# Salinity stress in the black-chinned tilapia Sarotherodon melanotheron

N'Golo Ouattara, Georgina Rivera-Ingraham, Jehan-hervé Lignot

► To cite this version:

N'Golo Ouattara, Georgina Rivera-Ingraham, Jehan-hervé Lignot. Salinity stress in the black-chinned tilapia Sarotherodon melanotheron. Journal of Experimental Zoology Part A: Ecological and Integra-tive Physiology, 2024, 341 (5), pp.553-562. 10.1002/jez.2798 . hal-04803189

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# Salinity stress in the black-chinned tilapia *Sarotherodon melanotheron*

N'Golo Ouattara<fnref n="1" />

Georgina A. Rivera-Ingraham<fnref n="2" />

Jehan-Hervé Lignot<fnref n="2" /> ID

## Correspondence

Jehan-Hervé Lignot, UMR9190-MARBEC, IRD-Ifremer-CNRS-Université de Montpellier, Montpellier, France.

Email: jehan-herve.lignot@umontpellier.fr

## Funding information

Marie-Curie IF (FP7-PEOPLE-2013-IEF), Grant/Award Number: 622087-"IAS-Life"; Amruge French Government Scholarship

## Abstract

Physiological and morphological acclimation capacities of black-chinned tilapia, *Sarotherodon melanotheron* were studied from fish to gill cell level when fish are maintained in freshwater, seawater, and hypersaline conditions. Fish osmoregulatory capacity, gill ionocyte morphology, osmo-respiratory compromise, O₂ consumption rate, branchial antioxidative defense, and cell apoptosis were considered. Captive juvenile tilapias were maintained in controlled freshwater conditions (FW: 0.4 ppt; 12 mOsm kg⁻<fnref n="1" />) or gradually transferred to seawater (SW: 32 ppt; 958 mOsm kg⁻<fnref n="1" />) and concentrated SW (cSW: 65 ppt; 1920 mOsm kg⁻<fnref n="1" />). After 15 days in these conditions, blood osmolality and chloride ion concentration were determined. Gill ionocyte density and morphology were measured using immunolabelled histological sections to specifically detect the sodium pump (NKA). Gill osmo-respiratory compromise was also calculated along with oxygen consumption rates from normoxic to hypoxic conditions from excised gills (indirect respirometry). Finally, catalase and caspase 3/7 activities were recorded from gill extracts. Results indicate that elevated salinity induces an osmotic imbalance and a profound morphological change with proliferating and hypertrophied ionocytes. This thickening of the gill interlamellar cell mass and the shortening of the lamellae induce a reduced osmo-respiratory ratio and reduced respiratory capacity under both normoxic and hypoxic conditions. Although salinity changes do not affect one of the major antioxidative defense mechanism, it strongly affects apoptosis that appears the most elevated in SW. However, in freshwater condition, fish can maintain their osmotic balance with a low ionocyte density, a low apoptotic level and a drastically reduced O₂ consumption in normoxic condition that is maintained in hypoxia. Therefore, S. melanotheron presents the typical functional remodeling due to environmental salinity changes ranging from FW to SW. However, elevated seawater induces major cellular stress inducing a profound gill morphofunctional dysfunctioning. While cell apoptosis is reduced, ionocyte proliferation is massively increased with impaired osmotic regulation and reduced O₂ consumption both in normoxic and hypoxic conditions.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2024 The Authors. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.

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<footnote n="1">¹Laboratory of Animal Biology and Cytology, Nangui Abrogoua University UFR-SN, Abidjan, Ivory Coast</footnote>

<footnote n="2">²UMR9190-MARBEC, IRD-Ifremer-CNRS-Université de Montpellier, Montpellier, France</footnote>
KEYWORDS

fish physiology, salinity stress, tilapia

# 1 | INTRODUCTION

Some species have adapted to varied environments with broad ecological tolerances. For example, divergent natural selective pressures from extreme aquatic environments (i.e., the continuum from freshwater to hypersaline seawater) can drive rapid phenotypic diversification in euryhaline species. Such parallel evolution can induce population differentiations with different morphotypes and ecotypes within the same species. Therefore, understanding how body or organ morphologies are influenced by physiological processes in plastic euryhaline species can unravel the limits of this plasticity (Li & Kültz, 2020; Weaver et al., 2016).

In euryhaline teleost fish, osmoregulatory processes are studied at different levels from the entire organism down to specific organs (gills, kidney, intestine), specialized cells (ionocytes, also known as chloride cells or mitochondria rich cells and accessory cells) and also at the molecular level (ionic pumps, transporters, water channels). The use of fish transferred to different saline media and the subsequent mechanistic approaches using physiological, biochemical, and molecular analyses has brought major insights about the functionality of these exchanges with the external environment, especially at the gill level. Also, physiological tradeoffs, for example between osmoregulation and oxygen consumption (aka the osmorespiratory compromise), have been thoroughly studied (Blewett et al., 2022; Gilmour & Perry, 2018; Sardella & Brauner, 2007; Wood & Eom, 2021). For example, for the euryhaline Atlantic killifish (*Fundulus heteroclitus*), those acclimated to freshwater present, compared to those acclimated to brackish or marine conditions, a decrease in hypoxia tolerance associated with a reduced respiratory surface area in the gills and corresponding lower oxygen extraction efficiency (Giacomin et al., 2019).

