Effects of Salinity on Gills’ Chloride Cells, Stress Indices and Gene Expression of Asian Seabass (Lates Calcarifer, Bloch, 1790)

A two-week research was carried-out to assess water salinity (WS) effects including 0, 15, 35 and 50 ‰ on osmoregulatory mechanisms and stress indices in Asian sea bass (34.4 g) juveniles. Except for �sh reared at 50 ‰ , in the other treatments gradually decreased to the prescribed WS during a 10-day period (-5 ‰ a day). After 10-day of acclimation period, �sh reared at the prescribed WS for two weeks. Fish reared at 15 and 35 ‰ had higher chloride cell (CC) counts in the interlamellar region. The number of CC in the interlamellar region elevated with increment of WS up to 35 ‰ , but they were pronouncedly reduced in 50 ‰ group . The diameter of CC in the interlamellar region was not affected by WS. The least nucleus diameter of CC in the interlamellar region was observed in �sh reared at 15 ‰ (P < 0.05). The greatest and the least amounts of serum aspartate aminotransferase content were observed in �sh reared at freshwater and 15 ‰ , respectively. Fish reared at 35 ‰ had the highest serum sodium and potassium contents. Serum chloride content and total osmolality increased with increment of WS (P < 0.05). Serum cortisol and glucose contents gradually increased with elevation of WS up to 35 ‰ , then their contents remarkably decreased. The relative expression of insulin like growth factor-1 in the liver of �sh reared at 35 ‰ was strikingly higher than the other groups. The relative expression of HSP70 gene in fresh water group was pronouncedly elevated compared to other treatments. The relative expression of interleukin-1β in 15 and 35 ‰ groups was higher than the other groups; however, the relative expression of lysozyme gene in the liver of �sh reared at fresh water was pronouncedly lower than the other treatments. The results of this study suggested rearing L. calcarifer at 15 ‰ closer to the isosmotic point and better provide its welfare.


Introduction
Asian seabass (Lates calcarifer) is a protandrous sh and tolerate a wide range of salinities (Mozanzadeh et al., 2021).Because of several favorable characteristics such as high tolerance to sudden environmental changes, great growth and feed utilisation, ease of reproduction in captive condition and high fecundity it has been considered as a worldwide candidate for marine cage culture (Mathew, 2009).
Due to its euryhaline characteristics, this species is commercially cultured in different aquaculture systems such as earthen ponds or recirculating systems supplied with fresh or brackish water (0-20‰) and in marine cages.
Euryhaline sh species have a great capacity to maintain their homoeostasis in hypo-and hypersaline environments (Evans, 2008).Gills, kidneys and gut are the vital organs in euryhaline sh for the uptake and excretion of salt in hypoosmotic and hyperosmotic environments, respectively (Evans et al., 2005).
Chlorides cells (CC), mainly developed in gills, particularly developed for osmoregulation (Hirose et al., 2003).These mitochondrial rich cells have high energy requirement for osmoregulation and they contain speci c enzymes (e.g.Na + /K + -ATPase pump among the others) by which they can actively secrete NaCl (Evans, 2008).It has been con rmed that hyperosmotic environments induce the proliferation of CC (Caberoy & Quinitio 2000;Carmona et al., 2004), but hypoosmotic condition reduce CC counts in the gills of sh (Fielder et al., 2007).Besides counts, it has been con rmed that size and location of CC in gills and also the activity of Na + /K + -ATPase pump correlate with water salinity (WS) in euryhaline sh (Lin et al., 2003;Laiz-Carrión et al., 2005a;Fielder et al., 2007).It has been con rmed that the function and morphological properties of CC during WS challenges modi ed by endocrine system are mainly catecholamines and growth hormone (GH) (Sakamoto & Hirano, 1993;Sakamoto & McCormick, 2006;Jiang et al., 2008).In this sense, it has been con rmed that increment of plasma cortisol induced CC proliferation and enhanced Na + , K − -ATPase activity in the gills of Mozambique tilapia (Oreochromis mossambicus, Jiang et al., 2008).Furthermore, GH and insulin-like growth factor I (IGF-1) seem to act synergistically with cortisol in the seawater acclimation process by affecting the activity of Na + , K + -ATPase pump in CC (Madsen, 1990;McCormick et al., 1991;Madsen & Bern, 1993;Sakamoto & Hirano, 1993;McCormick, 1996;Seale et al., 2002;Sakamoto & McCormick, 2006).In addition, the amounts of IGF-1 mRNA transcription increased in the liver of coho salmon (Oncorhynchus kisutch) during smolting process (Duan et al., 1995).
Thus, physiological processes also may affect by WS in euryhaline sh such as immunocompetence (El-Leithy et al., 2019) and stress indices (Deane et al., 2004).For example, Gu et al. (2018) reported that acute exposure to hypersaline condition pronouncedly reduced expression of the immune-related genes (e.g.interleukin 1-receptor type 2) in the gill cells of Japanese eels (Anguilla japonica).In addition, upregulation of heat shock protein 70 (HSP70) gene transcript has been observed in Yellow Perch (Perca avescens) under salinity treatment (Eissa et al., 2017).The above-mentioned authors also demonstrated a strong relationship between IGF-1 and HSP70 genes expression under salt treatment, which suggested them as valuable stress biomarkers in sh.
In this regard, in this study we aimed to assess the in uences of different WS on osmoregulatory and physiological responses in L. calcarifer by considering microscopic changes of CC in gills, serum electrolytes, stress indices and transcription of some immune and stress related genes.

