Effects of cortisol treatment on the salinity tolerance of Persian sturgeon, Acipenser persicus Juveniles
Sayedeh Ainaz Shirangi1, Mohammad Reza Kalbassi2*, Saber Khodabandeh3, Jehan-Herve Lignot4
1. Department of Biology, Faculty of Science, Gonbad-Kavous University, Gonbad, Iran
2. Department of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, Nor, Iran
3. Department of Marine Biology, Faculty of Marine Sciences, Tarbiat Modares University, Nor, Iran
4. University of Montpellier, UMR MARBEC, MARBEC (IRD – Ifremer – Univ. Montpellier - CNRS), Place EugèneBataillon, 34095 Montpellier Cedex 5, France
* Corresponding author’s E-mail: firstname.lastname@example.org
Persian sturgeon, Acipenser persicus juveniles are annually produced and released (2-3g body weight) to the estuaries surrounding the southern parts of the Caspian Sea by hatchery centers. However, juveniles cannot withstand the salinity stress and the majority of them die before entering the Caspian Sea. In order to improve the salinity acclimation in Persian sturgeon juveniles weighing less than 2 g (1.62 ± 0.27 g; 7.4 ± 0.58 cm), the fish were treated with cortisol bath method using three different concentrations; 3, 5 and 7 mg L-1 for 24 h in freshwater. The fish were then directly transferred from freshwater to the Caspian Sea water (11‰) and sampled after 1, 4 and 9 days post-transfer. Daily mortality was recorded. Plasma osmolality, changes in the abundance and size of the Na+-K+-ATPase immunoreactive fluorescence chloride cells within the gill epithelium of the juveniles were determined. In spite of lower mortality in fish treated with higher concentrations of cortisol, plasma osmolality, chloride cells number and size in treated fish exhibited the same trends as the control (untreated) fish after 9-day post-transfer. In conclusion, cortisol treatment alone, is not directly implicated in osmoregulation and had no effect on Persian sturgeon juveniles to improve their salinity tolerance.
Key words: Persian sturgeon, cortisol, salinity tolerance, osmotic improvement, chloride cell.
Persian sturgeon, Acipenser persicus, is the most common sturgeon species found along the Southern Caspian Sea coastline (Billard & Lecointre 2001; Pourkazemi 2006). This euryhaline fish breeds in freshwater (FW), and its main spawning grounds have been limited along the Iranian coastline (Abdolhay 1997). However, this species is facing serious depletion in the Caspian Sea and its nearby rivers due to overfishing, habitat loss and environmental degradation (Birstein et al. 1997; Billard & Lecointre 2001). Since the Persian sturgeon has been considered as ‘Critically Endangered species (Gesner et al. 2010), juveniles are annually produced by hatchery centers of the Iranian Fisheries Organization and released (2-3 g body weight) to the estuaries surrounding the southern parts of the Caspian Sea. However, it does not appear as an efficient way to restock sturgeons. Juveniles cannot withstand the salinity stress when directly transferred from FW to the Caspian Sea water (CSW) and the majority of them released into the estuaries die before entering the sea (Abdolhay & BardaranTahori 2006; Kazemi et al. 2006; Mosafer Khorjestan et al. 2009; Bakhshalizadeh et al. 2011). Among different parameters affecting ion-regulatory system (environmental salinity, feeding, activity, injury, reproductive state and a variety of stressors), the neuroendocrine system is the major means for modulating the osmo-regulatory ability of teleost fish (Bern & Madsen 1992).
Many studies have reported that alterations in the salinity and hormones such as cortisol, growth hormone (GH) and prolactin can stimulate expression and activity of different ion and electrolyte transporters in the chloride cells, such as Na+-K+- ATPase (NKA) and Na+-K+-Cl-(NKCC) co-transporter (Pelis & McCormick 2001; Mancera et al. 2002; Singer et al. 2003; Lin et al. 2004; Tse et al. 2006; Khodabandeh et al. 2009).
