detected a slightly increased noradrenaline synthesis and a down-regulated expression of dopamine-receptor genes without effect on plasma cortisol levels. In conclusion, the changes in serotonergic neurochemistry and selected gene-expression profiles, together with the initial plasma cortisol variation, indicate an acute response and a subsequent recovery phase with signs of habituation after 10 days of daily exposure to handling. Based on the basal expression patterns of particular genes and their significant regulation upon handling conditions, we suggest a group of genes as potential biomarkers that indicate handling stress on the brain monoamine systems.

Inadequate oxygen saturation can induce stress responses in fish and further affect their immunity. Pikeperch, recently introduced in intensive aquaculture, is suggested to be reared at nearly 100% DO (dissolved oxygen), yet this recommendation can be compromised by several factors including the water temperature, stocking densities or low circulation. Herein, we aimed to investigate the effect of low oxygen saturation of 40% DO (±3.2 mg/L) over 28 days on pikeperch farmed in recirculating aquaculture systems. The obtained data suggest that—although the standard blood and health parameters did not reveal any significant differences at any timepoint—the flow cytometric analysis identified a slightly decreased proportion of lymphocytes in the HK (head kidney) of fish exposed to hypoxia. This has been complemented by marginally downregulated expression of investigated immune and stress genes in HK and liver (including FTH1, HIF1A and NR3C1). Additionally, in the model of acute peritoneal inflammation induced with inactivated Aeromonas hydrophila, we observed a striking dichotomy in the sensitivity to the low DO between innate and adaptive immunity. Thus, while the mobilization of myeloid cells from HK to blood, spleen and peritoneal cavity, underlined by changes in the expression of key proinflammatory cytokines (including MPO, IL1B and TNF) was not influenced by the low DO, hypoxia impaired the influx of lymphocytes to the peritoneal niche in the later phases of the immune reaction. Taken together, our data suggest high robustness of pikeperch towards the low oxygen saturation and further encourage its introduction to the intensive aquaculture systems.

Analyses on the cellular level are indispensable to expand our understanding of complex tissues like the mammalian heart. Single-nucleus sequencing (snRNA-seq) allows for the exploration of cellular composition and cell features without major hurdles of single-cell sequencing. We used snRNA-seq to investigate for the first time an entire adult mammalian heart. Single-nucleus quantification and clustering led to an accurate representation of cell types, revealing 24 distinct clusters with endothelial cells (28.8%), fibroblasts (25.3%), and cardiomyocytes (22.8%) constituting the major cell populations. An additional RNA velocity analysis allowed us to study transcription kinetics and was utilized to visualize the transitions between mature and nascent cellular states of the cell types. We identified subgroups of cardiomyocytes with distinct marker profiles. For example, the expression of Hand2os1 distinguished immature cardiomyocytes from differentiated cardiomyocyte populations. Moreover, we found a cell population that comprises endothelial markers as well as markers clearly related to cardiomyocyte function. Our velocity data support the idea that this population is in a trans-differentiation process from an endothelial cell-like phenotype towards a cardiomyocyte-like phenotype. In summary, we present the first report of sequencing an entire adult mammalian heart, providing realistic cell-type distributions combined with RNA velocity kinetics hinting at interrelations.

The teleost head kidney is a highly relevant immune organ, and myeloid cells play a major role in this organ’s innate and adaptive immune responses. Because of their complexity, the early phases of the innate immune reaction of fish against bacteria are still poorly understood. In this study, naïve rainbow trout were stimulated with inactivated A. salmonicida and sampled at 12 h, 24 h and 7 d poststimulation. Cells from the head kidney were magnetically sorted with a monoclonal antibody mAB21 to obtain one (MAb21-positive) fraction enriched with myeloid cells and one (MAb21-negative) fraction enriched with lymphocytes and thrombocytes. The gene expression pattern of the resulting cell subpopulations was analysed using a panel of 43 immune-related genes. The results show an overall downregulation of the complement pathway and cytokine production at the considered time points. Some of the selected genes may be considered as parameters for diagnosing bacterial furunculosis of rainbow trout.

The recent development and broad application of sequencing techniques at the single-cell level is generating an unprecedented amount of data. The different techniques have their individual limits, but the datasets also offer unexpected possibilities when utilized collectively. Here, we applied snRNA-seq in whole adult murine hearts from an inbred (C57BL/6NRj) and an outbred (Fzt:DU) mouse strain to directly compare the data with the publicly available scRNA-seq data of the tabula muris project. Explicitly choosing a single-nucleus approach allowed us to pin down the typical heart tissue-specific technical bias, coming up with novel insights on the mammalian heart cell composition. For our integrated dataset, cardiomyocytes, fibroblasts, and endothelial cells constituted the three main cell populations accounting for about 75% of all cells. However, their numbers severely differed between the individual datasets, with cardiomyocyte proportions ranging from about 9% in the tabula muris data to around 23% for our BL6 data, representing the prime example for cell capture technique related bias when using a conventional single-cell approach for these large cells. Most strikingly in our comparison was the discovery of a minor population of cardiomyocytes characterized by proliferation markers that could not be identified by analyzing the datasets individually. It is now widely accepted that the heart has an, albeit very restricted, regenerative potential. However there is still an ongoing debate where new cardiomyocytes arise from. Our findings support the idea that the renewal of the cardiomyocyte pool is driven by cytokinesis of resident cardiomyocytes rather than differentiation of progenitor cells. We thus provide data that can contribute to an understanding of heart cell regeneration, which is a prerequisite for future applications to enhance the process of heart repair.

Aquaculture management involves regular handling procedures, but these can evoke stress responses in farmed fish. We compiled an extensive list of published parameters that indicate the most likely handling-induced physiological deviations from the norm. However, since these parameters are based almost exclusively on studies of rainbow trout and Atlantic salmon, we conducted a handling-challenge experiment with maraena whitefish ( Coregonus maraena ). This salmonid fish was sampled at either 3 or 24 h after a single 1-min handling or after 10 days of daily repeated 1-min handling. The cortisol levels were strongly elevated in some individuals at 3 h after the single handling challenge, but these elevations were not significantly different between the challenged and control cohorts. The phagocytic capacity of myeloid head-kidney cells stimulated with fluorophore-labeled, inactivated Aeromonas salmonicida was significantly decreased in maraena whitefish at 3 h after the handling challenge compared to control fish. Microarray analysis of head-kidney samples from the challenged and control fish revealed 12 differentially expressed genes at 3 h and 70 at 24 h after the single handling episode, but only 5 differentially expressed genes after 10 days of repeated daily handling. The identified genes were assigned to numerous stress- and immune-relevant functional pathways, including “glucocorticoid receptor signaling” (3 h post-challenge), “HIF1A signaling” (24 h post-challenge), or “complement system” (10 days of repeated challenge). Our data reveal the tight interconnection of immune and stress pathways in the head kidney of maraena whitefish and corroborate several parameters previously found regulated in other tissues of handling-stressed rainbow trout. These findings indicate that handling may compromise the health and welfare of maraena whitefish in aquaculture.