Research design
The current study was run in the aquatic research laboratory of Persian Gulf University, Bushehr, Iran.The juveniles of Asian seabass were purchased from Ramooz company (Delvar, Bushehr) and transferred into the lab.They were stocked into a 1000 L berglass tank.One hundred and eighty L. calcarifer (initial weight = 34.4± 0.4 g) juveniles were randomly distributed into twelve 250-L cylindrical polyethylene tanks (15 specimens in each tank) that supplied with hypersaline (50‰) that pumped from Persian Gulf.The selected WS treatments were fresh water (0‰), 15, 35 and 50‰.Except for the 50‰ group, WS in the other treatments gradually decreased to the prescribed WS during a 10-day period (-5‰ per a day).After 10-day of acclimation period, sh were reared at the prescribed WS for two weeks.The husbandry system was supplied with sand-ltered and disinfected water with the prescribed salinities.The prescribed WS were adjusted by diluting hypersaline water (50‰) of Persian Gulf with fresh water in 1000-L polyethylene tanks and pumped into the system.The mean values (mean ± standard deviation) for temperature, pH and dissolved oxygen were 25.0 ± 2.0°C, 7.9 ± 0.2 and 80% saturation level, respectively and photoperiod was 12L: 12D (Light: Darkness).About 70% of water of system was exchanged with new water daily.Fish were fed twice daily (0800 and 1600) with a commercial diet (size 3.0 mm, 500 g kg − 1 protein, 160 g kg − 1 lipid, Beyza Feed Mill 21, Shiraz, Iran) to visual satiation for two weeks.

Sampling
After nishing two-weeks of the husbandry trial, six specimens of each tank were anesthetized with 2phenoxyethanol and blood samples was collected from their caudal vein with syringes.Five h after blood coagulation, blood samples were centrifuged (4000 g, 10 min, at room temperature) and serum was separated and was kept at − 80°C until their analysis.After blood collection, the same sh were sacri ced with an overdose (1000 mg L − 1 ) of the anesthetic to dissect gills and the liver.The dissected gill was xed in formalin 10% for 24 h and then transferred to 70% ethanol until examination and the liver samples were snap-frozen by using liquid nitrogen then kept in -80°C until their analysis.

