Response Of Marine Medaka CRH System To Seawater Acidification

As carbon dioxide（CO₂）emissions into the atmosphere continue to increase,the oceans absorb large amounts of CO₂ resulting in a rise in surface CO₂,a rise in partial pressure,and a decrease in seawater pH resulting in ocean acidification.Ocean acidification can have a huge impact on the physiology and behavior of marine organisms.The hypothalamus,a neuroendocrine organ,synthesizes and releases corticotropin-releasing-hormone（CRH）after fish stress,which is important for maintaining endostasis.During stress,CRH,a key neuroendocrine factor,is widely distributed in the central nervous system,mainly in the hypothalamus,and plays an important role in stress regulation and regulates HPI axis activity.CRH and Corticotropin-releasing-hormonebindingprotein（Crhbp）,and Corticotropin-releasing-hormone receptor 1（Crhr1）are collectively known as the CRH system,which regulates endocrine and stress responses.The gills of fish are the main organ of respiration and the main site for sensing changes in the external water environment.The gills can also sense environmental stress and secrete CRH when stress occurs,while the gills have a large number of acid-base ion transporters that enable fish to sense acidification and perform acid-base regulation.In this paper,the model organism Oryzia melastigma was used to test the response of the CRH system to simulated ocean acidification,and a series of experiments were conducted.This paper is divided into three main parts:the first part uses immunohistochemistry for fluorescence immunohistochemistry of Crh proteins on gills,fluorescence quantitative PCR（qPCR）for CRH system-related genes crh,crhbp,crhr1and for acid-base homeostasis ion channel-related genes nhe3,rhcg1,ae1 to investigate the effects of acute acidification stress（2 h,4 h）The second part is to investigate the effect of acute acidification stress（2 h,4 h）on the CRH system and acid-base homeostasis ion channels in the gill of medaka;the second part is to investigate the effect of acute acidification stress（2 h,4 h）on the CRH system in the brain of medaka by using immunohistochemistry for fluorescence immunohistochemistry of Crh protein in the hypothalamus and qPCR for CRH system-related genes crh,crhbp,crhr1;the third part is to perform Crh In the third part,we performed immunohistochemistry,western-Blot protein quantification,high performance liquid chromatography-mass spectrometry（LC-MS）for cortisol concentration determination,and qPCR for crh,crhbp,and crhr1,to investigate the effects of chronic acidification stress for 7 d on the CRH system and HPI axis in medaka brain.1.Effects of acute acidification stress（2 h,4 h）on CRH system and acid-base homeostasis ion channels in the gill of medaka fishThe gill is the main organ of fish to perceive external environmental changes,and ocean acidification will lead to acute stress response in medaka.Medaka fish were divided into control（440 ppm CO₂）,1000 ppm CO₂ and acidified 1800 ppm CO2acidification groups,maintaining pH at 8.3±0.1（440 ppm CO₂）,7.9±0.1（1000ppm CO₂）and 7.6±0.1（1800 ppm CO₂）.A green fluorescent expression signal of Crh protein was detected at the end of the gill lamellae after 2 h of acidification compared to the control,and no Crh protein expression signal was yet detected in the gill arch filaments and gill rakers.The main distribution of Crh signal on the end of gill lamellae was located in the intracellular cytoplasm.In the control group,no significant Crh protein expression signal was detected;after 4 h of acidification,Crh protein expression signal was also detected at the end of gill lamellae,but the main distribution of the signal was in the intracellular cytoplasm and extracellular,and the extracellular secretion signal mainly showed a dotted distribution.The expression of crh mRNA and crhbp mRNA increased with the increase of acidification concentration.crhbp acted as a ligand for crh and acted as a negative feedback regulator,reducing the synthesis of crh mRNA and inhibiting the secretion of crh-induced adrenalotropic hormone（ACTH）.The expression of anion transporter ae1b mRNA decreased after 4 h of acidification,and the expression of rhcg1 mRNA decreased with the increase of acidification concentration,presumably,the cell’s ability to secrete HCO₃^-/H⁺decreased and the ability to secrete NH₃⁺increased after acidification,representing the accumulation of acidic ions in the cell.concentration increases,leading to an increase in intracellular pH.This section shows that ocean acidification can lead to activation of the CRH system on gills and secretion of Crh proteins corresponding to simulated ocean acidification,while acidification affects the expression of genes related to acid-base homeostasis ion channels.2.Effects of acute acidification stress（2 h,4 h）on the CRH system in the brain of medakaAccording to the immunohistochemical results,there was no significant difference in the distribution of Crh signals between the acidification group and the control group,only the middle hypothalamus had an extracellular secretory signal of Crh,and there might be a circadian rhythm of secreting Crh therefore a small amount of extracellular secretory signal.The results suggest that the hypothalamus did not activate the CRH system in response to acute acidification stress in medaka.3.Effects of chronic acidification stress for 7 d on the CRH system and HPI axis in the brain of medaka fishThe HPI axis is an important neurosecretory pathway that regulates endostasis in the fish organism.Stress activates Crh-secreting neurons in the hypothalamus,causing the hypothalamus to secrete Crh to induce the pituitary gland to secrete ACTH,which enters the renal interstitium to induce the production of the glucocorticoid cortisol,the end product of the HPI axis that regulates endostasis in response to stress.To investigate the response of the CRH system to chronic acidification stress in medaka fish for 7 d,acidification experiments were conducted for 7 d of acidification.The results showed that after 7 d of acidification,intra-cytoplasmic signals as well as extracellular secretory signals of Crh were detected in the anterior,middle and posterior lobes of the hypothalamus in the acidification-treated group compared to the control group,where the location of signals was mainly located extracellularly.western-Blot showed that the expression of Crh protein increased after acidification,which indicated that the hypothalamus activated the CRH system and secreted Crh after 7 d of acidification.In contrast,the mRNA expression of crh,crhbp,and crhr1 as well as cortisol concentration significantly decreased with increasing acidification concentration.Together,we found that acute acidification stress induced crh secretion and local endostasis regulation in the gill,when the hypothalamus was not involved in the response to stress.At the same time,acidification also induced changes in mRNA of genes related to gill acid-base homeostasis ion channels.After 7 d of acidification,the hypothalamic CRH system was activated,and CRH neurons secreted Crh and activated the HPI axis for negative feedback regulation.