Maladaptive stress responses are associated with reprogramming of the immune system. A comprehensive panel of biomarkers provides a reliable source of information for evaluation of the actual immune status and the impact and severity of a particular environmental challenge. Biomarkers are substances or characteristics that indicate an upcoming or persistent condition (1), as they reflect the “interaction between a biological system and an environmental agent of chemical, physical, or biological origin” (2, 3). In non-mammalian model species, such as teleost fishes, gene profiling has been widely used to search for parameters that are sensitive to environmental disturbances and that might be suitable for use as potential diagnostic markers for disease and distress (4–6). Different methodologies allow the screening of either preselected genes (quantitative PCR, qPCR) or whole transcriptomes (microarray, RNA sequencing) in different tissues and/or under different challenge conditions. The simultaneous assessment of multiple parameters, preferably from different analysis platforms, allows the drawing of realistic conclusions about unsuitable husbandry conditions and environmental stressors that may exceed the adaptive capacity of the examined individuals (7, 8). The recording of gene expression markers in conjunction with other molecular markers, such as plasma hormones or metabolites, behavioral features, growth rates, feed intakes, or parasite infestations (7), can reflect potential alterations at the different levels of the fish's biological response (9, 10).

Stressors in aquaculture can impair the immune defenses of fish, thereby facilitating the appearance of diseases (11). Handling stress, in particular, has been intensively investigated in the salmonids rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar, as these species have been domesticated for decades and are therefore more resilient to anthropogenic stress (12–14). These studies have provided an extensive list of a wide variety of parameters that change after typical handling procedures, such as chasing and netting, exposure to air, and transfer to (confinement) tanks (Table 1). The investigated parameters include body scores (15), concentrations of osmolytes and metabolites (e.g., hormones, carbohydrates, lactate, nucleotides, free fatty acids, and sialic acids) (16–21), blood cell counts (16, 22), enzyme activities (23–25), behavioral characteristics (23), susceptibility to parasites (26), and a long and diverse list of tissue-specific transcripts recorded via qPCR (27–29) or transcriptomic approaches (30–34).

By contrast, other salmonid species, such as the maraena whitefish Coregonus maraena, are non-domesticated, as they have been kept in aquaculture for only a few generations and on a comparatively small scale (35). These other fish species respond inappropriately to unavoidable aquaculture procedures, including transportation and tank cleaning, and only sparse reports are available regarding their physiological reaction patterns. However, a common response among all vertebrates is the immediate activation of the stress axis by an adequate stimulus. This activation is initiated in the brain and leads to the release of stress hormones into the bloodstream by specialized endocrine tissues (Figure 1). The chromaffin and interrenal cells of the head kidney, in particular, are in charge of secreting catecholamines and glucocorticoid hormones (e.g., cortisol), the main endocrine effectors of the stress response (11, 36, 37).

In a previous study, we analyzed the monoaminergic response to acute and repeated exposure to handling in the brain of maraena whitefish (38). In the present study, we recorded the effect of the same challenge, but with a focus on the phagocytic capacity of myeloid head-kidney cells and the transcriptomic response of the head kidney.

The stress-induced reprogramming of the immune system may be reflected by delayed wound healing, accelerated pathogenesis, decreased vaccine protection, and modulated autoimmune diseases (42, 43). In the teleostean head kidney, the neuroendocrine “stress” axis and the immune system are interlinked by several hormones, including adrenaline, noradrenaline, and the glucocorticoid cortisol, which are released by the chromaffin and interrenal cell populations of this organ. In the present study, we observed elevated cortisol levels in only a few maraena whitefish individuals at 3 h after the handling challenge, and we interpret this as possibly a subsiding cortisol response. Other researchers have made similar observations in salmonid fish after handling stress (21, 25, 33), giving us reason to assume that the cortisol levels in our fish had likely peaked earlier than 3 h post-challenge. Nevertheless, the pathway analysis of differentially regulated genes in maraena whitefish 3 h after the challenge suggests an activation of glucocorticoid receptor signaling, which is broadly considered as anti-inflammatory, antiproliferative, and proapoptotic (44).

The release of stress hormones initiates a cascade of secondary messengers, which alters the expression of a panel of genes (45, 46). These expression patterns also modulate the function of immune cells, including monocytes/macrophages, granulocytes, and lymphocytes, in the head kidney (47–49). For this reason, we investigated the impact of the experimental handling procedure on the transcriptome of the head kidney and the consequences of handling on innate immune functions in maraena whitefish. Our list of DE genes at 24 h after handling shares a remarkable degree of overlap with the previously published lists on handling-induced expression profiles (30–34), despite the previous use of tissues other than the head kidney from the related species rainbow trout. Microarray studies on the liver (32) or brain (30) of handling-stressed rainbow trout revealed increased transcript levels of genes coding for matrix metalloproteinases (MMP), arginase (ARG), SRY-box transcription factors (SOX), the heat-shock gene DNAJC5, and four genes associated with myosin (MYO), which we also found upregulated 24 h after handling in the head kidney of maraena whitefish. The upregulation of nine features associated with MHC-class I molecules in the liver of handling-stressed rainbow trout (31) agrees with the increased levels of TAPBP (encoding the MHC-class I antigen-processing molecule tapasin) in the present study. The increased hepatic expression of genes coding for a G-protein-coupled signaling factor in an RNA-seq analysis of handling-stressed rainbow trout (34) corresponds to the upregulation of GRP4 in maraena whitefish. These DE genes could prove to be suitable biomarkers for evaluating inappropriate handling of salmonids and for monitoring the advances in domestication processes of salmonid species.