Histological studies and serum biochemical assessments
A classic histology along with hematoxylin and eosin staining methods were done for evaluating the histoarchitecture of CC in the gills samples using an Olympus BH-2 photomicroscope.A computerized microscopic image analyzer (Digimizer 4.1.1)was used to determine histomorphometric parameters of gills including number of chloride cells (CC) in the interlamellar region, diameter of CC and their nucleus in the interlamellar region of 10 sh per treatment were evaluated.

Evaluation of relative expression of growth and immune-related genes in the liver
Total RNA of the liver samples was extracted by a commercial kit (Roche, Manheim, Germany) as described by the manufacturer instructions and then were treated with DNase I to remove contaminating genomic DNA.RNA quantity and purity were assessed by measuring the A260/A280 ratio using Spectrophotometer (Biotech photometer WPA), and the RNA quality was assessed through electrophoresis on a 1% agarose gel.Elongation factor 1α was applied for internal housekeeping gene as used a suitable reference gene by Mohd-Shaharuddin et al. ( 2015) and Paria et al. (2015) in previous studies on Asian seabass.A cDNA synthesis Kit (Cinna gene, Tehran, Iran) and primers (oligo(dT), Random Hexamer (Metabion)) following the manufacturer's instructions was applied for synthesizing cDNA.The expression of insulin-like growth factor I (IGF-1), interleukin 1β (IL-1β), heat shock protein 70 (HSP70) and lysozyme in the liver of L. calcarifer juveniles were assessed by quantitative real-time PCR assays in triplicate (Table 1).A real-time PCR machine (Rotor Gene-3000, Sydney, Australia) was applied to assess the expressions of these genes as described by Zeynali et al. (2020).The Ct of Ef1a under the in uence of salinity did not change signi cantly between different treatments (P < 0.01).Data analysis of the real-time PCR was performed in triplicate with Rotor-Gene, RG-3000 (Australia) software.The relative expression levels of candidate genes were calculated using the 2-ΔΔCt method (Livak & Schmittgen, 2001).

Results
The survival rate in all groups was 100%.The histological study of the gill demonstrated that sh reared at 15 and 35‰ had more CC counts in the interlamellar region (P < 0.05, Table 2, Fig. 1, 2).The number of CC in the interlamellar region increased with increasing WS up to 35‰, but they decreased in sh reared at 50‰.The diameter of CC in the interlamellar region was not affected by WS.The nucleus diameter of CC in the interlamellar region in sh reared at 15‰ was lower than the other groups (P < 0.05).Serum ALP and ALT contents did not change in sh reared at different WS (Table 3).Fish reared at freshwater and 15‰ had the highest while lowest serum AST content, respectively (P < 0.05).Abbreviations: ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase.
Fish reared at 35‰ had the highest serum sodium and potassium contents and sh reared at 15 and 50‰ showed the lowest values (Table 4).Serum chloride content and total osmolality increased with increment of WS (P < 0.05).
Water salinities (‰) Serum cortisol (Fig. 3a) and glucose (Fig. 3b) levels gradually increased with elevation of WS up to 35‰, then their contents remarkably decreased in serum of sh reared at 50‰.
The relative expression of IGF-1 in the liver of sh reared at 35‰ was signi cantly higher than the other groups, but sh reared at fresh water and 15‰ showed the lowest liver IGF-1 gene expression (Fig. 4a).
The relative expression of IL-1β in the liver of sh reared at 15 and 35‰ was higher than at fresh water and 50‰ (Fig. 4b).The relative expression of HSP70 gene in sh reared at fresh water was pronouncedly higher than other treatments (Fig. 4c); however, the relative expression of lysozyme gene in the liver of sh reared at fresh water was remarkably lower than those reared at salt waters (Fig. 4d).

