Gills Of Milkfish Research Articles

Salinity effects on expression and localization of aquaporin 3 in gills of the euryhaline milkfish (Chanos chanos).

Milkfish (Chanos chanos) are important euryhaline fish in Southeast Asian countries that can tolerate a wide range of salinity changes. Previous studies have revealed that milkfish have strong ion regulation and survival abilities under osmotic stress. In addition to ion regulation, water homeostasis in euryhaline teleosts is important during environmental salinity shifts. Aquaporins (AQP) are vital water channels in fish, and different AQPs can transport water influx or outflux from the body. AQP3 is one of the AQP channels, and the function of AQP3 in the gills of euryhaline milkfish is still unknown. The aim of this study was to investigate the expression and localization of AQP3 in the gills of euryhaline milkfish to contribute to our understanding of the physiological role and localization of AQP3 in fish. The AQP3 sequence was found in the milkfish next-generation sequencing (NGS) database and is mainly distributed in the gills of freshwater (FW)-acclimated milkfish. Under hypoosmotic and hyperosmotic stress, the osmolality of milkfish immediately shifted, similar to the aqp3 gene expression. Moreover, the abundance of AQP3 protein significantly decreased 3 h after transferring milkfish from FW to seawater (SW). However, there was no change within 7 days when the milkfish experienced hypoosmotic stress. Moreover, double immunofluorescence staining of milkfish gills showed that AQP3 colocalized with Na+ /K+ ATPase at the basolateral membrane of ionocytes. These results combined indicate that milkfish have a strong osmoregulation ability under acute osmotic stress because of the quick shift in the gene and protein expression of AQP3 in their gills.

Rapid response of osmotic stress transcription factor 1 (OSTF1) expression to salinity challenge in gills of marine euryhaline milkfish (Chanos chanos).

Euryhaline teleosts can survive in environments with different salinities. Cortisol is an important hormone for acclimation to seawater (SW) of euryhaline teleosts. Osmotic stress transcription factor 1 (OSTF1), also called the transforming growth factor-beta stimulated clone 22 domain 3 (tsc22d3), was first reported in tilapia as an acute response gene and protein under hyperosmotic stress, and it is regulated by cortisol. To date, most studies on OSTF1 have focused on freshwater inhabitants, such as tilapia, medaka, and catadromous eel. The expression of OSTF1 and the correlation between OSTF1 and cortisol in marine inhabitant euryhaline teleosts, to our knowledge, remain unclear. This study reveals the changes in the expression levels of branchial OSTF1, plasma cortisol levels, and their correlation in the marine inhabitant milkfish with ambient salinities. The two sequences of milkfish TSC22D3 transcripts were classified as OSTF1a and OSTF1b. Both genes were expressed universally in all detected organs and tissues but were the most abundant in the liver. Similar gene expression levels of ostf1a and ostf1b were found in SW- and fresh water (FW)-acclimated milkfish gills, an important osmoregulatory organ. Within 12 hours of being transferred from FW to SW, the gene expression level of ostf1b increased significantly (4 folds) within 12 h, whereas the expression level of ostf1a remained constant. Moreover, cortisol levels increased rapidly after being transferred to a hyperosmotic environment. After an intraperitoneal injection of cortisol, the gene expression levels of ostf1a and ostf1b were elevated. However, under hyperosmotic stress, ostf1a gene expression remained stable. Overall, the results revealed that ostf1b was the primary gene in milkfish responding to hypertonic stress, and cortisol concentration increased after the transfer of milkfish from FW to SW. Furthermore, cortisol injection increased the expression of ostf1a and ostf1b. As a result, factors other than cortisol may activate ostf1b in milkfish gills in response to an environmental salinity challenge.