Cortisol is a major corticosteroid with several physiological roles related to osmoregulation, respiratory and intermediary metabolism, growth, stress and immune function (Wendelaar Bonga 1997; Mommsen et al. 1999). In euryhaline teleost this hormone acts on several osmoregulatory organs such as gills, gut, kidney and urinary bladder (Mancera et al. 1994). Cortisol has a ‘dual osmoregulatory’ role. In addition to sea water (SW)-adapting ability in cooperation with GH/IGF-I axis, it improves the FW-adapting ability in cooperation with prolactin in some teleost fish (Bisbal & Specker 1991; McCormick 2001). Cortisol alone or most probably with GH/IGF-I enhance salinity tolerance after transfer from hypo- to hyper-osmotic medium via stimulating ion secretion as well as decreasing plasma ion levels and osmolality in SW-adapted teleosts. At the gill level, this effect is due to increased chloride cells number and size, enhanced NKA activity, NKA α -subunit and NKCC co-transporter expression induced by cortisol/cortisol with GH treatment (McCormick 1995, 2001; Madsen et al. 1995; Pelis & McCormick 2001; Mancera et al. 2002; Laiz-Carrión et al. 2003). It has been demonstrated that artificially-increased cortisol levels help to counteract osmotic stress when fish are transferred between two different osmotic media by decreasing the plasma ion concentrations in some teleost such as gilthead sea bream, Sparus aurataL., Sockeye salmon Oncorhynchus nerka and common carp, Cyprinus carpio (Mancera et al. 1994; Ban 2002; Abo Hegab & Hanke 1984). Also, this hormone induces elevated NKA activity in Sockeye salmon, O. nerka, common carp, C. carpio, Atlantic salmon, Salmo salar, parr and Juvenile Senegalese sole, Solea senegalensis, (Ban 2002; Abo Hegab & Hanke 1984; Pelis & McCormick 2001; Arjona et al. 2011). Furthermore, exogenous cortisol can affect the branchial chloride cells number and size to improve osmoregulatoy ability.
For Atlantic salmon, S. salar, Persian sturgeon, A. persicus, and beluga sturgeon, Huso huso, chloride cells number is increased by cortisol treatment in FW (Pelis & McCormick 2001; Khodabandeh et al. 2009; Mazloumi et al. 2015). Improvement of the hypo-osmoregulatory ability with cortisol occurred in the coastal cutthroat trout, Oncorhynchus clarki, parr (Morgan & Iwama 1996), in the Atlantic salmon, S. salar (Bisbal & Specker 1991) and the rainbow trout, Oncorhynchus mykiss (Madsen 1990). However, osmoregulation mechanism in some other species such as euryhaline tilapia, Oreochromis mossambicus, Odontesthes bonariensis and pejerrey, Odontesthes bonariensis, are not affected by hormonal treatment either in hypo or hyper-osmotic media (Abo Hegab & Hanke 1984; Tsuzuki et al. 2007).
Hence, the objective of the present study is to investigate the role of exogenous hormonal treatment to enhance the osmoregulation of juvenile Persian sturgeon less than 2 g via monitoring daily mortality, plasma osmolality, and changes in the abundance and localization of the NKA within the gill epithelium of Persian sturgeon following treatment with cortisol.
Although some studies have confirmed osmotic improvement of cortisol-treated sturgeon juveniles weighing 2-3 g in Iran (Khodabandeh et al. 2009; Khoshnood et al. 2010; Fakharzadeh et al. 2011; Mazloumi et al. 2015) but simultaneous interaction of the cortisol treatment and salinity stress for fish less than 2 g is still needed.
MATERIALS AND METHODS
Experimental rearing conditions and fish sampling
In order to improve the salinity acclimation in Persian sturgeon juveniles less than 2 g (1.62 ± 0.27 g) in weight and 7.4 ± 0.58 cm in length, the fish were treated with cortisol (hydrocortisone, Sigma) bathing method using three different concentrations; 3, 5 and 7 mg L-1 for 24 h in FW. They were then directly transferred to the CSW (11‰). So that, the fish were randomly distributed into eight fiberglass tanks filled with aerated FW (60 liters). After 72 h adaptation period, fish were transferred to the experimental tanks. Six tanks were used for hormonal treatments with 2 replicates (2 tanks for each treatment) and two tanks for fish abruptly transferred from FW to CSW as control group. 40 individuals were considered for each tank. The fish were then sampled after 1, 4 and 9 days post-transfer to the CSW.
Daily mortality was recorded. Plasma osmolality, alterations in the number and size of the NKA immunoreactive chloride cells within the gill epithelium of Persian sturgeon juveniles were determined following treatment with cortisol and transfer to the CSW.