Discussion
In many teleosts, changes in counts and/or dimension of CC mainly correlated to acclimation to WS throughout ontogenic development (Hiroi & McCormick, 2007).The ndings of the present research indicated that the CC morphology in the gills remarkably responded to different WS.The number of CC in the interlamellar region pronouncedly increased in sh with increment of WS especially in sh reared at 35‰, then decreased at 50‰.It was noticed that, the proliferation of CC particularly in sh reared at 35‰ was coincided with elevation of serum cortisol level and up-regulation of the liver IGF-1 gene.These results suggesting the direct in uence of these hormones on osmoregulation of L. calcarifer by modifying the morphology of CC in the gills.Furthermore, the augmentation of CC counts indicating the greater requirement for ionoregulation through these cells for keeping homeostasis.The amount of liver enzymes in body's uids is valuable biomarkers of sh welfare and health in response to stressful condition (Wagner & Congleton, 2004).In the present study, the amount of serum ALP and ALT did not change in sh reared at different WS, but sh reared at fresh water or 15‰ had the highest and least amount of plasma AST.These results indicated that sh reared at freshwater may be metabolized amino acids derived from proteolysis or used exogenous amino acids pool as a fuel source for gluconeogenic activity to cope with stressful condition; meanwhile sh reared at isosmotic condition (15‰) underwent the least stress.In accordance with these ndings, Farshadian et al., (2018) revealed that the value of ALP was not affected in yellow ne seabream reared at 5‰ and 35‰.
In this study, serum cortisol increased in sh reared at 35‰ that was in concomitant with hyperglycemia and the up-regulation of the liver IGF-1 gene that may be correlated with increase of CC in the interlamellar region.It should be mentioned that the concentrations of serum Na + and K + also increased in sh reared at 35‰ that may as consequence of increase in CC counts in this group.These ndings indicated that cortisol and IGF-1 can synergically enhance the salinity resistance in L. calcarifer.In this sense, it has been reported that cortisol can act directly on gills to augment Na + , K + -ATPase activity and CC density (Madsen & Bern, 1993).The increment of serum glucose in sh reared at 35‰ may be related to the increasing transfer of metabolites as a stored fuel source to deal with stress and to satisfy the energy demand for higher Na + , K + -ATPase activity in CC under the regulation of adrenalin and cortisol hormones (Wendelaar-Bonga, 2011).
The determination of the amounts of blood electrolytes, especially Na + , K + and Cl − , and osmolality and ion levels after changes in WS can provide information regarding the ionoregulatory ability and also successful acclimation of sh in a saline environment (Stewart et al., 2016).In the current research, the amounts of serum Na + and K + pronouncedly enhanced in sh reared at 35‰ that was associated with enhancing serum cortisol in this group indicating the increment tightening the junction between polygonal pavement cells in order to limit passive salt gain or loss during SW or FW acclimation, respectively (Chasiotis et al., 2012).However, the levels of these ions in sh reared at 35‰ were higher than those reared at 15 and 50‰.In this context, because of any correlation among result of Na with other ions and osmolality, the signi cant difference in the metioned parameter could probably be attributed to an error in Na + evaluation in the lab.In our study, the serum osmolality and Cl − linearly enhanced with elevation of WS suggesting strong osmoregulatory ability of this species as also reported The HSP family mainly functions as