Introduction
T he stress responses in teleost involve a variety of complex physiological processes in the organism, including alterations of immune functions [1]. Activation of hypothalamus-pituitary-interrenal (HPI) and hypothalamus-sympathetic-chromaffin (HSC) axes following acute stress plays a substantial role in adaptation to challenges fish encounter. Besides a clear role in stress responses, the head kidney has crucial functions in fish immunity, and due to its organization enables direct signaling between the endocrine and the immune system [2] . A more detailed knowledge about differential effects of acute stress on the immune system in fish is still scarce. The study intends to show that distress and eustress have different downstream effects on immune responses in koi carp.
Materials and methods
In total 60 juvenile koi carp trained to receive mosquito larvae as a daily feed reward were kept either in a group tank, from which they were sampled immediately, or kept in pairs in 50 L aquaria surrounded curtains, where they underwent experimental treatments after acclimatization. The treatments were as follows – opening curtains and lids of aquaria (C) as a control, receiving the feed reward (F) as a positive stressor, or netting the fish above the water surface for one minute (A) as a negative stressor. The fish were sampled 10, 30 or 60 minutes after each treatment. Each group consisted of 6 fish.
During sampling, blood, brain (including pituitary) and head kidneys were collected.
Blood analysis included measuring plasma glucocorticoid levels (cortisol, cortisone, corticosterone), as well as glucose and lactate. The plasma parameters were then analyzed using ANOVA . Gene expression analyses of HPI-related genes in the pituitary gland, immune-related genes , as well as genes related to isotocin and vasotocin signalling in the head kidney were analyzed by qPCR. Furthermore, the gene expression data was subjected to a p rincipal c omponent analysis (PCA), as well as bootstrapped (n=2000) and used for ElasticNet regression allowing treatment classification.
Results
The analysis of the p lasma steroid values revealed that one minute air exposure caused a significant increase in cortisol levels in fish 30 min after treatment. The gene expression analysis of the pituitary gland suggests that both treatments, feed rewarding and air exposure, are perceived as stressors, however they are processed differently. The head kidney transcriptome profile shows that acute eustress and distress induce differential immune-related responses. The p rincipal c omponent a nalysis revealed that HPI-axis and immune genes have the highest impact on the outcome. Moreover, the ElasticN et regression analysis of the gene expression of both pituitary and head kidney was able, with high accuracy, to classify the different treatments and indicate genes with stronger association to certain treatments.
Acknowledgements
This study was supported supported by the Bridge program (project no. 40B2-0_180864) through the Swiss National Science Fonds (SNSF) and the Innosuisse.
[1] Wendelaar Bonga, S. E. 1997. The stress response in fish. Physiological Reviews. 77. 591–625. 10.1152/physrev.1997.77.3.591.
[2] Weyts, F.A.A.; Cohen, N.; Flik, G.; Verburg-van Kemenade, B.M.L. 1999. Interactions between the immune system and the hypothalamo-pituitary-interrenal axis in fish. Fish & shellfish immunology. 9. 1–20. 10.1006/fsim.1998.0170.