Hypothermal Effects on Energy Supply for Ionocytes in Gills of Freshwater- and Seawater-Acclimated Milkfish, Chanos chanos

The milkfish, Chanos chanos, is an important aquaculture species that can be cultured in freshwater (FW) and seawater (SW) ponds because of its high euryhalinity. In winter, cold snap leading to high mortality of this tropical species is a critical issue for the aquaculture industry in Taiwan. Under hypothermal stress, however, changes in energy supply for osmoregulation of this euryhaline species is intriguing. In this study, we used an antibody against glycogen phosphorylase (GP) to identify glycogen-rich (GR) cells distributed adjacent to the mitochondria-rich (MR) ionocytes in milkfish gills. Glucose transporter 1 (GLUT1), which plays a major role in energy supply, was also identified and localized in GR cells. Moreover, the expression of indicators of aerobic metabolism and energy production, citrate synthase (CS) and cytochrome c oxidase (COX), were analyzed in gills of FW- and SW-acclimated milkfish to reveal different strategies of energy utilization under hypothermal stress. When exposed to a low-temperature environment, SW individuals used branchial glycogen and lactate to match the energy demands of aerobic metabolism in ionocytes, and elevated aerobic capacity to support and maintain normal functions in gills. However, branchial glycogen mainly localized in the GR cells of FW milkfish was not utilized under hypothermal stress. Meanwhile, a similar level of branchial COX activity and COXIV protein abundance at low temperatures between FW and SW milkfish indicated similar energy requirements in gills. This suggested that another source, but not branchial glycogen, maintained the energy demand in FW milkfish. The present study illustrated differential energy supply strategies in gills between SW- and FW-acclimated milkfish for osmoregulation under low temperatures.

Differential expression of two ATPases revealed by lipid raft isolation from gills of euryhaline teleosts with different salinity preferences.

In euryhaline teleosts, Na+, K+-ATPase (NKA) and V-type H+-ATPase A (VHA A) are important ion-transporters located in cell membrane. Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol, sphingolipids, and proteins (e.g., flotillin). Flotillin is a LR-associated protein, commonly used as the LR marker. Previous mammalian studies showed that LR may play a crucial role in ion exchanges. Meanwhile, studies on mammals and rainbow trout showed that NKA were found to be present mainly in LR. However, little is known about LR in fish. Therefore, the present study aimed to investigate the involvement of branchial LR in osmoregulation of tilapia and milkfish, two euryhaline teleosts with different salinity preferences, by (i) extracting LR from the gills of euryhaline teleosts; (ii) detecting the abundance of LR marker protein (flotillin-2) and ion-transporters (NKA and VHA A) in branchial LR and non-LR of fresh water- and seawater-acclimated milkfish and tilapia. The results indicated that the protein abundance of LR marker, flotillin-2, changed with environmental salinities in branchial LR of tilapia. In addition, flotillin-2 and NKA were only found in LR in both tilapia and milkfish gills, while VHA A were mainly present in non-LR. Relative protein abundance of NKA was found to be significantly higher in gills of freshwater milkfish and seawater tilapia, while VHA A was significantly higher in gills of freshwater tilapia and milkfish. This study illustrated differential distribution and salinity-dependent expression of NKA and VHA A in cell membrane of gill tissues of euryhaline teleosts with different salinity preferences.

The Presence of Microplastics in Water, Sediment, and Milkfish (Chanos chanos) at the Downstream Area of Citarum River, Indonesia