molecular chaperones in cells to prohibit protein disruption, regulate protein homeostasis and contribute in refolding of misfolded proteins.They are also implicated in the general protection of stressed cells (Basu et al., 2002).Our ndings demonstrated that sh reared at freshwater has higher liver HSP70 gene expression that was coincided with the highest liver AST content compared to other groups suggesting this treatment was under stressful condition.These results indicate a direct role of the stress protein in salinity tolerance by L. calcarifer.The key role of HSP70 in the adaptation of sh to changes of WS has been well documented (Smith et al., 1999).For example, hypoor hyperosmotic shock enhanced the branchial expression of HSP70 in the silver sea bream (Sparus sarba, Deane et al., 2004).The authors of the above-mentioned study revealed that the activity and mRNA levels of HSP70 were lower around isosmotic WS that was attributed to the best growth performance in silver sea bream.
It has been con rmed that there is a direct relationship between immune-related genes and environmental salinity in sh (Gu et al., 2018).In ammatory-related genes (pro-in ammatory cytokines) such as IL-1β enable the organisms in responding to stress condition by inducing neutrophil chemo-attractant ability and their migration toward in ammatory sites (Uribe et al., 2011).In the present study, rearing sh at 15 and 35‰ induced liver IL-1β up-regulation suggesting changes in WS can modify immune responses in this species.Similar to our ndings, proliferation of leucocytes and their activities after acute salinity change were found in pipe sh (Syngnathus typhle) (Birrer et al., 2012).Furthermore, El-Leithy et al. (2019) reported that levels of IL-1β, IL-8, and cc-chemokine were higher in the liver of Nile tilapia (Oreochromis niloticus) reared at 16‰ compared to groups reared at 20‰ suggesting pro-in ammatory respond in sh reared at 16‰.In contrast, Choi et al. (2012) reported that rapid decreases in salinity, did not affect splenic leucocytes IL-1β transcription in Nile tilapia.In addition, chronic hyperosmotic stress in striped cat sh (Pangasianodon hypophthalmus, Sauvage), inhibited kidneys' toll-like receptors expression suggesting immune-suppressive effects of salinity stress (Schmitz et al. 2017).
Lysozyme possesses a direct antibacterial effect by splitting peptidoglycan layers of Gram-positive bacteria and act as an opsonin that trigger phagocytes to destroy Gram-negative bacteria (Yano, 1996).In the present study, Lysozyme gene expression down-regulated in the liver of sh reared at freshwater that was associated with up-regulation of the liver HSP70 in this group suggesting immunosuppressive effects of hypoosmotic stress.In this sense, Yada et al. (2012) reported that hyperosmotic condition increased LZ gene expression in the gills of Atlantic salmon (Salmo salar).Furthermore, it has been reported that increasing WS enhanced serum/plasma lysozyme activity in brown trout (Marc et al., 1995) (Mozanzadeh et al., 2021).These results indicate that WS can directly affect sh immunocompetence by affecting immune-related genes, chaperones as well as endocrine system especially catecholamines and GH.
In conclusion, the ndings of this study indicated that changes in WS pronouncedly alter the histoarchitecture of CC of gills maybe through stress response pathway (e.g.cortisol) and IGF-1 also synergically modi ed these responses.Immune-related genes also triggered by intermediate WS (15 and 35‰), suggesting mediatory role of WS in sh immunity.Finally, rearing L. calcarifer at intermediate salinities (e.g.15‰) is suggested because of lower concentration of AST in the liver and this salinity is closer to its isotonic point compared to the other salinities.