Medium and blood osmolality
From each collected fish, blood samples were taken from the caudal vein, just behind the anal fin, with an insulin syringe. On average, 50 µL of blood was collected per fish.
After blood centrifugation (6000g for 10 min), blood plasma was collected. Batches of 10 individuals were pooled in order to determine plasma osmolality. Medium and blood plasma osmolality (triplicates of 15μL each) were determined using an Osmomat 030 osmometer (GANOTEC, Germany).
For fluorescent microscopy, fish gills were excised and fixed in Bouin’s fixative solution for 48 hours (4 fish per treatment).
They were then washed in 70° alcohols, dehydrated in an ascending series of ethanol, and processed for embedding in ParaplastX-TRA® (Sigma-Aldrich, P3808).
Sections (4 µm) cut on a Leitz Wetzlar microtome were collected on glass slides and were stained using the classical Masson's Trichrome staining protocol (Martoja & Martoja-Pierson, 1967) or directly used for immunolabeling.
For immunolabeling, sections were immersed in Histoclear (Histological Clearing Agent, Agar, R1345) twice for 5 min to dewaxing, in butanol for 5 min and hydrated through a graded series of ethanol. Slide rinse was performed using a solution of 10 mM Phosphate-buffered saline (PBS), 150 mM NaCl and 0.01% Tween 20, pH 7.3, for 10 min and then treated with 50 mmol NH4Cl in PBS, pH 7.3 for 5 min to mask free aldehyde groups of the fixative. The sections were then incubated for 10 min with a blocking solution (BS) containing 1% bovine serum albumin (BSA) and 0.1% gelatine in PBS, followed by incubation for 2 hours at room temperature in a wet chamber with the primary monoclonal antibody (α5) to Na+, K+-ATPase (Hybridoma Bank, University of Iowa) diluted in PBS at 10 µg mL-1 covering the sections. Control sections were incubated in PBS without the primary antibodies.
After three extensive 5-min washes in PBS to remove unbound antibody, the sections were incubated for 1 hour with the secondary antibody, donkey-anti mouse; Alexa Fluor® 488 (Invitrogen, Life Technologies), 10 µg mL-1. Following extensive washing in PBS (6 times five-min washes), sections were mounted in 80% glycerine, 20% PBS plus 2% N-propyl-gallate to retard photobleaching (ImmunoHistoMount, Aqueous-based Media, Santa Cruz Bio-technology, USA). Stained and labelled sections were all examined with a Leitz Diaplan microscope equipped with a special filter for fluorescence and associated with a Leica DC 300 F digital camera and its FW 4000 I software (Leica Microsystems, Rueil-Malmaison, France).
Chloride cell morphometry
The number and size of chloride cells located in the filaments and along the base of the lamellae were evaluated on histological and immunofluorescent gill sections of the juveniles kept in CSW without hormonal treatment and treated with different concentrations of cortisol (4 individuals for each experimental condition). Gill sections were photographed (3 slides for each individual and 5 photographs for each slide) at the same magnification (40x). Morphometrically measures were obtained using the freeware Image J (http://rsbweb.nih.gov/ij/). The number of chloride cells / mm2 filament and the size of 10 chloride cells per section (µm2) were calculated (only chloride cells sectioned through their nucleus were considered).
Statistical analyses were performed using SPSS (version 23). Data was analyzed by one-way analysis of variance (ANOVA) and post-hoc Tukey’s HSD tests (P < 0.05) to compare the different concentrations of cortisol treatment to CSW transfer at the different times after transfer to CSW.
Daily mortality rates recorded for fish transferred from FW to CSW differed between control group and fish treated with cortisol bath with three different concentrations (Fig. 1). Higher mortality rate could be observed for untreated fish with cortisol bath compared to treated fish. It seemed that fish treated with higher concentrations of cortisol show higher survivability during the whole experimental period.
Therefore, cortisol treatment with higher concentrations has effect on mortality rates in Persian sturgeon juveniles weighing less than 2 g.