Citarum River is the largest natural stream in West Java, Indonesia, flowing across an area of 6614 km2. About 3000 industries discharge their wastewater into the stream, affecting almost 19 million people who live along the river. Considering the perseverance and the prospective toxicity of microplastics (MPs), investigating their concentrations in this river is critical to help illustrate the exposure of the risks to the residents of the area and beyond. This study was focused on identifying the MPs concentrations in the water, sediment, and milkfish (Chanos chanos). A volume-reduce method by using manta trawl was used to take water samples. Sediment and milkfish samples were taken using a grab sampling method. Digestion of fish was using Fenton oxidation method according to weighted ratio (1:5) and H2O2 30% (w/v). The average MPs concentration in the river was 0.0574 ± 0.025 particles/m3; in the seawater ponds 3.000 ± 2.645 particles/L; and in the mixed-water ponds, where the water from the river and the sea were mixed, 0.666 ± 0.577 particles/L. The average MPs concentration in the sediment of Citarum River was 16.666 ± 0.577 particles/100 g; in the seawater ponds 13.335 ± 1.527 particles/100 g; in the mixed-water ponds 11.665 ± 0.577 particles/100 g; and in the seawater 3.335 ± 0.331 particles/100 g. The average of MPs concentration in the gut and gills of milkfish in the seawater ponds was 2.666 ± 2.333 particles/fish, and in the mixed-water ponds was 1.166 ± 0.983 particles/fish. The average of MPs concentration in the milkfish tissues taken from the sea was 1.333 ± 0.577 particles/fish; and for the ones taken from the mixed-water ponds, the concentration was 1.111 ± 0.838 particles/fish. Using the Kruskal Wallis test to generate statistical analysis, there is a significant difference between the MPs concentrations in the water and sediment samples of Citarum River based on their locations (p value = 0.024 and 0.032 < 0.05). The most dominant plastic polymers in the samples were polyethylene (PE) and polypropylene (PP). There is no correlation between the level of MPs concentrations in water and sediments and the level of MPs concentrations in milkfish. However, the existence of microplastics in every sample that came from different points in the sampling area should sound an alarm, either to the local government or residents.

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 Cchsp90β, 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.

Occurence of Microplastics in Water, Sediment and Milkfish (Chanos chanos) in Citarum River Downstream (Case Study: Muara Gembong)

Microplastics in aquatic environment can possibly enter into the aquatic organism, so this study aims to identify the microplastic concentrations in water, sediment, and milkfish. The samples are taken in downstream Citarum river at Muara Gembong. Water samples were taken using a manta trawl pulled by a boat while sediment and milkfish samples were taken using the grab sampling method. Organic removal and cleaning was conducted by using the Fenton Oxidation method which use Fe and H2O2. The average of microplastic concentrations in river water, seawater ponds, mixed water, and seawater consecutively are 0.0574 ± 0.025 particles/m3, 3.000 ± 2.645 particles/L, 0.666 ± 0.577 particles/L and 1.333 ± 1.155 particles/L. The average of microplastic concentrations in sediment sea water pond, sediment mixed water pond and sea sediment samples consecutively are 3.666 ± 0.577 particles/20g, 2.667 ± 1.527 particles/20g, 2.333 ± 0.577 particles/20g and 0.667 ± 1.154 particle/20g. The average of microplastic concentrations in the gut and gills of milkfish in sea ponds and mixed ponds consecutively are 2.333 ± 2.266 particles/fish and 2.222 ± 3.768 particles/fish. The average of microplastic concentrations in milkfish tissues in the sea and mixed water ponds are 1.333 ± 1.000 particles/fish and 1.111 ± 1.167 particles/fish.

Cortisol regulation of Na+, K+-ATPase β1 subunit transcription via the pre-receptor 11β-hydroxysteroid dehydrogenase 1-like (11β-Hsd1L) in gills of hypothermal freshwater milkfish, Chanos chanos

