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Relatively warm environments caused by global warming enhance the productivity of aquaculture activities in tropical/subtropical regions; however, the intermittent cold stress (ICS) caused by negative Arctic Oscillation can still result in major economic losses. In contrast to endotherms, ectothermic fishes experience ambient temperature as an abiotic factor that is central to performance and survival. Therefore, the occurrence of extreme temperatures caused by climate change has ignited a surge of scientific interest from ecologists, economists and physiologists. In this study, we test the transgenerational effects of rearing cold-experienced (CE) and cold-naive (CN) strains of tropical tilapia. Our results show that compared to CN tilapia, the CE strain preferentially converts carbohydrates into lipids in liver at a regular temperature of 27 °C. Besides, at a low temperature of 22 °C, the CE strain exhibits a broader aerobic scope than CN fish, and their metabolite profile suggests a metabolic shift towards the utilization of glutamate derivatives. Therefore, in response to thermal perturbations, this transgenerational metabolic adjustment provides evidence into the adaptive trade-off mechanisms in tropical fish. Nevertheless, global warming may result in less thermal variation each year, and the stabilized ambient temperature may cause tropical tilapia to gradually exhibit lower energy deposits in liver. In addition to those habitants in cold and temperate regions, a lack of cold exposure to multiple generations of fish may decrease the native cold-tolerance traits of subtropical/tropical organisms; this notion has not been previously explored in terms of the biological effects under anthropogenic climate change.

The neuroplastic mechanisms in the fish brain that underlie sex reversal remain unknown. Gonadotropin-releasing hormone 3 (GnRH3) neurons control male reproductive behaviours in Mozambique tilapia and show sexual dimorphism, with males having a greater number of GnRH3 neurons. Treatment with androgens such as 11-ketotestosterone (KT), but not 17β-estradiol, increases the number of GnRH3 neurons in mature females to a level similar to that observed in mature males. Compared with oestrogen, the effect of androgen on neurogenesis remains less clear. The present study examined the effects of 11-KT, a non-aromatizable androgen, on cellular proliferation, neurogenesis, generation of GnRH3 neurons and expression of cell cycle-related genes in mature females. The number of proliferating cell nuclear antigen-positive cells was increased by 11-KT. Simultaneous injection of bromodeoxyuridine and 11-KT significantly increased the number of newly-generated (newly-proliferated) neurons, but did not affect radial glial cells, and also resulted in newly-generated GnRH3 neurons. Transcriptome analysis showed that 11-KT modulates the expression of genes related to the cell cycle process. These findings suggest that tilapia could serve as a good animal model to elucidate the effects of androgen on adult neurogenesis and the mechanisms for sex reversal in the fish brain.

The development of an ion regulatory mechanism for body fluid homeostasis was an important trait for vertebrates during the evolution from aquatic to terrestrial life. The homeostatic mechanism of Cl(-) in aquatic fish appears to be similar to that of terrestrial vertebrates; however, the mechanism in non-mammalian vertebrates is poorly understood. Unlike in mammals, in which the kidney plays a central role, in most fish species, the gill is responsible for the maintenance of Cl(-) homeostasis via Cl(-) transport uptake mechanisms. Previous studies in zebrafish identified Na(+)-Cl(-) cotransporter (NCC) 2b-expressing cells in the gills and skin as the major ionocytes responsible for Cl(-) uptake, similar to distal convoluted tubular cells in mammalian kidney. However, the mechanism by which basolateral ions exit from NCC cells is still unclear. Of the in situ hybridization signals of twelve members of the clc Cl(-) channel family, only that of clc-2c exhibited an ionocyte pattern in the gill and embryonic skin. Double in situ hybridization/immunocytochemistry confirmed colocalization of apical NCC2b with basolateral CLC-2c. Acclimation to a low Cl(-) environment increased mRNA expression of both clc-2c and ncc2b, and also the protein expression of CLC-2c in embryos and adult gills. Loss-of-function of clc-2c resulted in a significant decrease in whole body Cl(-) content in zebrafish embryos, a phenotype similar to that of ncc2b mutants; this finding suggests a role for CLC-2c in Cl(-) uptake. Translational knockdown of clc-2c stimulated ncc2b mRNA expression and vice versa, revealing cooperation between these two transporters in the context of zebrafish Cl(-) homeostasis. Further comparative genomic and phylogenetic analyses revealed that zebrafish CLC-2c is a fish-specific isoform that diverged from a kidney-predominant homologue, in the same manner as NCC2b and its counterparts (NCCs). Several lines of molecular and cellular physiological evidences demonstrated the cofunctional role of apical NCC2b and basolateral CLC-2c in the gill/skin Cl(-) uptake pathway. Taking the phylogenetic evidence into consideration, fish-specific NCC2b and CLC-2c may have coevolved to perform extra-renal Cl(-) uptake during the evolution of vertebrates in an aquatic environment.