Declarations
The survival rate in all groups was 100%.The histological study of the gill demonstrated that sh reared at 15 and 35‰ had more CC counts in the interlamellar region (P < 0.05, Table 2, Fig. 1, 2).The number of CC in the interlamellar region increased with increasing WS up to 35‰, but they decreased in sh reared at 50‰.The diameter of CC in the interlamellar region was not affected by WS.The nucleus diameter of CC in the interlamellar region in sh reared at 15‰ was lower than the other groups (P < 0.05).The survival rate in all groups was 100%.The histological study of the gill demonstrated that sh reared at 15 and 35‰ had more CC counts in the interlamellar region (P < 0.05, Table 2, Fig. 1, 2).The number of CC in the interlamellar region increased with increasing WS up to 35‰, but they decreased in sh reared at 50‰.The diameter of CC in the interlamellar region was not affected by WS.The nucleus diameter of CC in the interlamellar region in sh reared at 15‰ was lower than the other groups (P < 0.05).

Figure
Serum cortisol (Fig. 3a) and glucose (Fig. 3b) levels gradually increased with elevation of WS up to 35‰, then their contents remarkably decreased in serum of sh reared at 50‰.
The relative expression of IGF-1 in the liver of sh reared at 35‰ was signi cantly higher than the other groups, but sh reared at fresh water and 15‰ showed the lowest liver IGF-1 gene expression (Fig. 4a).
The relative expression of IL-1β in the liver of sh reared at 15 and 35‰ was higher than at fresh water and 50‰ (Fig. 4b).The relative expression of HSP70 gene in sh reared at fresh water was pronouncedly higher than other treatments (Fig. 4c); however, the relative expression of lysozyme gene in the liver of sh reared at fresh water was remarkably lower than those reared at salt waters (Fig. 4d).
in other euryhaline sh(Laiz-Carrion et al., 2005b; Saud et al., 2007;Herrera et al., 2009;Vargas-Chacoff et al., 2011).It has been suggested that these increased ions levels are due to elevated Na + /K + -ATPase activities mainly in gut and kidney for uptaking ions from gut uids and urine.In euryhaline teleosts, GH and IGF-1 have osmoregulatory effects and act on the gill is through changes in tissue responsiveness to cortisol through elevation the numbers of gill cortisol receptors(Shrimpton et al., 1995;Sakamoto & McCormick, 2006).Thus, GH and IGF-1 synergically along with cortisol appears to control gills' osmoregulatory function by affecting the activity of Na + /K + -ATPase, distribution and density of CC(Sakamoto & McCormick, 2006;Deane & Woo, 2009).In the current research, the expression of IGF-1 remarkably enhanced in the liver of sh reared at 35 and 50‰ indicating the key role of this hormone for maintaining homeostasis at hyperosmotic environments.In addition, up-regulation of liver IGF-1 gene in 35‰ group was associated with the increment of serum cortisol, which consequently enhanced CC in the interlamellar region in this group.Similarly, it has been found that liver IGF-1 expression increased by seawater exposure in black sea bream and Atlantic salmon(Deane & Woo 2005;Breves et al., 2017).

Table 2
Gills' chloride cells (CC) morphology of Lates calcarifer juveniles reared in water salinities (0, 15, 35 and 50 ‰) at the end of the trial.A different superscript in the same row denotes statistically signi cant differences (P < 0.05).

Table 3
Serum enzymes contents (U L − 1 ) (mean ± SE, n = 3 tank) of Lates calcarifer juveniles reared in water salinities (0, 15, 35 and 50‰) at the end of the trial.A different superscript in the same row denotes statistically signi cant differences (P < 0.05).
(Miyazaki et al., 1998)3)rement of CC counts in the gill laments was reported in different hyperosmotic-acclimated sh such as European sea bass (Dicentrarchus labrax,Varsamos et al., 2002), killi sh(Lima & Kültz, 2004), Adriatic sturgeon (Acipenser naccarii, Martínez-Álvares et al., 2005), Mozambique tilapia(Hiroi et al., 2005), fat snook (Centropomus parallelus,Sterzelecki et al., 2013).It should be mentioned that sh reared at 15‰ had the smallest nucleus diameter in CC suggesting these sh were at isosmotic condition and less osmotic stress pressure was on them; however, sh reared at other WS showed nucleus hypertrophy mas a result of hypo and/or hyperosmotic stress condition.Similarly, Laiz-Carrión et al., (2005a) revealed that the number and size of CC were remarkably enhanced in gilthead seabream reared at 5‰ and 60‰, whereas exposure of sh to intermediate WS (15‰ and 25‰) reduced their CC size as a result of lower need for ion pumps required in sh reared at isosmotic environments.Increment of CC size in the hypoand/or hyperosmotic environments indicates the increasing the permeability of cells' junctions and CC complexes for augmentation of Na + and Cl − turnover(Miyazaki et al., 1998).