The blood osmolality of fish transferred from FW to CSW, whether treated or untreated with cortisol, significantly increased over the post-transfer hours (P < 0.05) (Fig. 2). Compared to the control group, in fish treated with 3 and 5 mg L-1 cortisol, the highest blood plasma osmolality level was reached 4 days (96 h) after transfer to CSW. However, for the fish treated with 7 mg L-1 cortisol, blood osmolality was at the maximum level only 1 day (24 h) after transfer to the CSW. Only the fish treated with 3 mg L-1 cortisol exhibited significantly decreased plasma osmolality level to CSW osmolality 9 days post-transfer. These fish also displayed significantly lower osmolality values compared to the other two groups and control (P < 0.05). However, in other groups, it was remained at a higher level than CSW osmolality (40 to 45 mOsm Kg-1 higher) throughout the entire duration of the experiment.
Chloride cell morphometry
Chloride cells were identified using NKA immunofluorescent labelling and located along gill filaments and lamellae (Fig. 3). For cortisol treated fish at the all three different concentrations, immunoreactive chloride cells (primary filament or lamellae) number and size were not increased compared to the control group 9 days post-transfer to the CSW (Fig. 4A and 4B) (P < 0.05). Almost 1823 ± 113 chloride cells were observed per mm2 of the gill epithelium and the area of each chloride cell was measured ~124.78 ± 13.62 µm2 whether in control or in treated groups (Fig. 4A and 4B).
There are different ways of hormonal treatments by corticosteroids including bath, injections and live food enrichment. Injection or hormonal implantation is more acute methods but mostly used in large fish and is especially suitable for broodstocks (Madsen & Korsgaard 2004). Because of small sizes of the Persian sturgeon juveniles in this study and in agreement with the other studies (Khodabandeh et al. 2009; Khoshnood et al. 2010; Fakharzadeh et al. 2011; Mazloumi et al. 2015), it is suggested that the osmo or ion-regulatory problems can be solved using cortisol bath method with three different concentrations (3, 5 and 7 mg L-1).
Therefore, in this study, cortisol treatments have been performed with similar concentrations for Persian sturgeon juveniles weighing less than 2 g. In spite of lower mortality in fish treated with 5 and 7 mg L-1 cortisol, chloride cell number and size in treated fish in general displayed the same trends as the control group after 9-days transfer to the CSW.
Most of the studies have focused on the osmoregulatory readiness of sturgeons either in FW (Khodabandeh et al. 2009; Khoshnood et al. 2010; Mazloumi et al. 2015) or during short-acclimation (24 h) in CSW (maximum 7‰) (Fakharzadeh et al. 2011). However, there was no report on simultaneous interaction of hormonal treatment and salinity stress within long-term salinity acclimation (9 days) in the Persian sturgeon juveniles.
No mortality was observed in juvenile Senegalese sole, S. senegalensis, treated with cortisol implantation during 14 days acclimation in SW (38‰) (Arjona et al. 2011). Furthermore, for Persian sturgeons, it has been suggested that fish treated with 5 and 7mg L-1 cortisol exhibits a lower mortality compared to those treated with 3 mg L-1 or control group during 24 h acclimation in FW and diluted CSW (7‰) (Khodabandeh et al. 2009; Fakharzadeh et al. 2011). However, in the present study, mortality rates in both treated and control was lower within the 24 h post-transfer to the CSW, compared to the other studies. Nevertheless, all fish treated with three cortisol concentrations and control group displayed high and similar mortality.
Therefore, result of mortality rates exhibited no effect of cortisol treatment on osmoregulatory enhancement in Persian sturgeon juveniles. In the present study, only fish treated with 3 mg L-1 cortisol significantly lowered their plasma osmolality levels down to the Caspian Sea osmolality 9 days post-transfer to the CSW. This is similar to results reported from other teleosts exposed to the salinity challenges. However, the other fish (control group and those treated with 5 and 7 mg L-1) maintained their plasma osmolality at high level entire the whole experiments. In accordance with the present study, hormonal treatment with cortisol has not displayed influences on the plasma osmolality in gilthead sea bream, S. auruta L., Juvenile Senegalese sole, S. senegalensis, yearling Coho salmon, Oncorhynchuskisutch, channel catfish, Ictalurus punctatus and pejerrey, O. bonariensis, during transfer from FW to BW/SW (Redding et al. 1984; Mancera et al. 1994; Eckert et al. 2001; Tsuzuki et al. 2007; Arjona et al. 2011).