Hypothermal stress changes the balance of osmoregulation by affecting Na+, K+-ATPase (Na-K-ATPase) activity or inducing modulation to epithelium permeability in fish. Meanwhile, cellular concentrations of cortisol can be modulated by the pre-receptor enzymes 11β-hydroxysteroid dehydrogenase 1 and 2 (11β-Hsd1 and 2). In fish, increasing levels of exogenous cortisol stimulate Na+ uptake via specific interaction with cortisol. This study investigated cortisol effects on expression of Na-K-ATPase subunit proteins and activity in gills of milkfish under hypothermal stress and revealed that the plasma cortisol contents as well as gill 11β-hsd1l and na-k-atpase β1 mRNA abundance were decreased in fresh water (FW) milkfish. Meanwhile, in the seawater (SW) milkfish, the plasma cortisol contents and gill 11β-hsd1l and na-k-atpase β1 mRNA abundance was increased under hypothermal stress. On the other hand, the abundance of 11β-hsd2 mRNA increased in both FW and SW. In addition, 11β-hsd1l expression increased in FW milkfish but decreased in SW milkfish after cortisol injection. Accordingly, the results that gill Na-K-ATPase activity of FW milkfish was affected by environmental temperatures as well as cortisol-dependent Na-K-ATPase β1-subunit levels might be due to increased expression of 11β-hsd1l that elevated intracellular cortisol contents. In hypothermal SW milkfish, decreasing abundance of Na-K-ATPase β1 protein due to reduced expression of 11β-hsd1l was found after cortisol injection. Thus, under hypothermal stress, 11β-HSD1L in FW milkfish gills was used to modulate cortisol and the following effects on increasing the transcription of Na-K-ATPase β1 protein.

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 β1 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.

Histopatologi Insang Ikan Bandeng (Chanos chanos) yang tertangkap di Muara Tukad Badung, Bali

This study demonstrates the usefulness as a bio-indicator of milkfish (Chanos chanos), which are often caught in the TukadBadung Estuary Bali. Information regarding the histopathology gill organs of milkfish provides important early information for use in biomonitoring efforts to make ecosystems sustainable. The measurement results of heavy-metal Leads at Station III (0,0101 mg/L) exceeded the water quality standard threshold based on the KepMen Negara LH No. 51 Tahun 2004 standard for seawater quality (seawater biota). The results of the observation of the histopathological features of milkfish gills caught in Muara Tukad Badung revealed oedema, hyperplasia, hypertrophy fusion of the gill lamellae, Lifting ephitelium and telangiectasis.

Na+, K+-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na+, K+-ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1-β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1-β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1-β1 association in gill ionocytes of euryhaline teleosts.

Relative changes in the abundance of branchial Na+/K+‐ATPase α‐isoform‐like proteins in marine euryhaline milkfish (Chanos chanos) acclimated to environments of different salinities

Previous studies revealed that upon salinity challenge, milkfish (Chanos chanos), the euryhaline teleost, exhibited adaptive changes in branchial Na(+)/K(+)-ATPase (NKA) activity with different Na(+) and K(+) affinities. Since alteration of activity and ion-affinity may be influenced by changes in different isoforms of NKA alpha-subunit (i.e., the catalytic subunit), it is, thus, intriguing to compare the patterns of protein abundance of three major NKA alpha-isoform-like proteins (i.e., alpha1, alpha2, and alpha3) in the gills of euryhaline milkfish following salinity challenge. The protein abundance of three NKA alpha-isoform-like proteins in gills of milkfish reared in seawater (SW), fresh water (FW), as well as hypersaline water (HSW, 60 per thousand) were analyzed by immunoblotting. In the acclimation experiments, the SW group revealed significantly higher levels of NKA alpha1- and alpha3-like proteins than the FW or HSW group. Time-course experiments on milkfish that were transferred from SW to HSW revealed the abundance of branchial NKA alpha1-like and alpha3-like proteins decreased significantly after 96 and 12 hr, respectively, and no significant difference was found in NKA alpha2-like protein. Furthermore, when fish were transferred from SW to FW, the amounts of NKA alpha1- and alpha3-like proteins was significantly decreased after 96 hr. Taken together, acute and chronic changes in the abundance of branchial NKA alpha1- and alpha3-like proteins may fulfill the requirements of altering NKA activity with different Na(+) or K(+) affinity for euryhaline milkfish acclimated to environments of various salinities.

Changes induced in the gills of milkfish (Chanos chanos Forsskål) fingerlings after acute exposure to nifurpirinol (Furanace; P-7138).

Changes induced in the gills of milkfish (Chanos chanos Forsskål) fingerlings after acute exposure to nifurpirinol (Furanace; P-7138).