The present study provides in vivo evidence to prove the functional plasticity of monocarboxylate transporters (MCTs) in brains of vertebrates using zebrafish (Danio rerio) as a model. In the mammalian central nervous system (CNS), energy demands are largely met by oxidation of glucose. In recent studies, in addition to glucose, lactate is also considered an energy substrate for the CNS. Astrocytes were demonstrated to play an important role in transporting lactate as metabolic substrate from capillaries to neurons through monocarboxylate transporters (MCTs). The present study was to use zebrafish as an in vivo model to test the hypothesis of whether the various MCT homologs play differential roles in the development and functioning of the CNS. Using RT-PCR and double in situ hybridization coupling with immunocytochemical staining experiments, zebrafish MCTs1-4 were all found to be expressed in brains of embryos, and were further elucidated to be localized in both neurons and astrocytes. Loss-of-functions by morpholino knockdown further provided in vivo evidences to infer that zMCTs1, -2, and -4 may be involved in metabolite transport and functioning in the developing brain. Subsequent rescue experiments with capped mRNAs of specific isoforms further indicated that zMCT2 is an indispensable monocarboxylate-transporting route for CNS development and function in zebrafish. This information is essential for identifying proper candidates of MCT isoforms that are involved in the development and functioning of the CNS.

Strong underwater acoustic noise has been known that may cause hearing loss and actual stress in teleost. However, the long-term physiological effects of relatively quiet but continuously noise on fish were less understood. In present study, milkfish, Chanos chanos , were exposed to the simulated-wind farm noise either quiet (109 dB re 1 μPa/125.4 Hz; approx. 10–100 m distant from the wind farm) or noisy (138 dB re 1 μPa/125.4 Hz; nearby the wind farm) conditions for 24 h, 3 days and 1 week. Comparing to the control group (80 dB re 1 μPa/125.4 Hz), the fish exposed to noisy conditions had higher plasma cortisol levels in the first 24 h. However, the cortisol levels of 24 h spot returned to the resting levels quickly. The fish exposed under noisy condition had significantly higher head kidney star (steroidogenic acute regulatory) and hsd11b2 (11-β-hydroxysteroid dehydrogenase 2) mRNA levels at the following treatment time points. In addition, noise exposure did not change hypothalamus crh (Corticotropin-releasing hormone) mRNA levels in this experiment. The results implied that the weak but continuously noise was a potential stressor to fish, but the impacts may be various depending on the sound levels and exposure time. Furthermore, this study showed that the continuous noise may up-regulate the genes that are related to cortisol synthesis and possibly make the fish more sensitive to ambient stressors, which may influence the energy allocation appearance in long-term exposures.

Background: Cephalopods have evolved strong acid–base regulatory abilities to cope with CO2 induced pH fluctuations in their extracellular compartments to protect gas transport via highly pH sensitive hemocyanins. To date, the mechanistic basis of branchial acid–base regulation in cephalopods is still poorly understood, and associated energetic limitations may represent a critical factor in high power squids during prolonged exposure to seawater acidification. Results: The present work used adult squid Sepioteuthis lessoniana to investigate the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids. Routine metabolic rates, NH4 + excretion, extracellular acid–base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168 h. Metabolic rates were significantly depressed by 40% after exposure to pH 7.3 conditions for 168 h. Animals fully restored extracellular pH accompanied by an increase in blood HCO3 � levels within 20 hours. This compensation reaction was accompanied by increased transcript abundance of branchial acid–base transporters including V-type H + -ATPase (VHA), Rhesus protein (RhP), Na + /HCO3 � cotransporter (NBC) and cytosolic carbonic anhydrase (CAc). Immunocytochemistry demonstrated the sub-cellular localization of Na + /K + -ATPase (NKA), VHA in basolateral and Na + /H + -exchanger 3 (NHE3) and RhP in apical membranes of the ion-transporting branchial epithelium. Branchial VHA and RhP responded with increased mRNA and protein levels in response to acidified conditions indicating the importance of active NH4 transport to mediate acid–base balance in cephalopods. Conclusion: The present work demonstrated that cephalopods have a well developed branchial acid–base regulatory machinery. However, pelagic squids that evolved a lifestyle at the edge of energetic limits are probably more sensitive to prolonged exposure to acidified conditions compared to their more sluggish relatives including cuttlefish and octopods.

