Responses of medaka (Oryzias latipes) ammonia production and excretion to overcome acidified environments.

Anthropogenic acidification of water is an on-going environmental disaster for freshwater fishes. Fishes rely on ammonia excretion to eliminate the excess acid and mitigate the harmful effects; however, it remains largely unknown how ammoniagenesis occurs and is coordinated with ammonia excretion upon acidic stress. Medaka (Oryzias latipes) was used to examine the effects of acidic stress on ammonia production and excretion. We reveal an undiscovered ammonia-producing cell type that is rich in glutaminase (GLS) and located adjacent to the ammonia-excreting ionocytes, Na+/H+ exchanger (NHE) cells, in the gills. The gills, comparing with other ammoniagenetic organs, is the quickest to respond to the acidic stress by triggering GLS-dependent ammonia production. The unique division of labor between GLS and NHE cells in the gills allows medaka to simultaneously upregulate GLS activity and ammonia excretion shortly after exposure to acidic environments. Pharmacological experiment with a GLS inhibitor abolished the activated ammonia excretion, further suggesting the essential role of the unique feature in the responses to acidic stress. Our study shades light on a novel physiological mechanism to timely and efficiently mitigate adverse effects of acidification, providing a new way to assess the impact of on-going environmental acidification on fish.

Cortisol and glucocorticoid receptor 2 regulate acid secretion in medaka (Oryzias latipes) larvae.

Freshwater fish live in environments where pH levels fluctuate more than those in seawater. During acidic stress, the acid-base balance in these fish is regulated by ionocytes in the gills, which directly contact water and function as an external kidney. In ionocytes, apical acid secretion is largely mediated by H+-ATPase and the sodium/hydrogen exchanger (NHE). Control of this system was previously proposed to depend on the hormone, cortisol, mostly based on studies of zebrafish, a stenohaline fish, which utilize H+-ATPase as the main route for apical acid secretion. However, the role of cortisol is poorly understood in euryhaline fish species that preferentially use NHE as the main transporter. In the present study, we explored the role of cortisol in NHE-mediated acid secretion in medaka larvae. mRNA expression levels of transporters related to acid secretion and cortisol-synthesis enzyme were enhanced by acidic FW treatment (pH 4.5, 2 days) in medaka larvae. Moreover, exogenous cortisol treatment (25 mg/L, 2 days) resulted in upregulation of nhe3 and rhcg1 expression, as well as acid secretion in 7 dpf medaka larvae. In loss-of-function experiments, microinjection of glucocorticoid receptor (GR)2 morpholino (MO) caused reductions in nhe3 and rhcg1 expression and diminished acid secretion, but microinjection of mineralocorticoid receptor (MR) and GR1 MOs did not. Together, these results suggest a conserved action of cortisol and GR2 on fish body fluid acid-base regulation.

Oxytocin Signaling Acts as a Marker for Environmental Stressors in Zebrafish.

The oxytocin system plays a role in stress responses and behavior modulation. However, the effects of oxytocin signaling on stress adaptation remain unclear. Here, we demonstrated the roles of oxytocin signaling as a biomarker under stress conditions in the peripheral tissues (the gills) and central nervous system (the brain). All the environmental stressors downregulated the expression of oxytocin receptors in the gills, and the alteration of the expression of oxytocin receptors was also found in the brain after the acidic (AC) and high-ammonia (HA) treatments. The number of oxytocin neurons was increased after double-deionized (DI) treatment. By transgenic line, Tg(oxtl:EGFP), we also investigated the projections of oxytocin neurons and found oxytocin axon innervations in various nuclei that might regulate the anxiety levels and aggressiveness of adult zebrafish under different environmental stresses. The oxytocin system integrates physiological responses and behavioral outcomes to ensure environmental adaptation in adult zebrafish. Our study provides insight into oxytocin signaling as a stress indicator upon environmental stressors.

Potential osmoprotective roles of branchial heat shock proteins towards Na+, K+-ATPase in milkfish (Chanos chanos) exposed to hypotonic stress.

In euryhaline teleosts, osmoregulatory mechanisms vary with osmotic stresses, and heat shock proteins (HSPs) play a central role in maintaining cellular homeostasis. The present study aimed to investigate the expression and potential roles of HSP70 and HSP90 in the gills of seawater (SW)- and freshwater (FW)-acclimated milkfish (Chanos chanos). Four HSP genes, including Cchsc70 (heat shock cognate 70), Cchsp70, Cchsp90α, and Cchsp90ß, were analyzed in milkfish gills. Among these genes, only the mRNA abundance of branchial Cchsp90α was significantly lower in the FW-acclimated than in the SW-acclimated milkfish. Immunoblotting showed no significant difference in the relative protein abundance of branchial HSP70 and HSP90 between the two groups. The time-course experiments (from SW to FW) showed that the protein abundance of HSP70 and HSP90 at the 3 h and 6 h post-transfer and then declined gradually. To further illustrate the potential osmoregulatory roles of HSP70 and HSP90, their interaction with Na+, K+-ATPase (NKA, the primary driving force for osmoregulation) was analyzed using co-immunoprecipitation. The results showed the interaction between HSP70, HSP90 and NKA after acclimation to SW or FW increased within 3 h; and then returned to normal levels within 7 days. To our knowledge, the present study was the first to demonstrate that the interaction between HSP70, HSP90 and NKA changes with hypotonic stress in euryhaline teleosts. Before the transfer, no interaction was detected. When transferred to FW from SW, the interaction of HSP70 and HSP90 with NKA were detected. The results suggested that HSP70 and HSP90 participated in the acute responses of osmoregulatory mechanisms to protect branchial NKA from hypotonic stress in milkfish.

Positive correlation of gene expression between branchial FXYD proteins and Na+/K+-ATPase of euryhaline milkfish in response to hypoosmotic challenges.

FXYD proteins are crucial regulators of Na+/K+-ATPase (NKA), which plays an important role in ion exchange by providing the driving force for other ion-transporting systems in the osmoregulatory organs, including the gills. In milkfish (Chanos chanos), gill NKA has been widely investigated and found to alter its expression (both mRNA and protein) and activity in response to environmental salinity changes. However, the expression and roles of the regulatory proteins of NKA, the FXYD proteins, in milkfish gills upon salinity challenge is not yet clear. Hence, this study illustrated the potential roles of milkfish branchial FXYD proteins in modulating NKA expression via identification and tissue distributions of FXYD proteins, as well as the effects of salinity on expression of gill fxyd and nka mRNA. Six milkfish FXYD proteins (CcFXYD) were identified. In milkfish gill, gill-specific Ccfxyd11 was the predominant member, followed by Ccfxyd9 and Ccfxyd8. Upon hypoosmotic challenges, increases in gill Ccfxyd11, Ccfxyd8, Ccnka α1, and Ccnka ß1 mRNA as well as significantly positive correlations were observed. Moreover, after acute salinity changes, expression of gill Ccfxyd11 and Ccnka was found to change with ambient salinity, and significant positive correlations were also exhibited between Ccfxyd11 and Ccnka α1. Overall, these results revealed close relationships between CcFXYD11 and CcNKA α1 in milkfish gills, highlighting the potential roles of CcFXYD11 in osmoregulation.