In many teleosts, changes in counts and/or dimension of CC mainly correlated to acclimation to WS throughout ontogenic development (Hiroi & McCormick, 2007). The findings 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 fish with increment of WS especially in fish reared at 35‰, then decreased at 50‰. It was noticed that, the proliferation of CC particularly in fish 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 influence 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. Similarly, remarkable increment of CC counts in the gill filaments was reported in different hyperosmotic-acclimated fish such as European sea bass (Dicentrarchus labrax, Varsamos et al., 2002), killifish (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 fish reared at 15‰ had the smallest nucleus diameter in CC suggesting these fish were at isosmotic condition and less osmotic stress pressure was on them; however, fish 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 fish to intermediate WS (15‰ and 25‰) reduced their CC size as a result of lower need for ion pumps required in fish reared at isosmotic environments. Increment of CC size in the hypo-and/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).
The amount of liver enzymes in body’s fluids is valuable biomarkers of fish 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 fish reared at different WS, but fish reared at fresh water or 15‰ had the highest and least amount of plasma AST. These results indicated that fish 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 fish reared at isosmotic condition (15‰) underwent the least stress. In accordance with these findings, Farshadian et al., (2018) revealed that the value of ALP was not affected in yellowfine seabream reared at 5‰ and 35‰.
In this study, serum cortisol increased in fish 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 fish reared at 35‰ that may as consequence of increase in CC counts in this group. These findings 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 fish 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 fish in a saline environment (Stewart et al., 2016). In the current research, the amounts of serum Na+ and K+ pronouncedly enhanced in fish 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 fish 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 significant 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 in other euryhaline fish (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 fluids 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 fish 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).
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 findings demonstrated that fish 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 fish to changes of WS has been well documented (Smith et al., 1999). For example, hypo- or 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 confirmed that there is a direct relationship between immune-related genes and environmental salinity in fish (Gu et al., 2018). Inflammatory-related genes (pro-inflammatory cytokines) such as IL-1β enable the organisms in responding to stress condition by inducing neutrophil chemo-attractant ability and their migration toward inflammatory sites (Uribe et al., 2011). In the present study, rearing fish at 15 and 35‰ induced liver IL-1β up-regulation suggesting changes in WS can modify immune responses in this species. Similar to our findings, proliferation of leucocytes and their activities after acute salinity change were found in pipefish (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-inflammatory respond in fish 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 catfish (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 fish 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), rainbow trout (Yada et al., 2001; Fast et al., 2002), Nile tilapia (Dominguez et al., 2005), sablefish (Kim et al., 2017), yellowfin seabream and Asian seabass (Mozanzadeh et al., 2021). These results indicate that WS can directly affect fish immunocompetence by affecting immune-related genes, chaperones as well as endocrine system especially catecholamines and GH.
In conclusion, the findings 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 modified these responses. Immune-related genes also triggered by intermediate WS (15 and 35‰), suggesting mediatory role of WS in fish 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.