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Glucose, a carbohydrate metabolite, plays a major role in the energy supply for fish iono- and osmoregulation, and the way that glucose is transported in ionocytes is a critical process related to the functional operations of ionocytes. Eighteen members of glucose transporters (GLUTs, SLC2A) were cloned and identified from zebrafish. Previously, Na+,K+-ATPase-rich (NaR), Na+-Cl−cotransporter-expressing (NCC), H+-ATPase-rich (HR), and glycogen-rich (GR) cells have been identified to be responsible for Ca2+uptake, Cl−uptake, Na+uptake, and the energy deposition, respectively, in zebrafish skin/gills. The purpose of the present study was to test the hypothesis of whether GLUT isoforms are specifically expressed and function in ionocytes to supply energy for ion regulatory mechanisms. On the basis of translational knockdown of foxi3a/ 3b (2 transcriptional factors related to the ionocytes' differentiation) and triple in situ hybridization/immunocytochemistry, 3 GLUT isoforms, zglut1a, - 6, and - 13.1, were specifically localized in NaR/NCC cells, GR cells, and HR cells, respectively. mRNA expression of zglut1a in embryos and adult gills were stimulated by the low Ca2+or low Cl−freshwater, which has been previously reported to upregulate the functions (monitored by epithelial Ca2+channel, NCC mRNA) of NaR/NCC cells, respectively while that of zglut13.1 was stimulated only by low Na+, a situation to upregulate the function (monitored by carbonic anhydrase 15a mRNA) of HR cells. On the other hand, ambient ion compositions did not affect the zglut6 mRNA expression. Taken together, zGLUT1a, -6, and 13.1, the specific transporters in NaR/NCC cells, GR cells, and HR cells, may absorb glucose into the respective cells to fulfill different physiological demands.

In zebrafish ( Danio rerio), six distinct Na+-K+-ATPase (NKA) α1-subunit genes have been identified, and four of them, zatp1a1a.1, zatp1a1a.2, zatp1a1a.4, and zatp1a1a.5, are expressed in embryonic skin where different types of ionocytes appear. The present study attempted to test a hypothesis of whether these NKA α1 paralogues are specifically expressed and function in respective ionocytes. Double fluorescence in situ hybridization analysis demonstrated the specific expression of zatp1a1a.1, zatp1a1a.2, and zatp1a1a. 5 in NKA-rich (NaR) cells, Na+-Cl−cotransporter (NCC)-expressing cells, and H+-ATPase-rich (HR) cells, respectively, based on the colocalization of the three NKA α1 genes with marker genes of the respective ionocytes (epithelial Ca2+channel in NaR cells; NCC in NCC cells; and H+-ATPase and Na+/H+exchanger 3b in HR cells). The mRNA expression (by real-time PCR) of zatp1a1a.1, zatp1a1a.2, and zatp1a1a.5 were, respectively, upregulated by low-Ca2+, low-Cl−, and low-Na+freshwater, which had previously been reported to stimulate uptake functions of Ca2+, Cl−, and Na+. However, zatp1a1a.4 was not colocalized with any of the three types of ionocytes, nor did its mRNA respond to the ambient ions examined. Taken together, zATP1a1a.1, zATP1a1a.2, and zATP1a1a.5 may provide driving force for Na+-coupled cotransporter activity specifically in NaR, NCC, and HR cells, respectively.

The vertebrate brain is a highly energy consuming organ that requires continuous energy provision. Energy metabolism of ectothermic organisms is directly affected by environmental temperature changes and has been demonstrated to affect brain energy balance in fish. Fish were hypothesized to metabolize lactate as an additional energy substrate during acute exposure to energy demanding environmental abiotic fluctuations to support brain functionality. However, to date the pathways of lactate mobilization and transport in the fish brain are not well understood, and may represent a critical physiological feature in ectotherms during acclimation to low temperature. We found depressed routine metabolic rates in zebrafish during acute exposure to hypothermic (18°C) conditions accompanied by decreased lactate concentrations in brain tissues. No changes in brain glucose content were observed. Acute cold stress increased protein concentrations of lactate dehydrogenase 1 (LDH1) and citrate synthase (CS) in brain by 1.8- and- 2.5-fold, paralleled by an increased pyruvate to acetyl-CoA transformation. To test the involvement of monocarboxylate transporters (MCTs) under acute cold stress in zebrafish, we cloned and sequenced seven MCT1-4 homologues in zebrafish. All drMCT1-4 are expressed in brain tissues and in response to cold stress drmct2a and drmct4a transcripts were up-regulated 5- and 3-fold, respectively. On the contrary, mRNA levels of drmct1a, -1b and -4b in zebrafish brain responded with a down regulation in response to cold stress. By expressing drMCTs in Xenopus oocytes we could provide functional evidence that hypothermic stress leads to a 2-fold increase in lactate transport in drMCT4b expressing oocytes. Lactate transport of other paralogues expressed in oocytes was unaffected, or even decreased during cold stress. The present work provides evidence that lactate utilization and transport pathways represent an important energy homeostatic feature to maintain vital functions of brain cells during acute cold stress in ectotherms.