Similar to the result of plasma osmolality, exogenous cortisol treatment alone could not evoke the complete differentiation and maturation of the gill chloride cells in CSW. Chloride cells number and size remained unchanged even 9 days after transfer from FW to the CSW.
Fig. 1. Daily mortality rates for CSW-acclimated Persian sturgeons without cortisol treatment (control) (A), cortisol treated sturgeon with 3 mg L-1 (B) 5 mg L-1 (C), 7 mg L-1 (D) during 9 days in CSW.
Fig. 2. Plasma osmolality for CSW-acclimated Persian sturgeons in control group (A), cortisol-treated sturgeon with 3 mg L-1 (B) 5 mg L-1 (C), 7 mg L-1 (D) during 9 days in CSW. The letters are used to indicate significant differences between FW and CSW-acclimated fish at different times post transfer (One-Way ANOVA test and post-hoc Tukey’s HSD tests, P<0.05). Data are expressed as mean ± SD (n = 10).
Fig. 3. Immunolocalization of Na+, K+-ATPase in the gills of CSW-acclimated Persian sturgeons in control group (A), cortisol-treated sturgeon with 3 mg L-1 (B) 5 mg L-1 (C), 7 mg L-1 (D) after 9 days in CSW. CC, chloride cell; F, gill filament; L, lamellae. Scale bar: 50 µm.
Fig. 4. Simultaneous effect of abrupt salinity transfer from FW to CSW (11‰) and cortisol treatment on chloride cell number (A) and area (B) in the gills CSW-acclimated Persian sturgeons after 9 days in CSW (One-Way ANOVA test and post-hoc Tukey’s HSD tests, P<0.05). Data are expressed as mean ± SD (n = 4).
This, together with an elevated plasma cortisol in the initial phase of SW adaptation, might synchronously stimulate the complete differentiation and maturation of SW chloride cells (Seidelin & Madsen 1999).
Furthermore, an anti-apoptotic action of IGF-I on gill chloride cells, permits cortisol to affect a greater number of partially or fully differentiated chloride cells to enhance salinity tolerance (Veillette & Young 2005). On the other hand, some other hormones mainly prolactin possesses antagonistic effect on cortisol in SW-acclimated fish (Seidelin & Madsen 1997). In hypoosmotic BW, prolactin reduces chloride cell number and size. Sakamoto & McCormick (2006) have suggested that the control of cell turnover (apoptosis and cell proliferation) in different osmoregulatory epithelia (e.g., gill and gastrointestinal tract) is a critical feature of the control of osmoregulation by prolactin. Therefore, it is hypothesized that the level of prolactin in CSW-acclimated fish remained highly and this hormone possesses inhibitory effect on cortisol- treated fish in CSW.
Plasma osmolality and the chloride cell alterations in general, exhibit similar trends, being affected by salinity, time and the interaction of salinity and time, but not by cortisol. In conclusion, this study show that exogenous cortisol at least alone, is not directly implicated in osmoregulation in Persian sturgeon juveniles less than 2 g in weight. Furthermore, effect of hormonal treatment on osmoregulation enhancement in the teleosts is species-specific. It is suggested that other factors perhaps, other hormones acting in concert with cortisol, may activate ion transport mechanisms or inhibit cortisol effect.
In addition to exogenous hormone treatment affecting salinity tolerance, the level of these hormones, mostly cortisol in plasma might be further considered. Because of salinity stress induced by transfer from FW to CSW in Persian sturgeon juveniles, it is possible that the plasma cortisol level increases autonomously. Therefore, artificially cortisol may not be effective for salinity acclimation.
A reason for this result may be due to endogenous cortisol and GH. Therefore, cortisol treatment conjunction with other hormones, as well as measuring plasma cortisol in treated and untreated fish could be suggested. More detailed research is necessary to understand the sequential roles of hormone treatment in salinity tolerance of sturgeon fish.
We are grateful to Tarbiat Modares University (Faculty of Marine Science), Iran and the University of Montpellier, France (MARBEC laboratory). We thank the Iranian fisheries organization for providing the endangered species. The field experiments on live fish were carried out with the expertise of Mr Abbas Alizadeh, head of ShahidBeheshti Artificial Sturgeon Propagation and Rearing Center (SAPRC), Rasht, Iran.