The transcriptional response to the handling procedure increased six-fold after 24 h (compared to the 3 h sampling made 21 h earlier) and included numerous genes that contribute to both stress and immune pathways. The pathway steroid hormone signaling in epithelial cells likely reflects the glucocorticoid-mediated physiological changes and is closely interrelated with the canonical “stress pathways” HIF1A signaling, unfolded protein response, endoplasmic reticulum stress pathway, and protein ubiquitination pathway. The restructuring of metabolic processes is reflected by differentially regulated pathways, such as glycolysis, that mobilize energetic resources. The pathways arginine degradation and signaling in epithelial cells and neutrophils indicate the proliferation and/or differentiation of phagocytic immune cells, such as macrophages and neutrophilic granulocytes. In this context, the pathways antigen presentation pathway, glutathione redox reactions, and phagosome maturation suggest that handling affects key processes involved in the detection and destruction of potentially invading pathogens. In line with this prediction, we observed a significant decrease in phagocytosis by the phagocytic cells from treated whitefish compared with the undisturbed controls. The finding of decreased phagocytosis capacity is worrisome because it may indicate an increased susceptibility to bacterial infection. The resistance to pathogens is apparently weakened in teleost fish following a disruptive stimulus, as has been concluded by other authors (50–52). Similar conclusions were drawn from studies that investigated the relationship between handling stress and parasite infestation (26).

The interconnection of stress and immune pathways early after the onset of a disturbing stimulus is also illustrated by the cytokines and transcription factors that were predicted to have caused the altered expression profiles in the treated maraena whitefish. At all the time points examined, members of the interleukin-2 family (IL2, IL4/13, IL15, and IL21) were presumably involved in the regulation of the handling-induced expression profiles. The respective interleukins are considered to share conserved pleiotropic functions with their mammalian orthologs (53); this would include the differentiation of T cells and B cells, which develop in the head kidney. Stress has been reported to influence the ratio of leukocyte populations in fish blood (7, 54, 55) and the results of our pathway analysis suggest that leukocyte proliferation was modulated in response to handling.

Notably, tumor necrosis factor alpha (TNF) presumably contributed to the expression profile at 24 h after the single handling. TNF is an early cytokine that emerges after a disruptive stimulus in various fish species (56). The list of transcription factors with predicted involvement in the expression profiles at 3 h and 24 h after handling included both typical stress-related factors (e.g., HSF1, CREB1, and ATF4) and classical immune-related factors (mainly STATs and Rel/NF-κB). FOXO1 is a transcription factor that regulates the availability of energy and the type of antibodies produced by B cells, so it may also have an ambivalent role in stress and immune pathways.

After 10 days of repeated daily handling, only a few genes were differentially regulated relative to undisturbed control fish. One of these was CD46, which encodes a transmembrane glycoprotein that protects cells from excessive complement activation in many vertebrates, including fish (57). The activity and expression levels of complement components in salmonid fish are induced by husbandry practices and environmental stress (58–60), and transcriptomic analyses on handling-stressed rainbow trout have also identified complement genes (complement factor H, C1q-like proteins, complement receptor) as regulated features (31, 33, 34).

The low number of differentially regulated genes after 10 days of repeated handling agrees with our previously reported results (38). The lack of changes in plasma cortisol concentrations and monoamine activities in the brain over a 10 day period of repeated handling suggested a habituation to handling procedures over time. The genes encoding dopamine receptors and monoamine synthesis enzymes could therefore be potential indicators of habituation in the brain. The present study extends the panel of indicators to additional genes in the head kidney and confirms that maraena whitefish might habituate to the daily handling conditions applied in this study.

A comprehensive panel of biomarkers provides a reliable source of information for evaluation of the actual biological status of fish and the impact and severity of a particular environmental challenge. This information is especially important for species that have only recently been introduced into intensive aquaculture and have not yet undergone breeding programs. A technical approach based on global transcriptome analysis and complemented by the quantification of metabolite-based parameters has not yet been published for coregonid species. The handling challenge tested in this study had already reduced the phagocytic activity of myeloid cells from maraena whitefish at only 3 h after the treatment, and this could increase disease susceptibility. The level of various genes involved in stress and immune pathways deviated from the norm 24 h after the challenging event. However, 10 days of repeated handling resulted in modulation of the concentrations of significantly fewer parameters. Taken together, the data indicate that relatively short handling procedures have a measurable impact on the physiology of maraena whitefish and should be minimized as much as possible to prevent increased disease susceptibility. Our data also show that habituation may decrease the activation of the stress axis and subsequent immune suppression. Therefore, close monitoring of the wellbeing of fish in aquaculture is essential to establish a balance between challenges that cause distress and subtler challenges that can lead to habituation.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/, GSE183125.

The animal study was reviewed and approved by Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern, Germany. Written informed consent was obtained from the owners for the participation of their animals in this study.

AR, UG, JM-R, and TG designed research. JM-R, RB, and MT performed stress experiments and sampled fish. JM-R, TV, and UG performed phagocytosis assays and flow cytometry analyses. DK, AR, and JM-R conducted microarray analyses. JM-R and AR performed statistical analyses and wrote the paper. UG reviewed and edited the manuscript. All authors have read and agreed to the final version of the manuscript.

JM-R has been funded by an inter-institutional PhD project of the FBN. The publication of this article was funded by the Open-Access Fund of the FBN.

The 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.

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