## 1.1 | Tilapias as model species to decipher osmoregulatory processes

Among Tilapias, some species such as the Nile tilapia, *Oreochromis niloticus*, live mostly in freshwater (FW) environments and is not very salt-tolerant. Others, such as the Mozambique tilapia, *Oreochromis mossambicus*, and the black-chinned tilapia, *Sarotherodon melanotheron* (also called lagoon tilapia), are euryhaline and, therefore, highly tolerant to salinity changes. The black-chinned tilapia holds the record of plasticity to salinity fluctuations in fish, adults being able to reproduce in salinities ranging from 0 to 120 ppt (Panfili et al., 2004). It is found in many different environments from FW to seawater (SW) and even hypersaline seawater (HSW) and has been used therefore as a study model for addressing plastic response to such fluctuations. This has been mainly done in terms of reproduction (Legendre et al., 2016; Panfili et al., 2004), behavior (Jennings, 1991) and

osmoregulation processes (Lorin-Nebel et al., 2012; Ouattara et al., 2009). Much research effort has been additionally done to supply these fields of study with molecular tools, namely transcriptomes (Avarre et al., 2014). For example, when S. melanotheron fish are transferred from FW to SW and HSW, drastic ionocyte proliferation occur along with enlargement of these cells (Lorin-Nebel et al., 2012; Ouattara et al., 2009). This increase in number and size occurs up to a hyper salinity of 70 ppt. At higher salinity, only a cell size increase occurs. It has also been documented that, 75% of preexisting ionocytes located in the yolk sac membrane of Mozambique tilapia embryos survive direct transfer from FW to SW, and that each of these ionocytes show a remarkable increase in size (Hiroi et al., 1999; Hiroi & McCormick, 2012). Also, cell turnover occurs since 50% of the initial number of ionocytes appeared after transfer to SW. Therefore, the small FW-type ionocyte can survive direct transfer from FW to SW and can be transformed to the larger SW-type ionocytes. Among these ionocytes, three different types were described along with accessory cells: single ionocytes with no apical pit (presumably immature cells), ionocytes with an apical pit in contact with the external water (mature cells), and cell complexes formed by ionocytes and accessory cells.

The function of these ionocytes also differs with the salinity of the external medium. For example, the activity of the Na⁺/K⁺-ATPase (NKA, also known as the sodium pump) in these cells is usually dependent to the external salinity. As already observed in fish such as the European seabass, *Dicentrarchus labrax*, the marbled spinefoot, *Siganus rivulatus*, and others, NKA activity over a large salinity gradient presents a U shape curve with maximal activity levels at extreme salinities (Saoud et al., 2007). It has, therefore, a key role for ion absorption in FW and ion secretion in SW (Hiroi & McCormick, 2007; Kültz et al., 1992). However, in S. melanotheron, NKA activity is lower in fish kept in FW and increased with salinity (Lorin-Nebel et al., 2012). Different transporters are at play within the gill ionocytes due to salinity changes. In FW, branchial ion uptake occurs through the sodium-chloride symporter, NCC, that is expressed on the apical surface of the ionocytes. Also in SW, ion secretion involves the cotransporter Na⁺/K⁺/2Cl⁻ (NKCC)NKCC that is co-expressed with NKA within the tubulo-vesicular network and along the basolateral membranes of the gill ionocytes. These different transporters have already been described in the European seabass (Lorin-Nebel et al., 2006), the Mozambique tilapia (Hiroi et al., 2005; Wu et al., 2003) and for S. melanotheron (Lorin-Nebel et al., 2012). Finally, the Cl⁻ channel CFTR that is not expressed in the gill ionocytes of S. melanotheron when fish are kept in FW, is expressed in the apical pit of the gill ionocytes in SW-acclimated fish (Lorin-Nebel et al., 2012). It demonstrates the key function of this protein in the secretion of ions in saline waters.

Profound morphological and functional remodeling occur in gill ionocytes due to salinity changes along with cell renewal (ionocyte
proliferation and apoptosis). These are essential mechanisms reshaping the gills to cope with salinity changes. However, the drastic proliferation and size increase of these ionocytes occurring in *S. melanotheron* with salinity induce profound gill remodeling with an interlamellar cell mass (ILCM) filling the space between the lamellae (Lorin-Nebel et al., 2012) and, therefore, compromising breathing capacities. ILCM has been documented in several fish species as a result of many factors such as hypoxia, temperature variations, environmental ammonia, or air exposure (LeBlanc et al., 2010; Mitrovic et al., 2009; Nilsson et al., 2012; Sinha et al., 2014; Turko et al., 2019). For example, it can rapidly grow as a response to high salinity, as seen for the Arctic grayling (Blair et al., 2017), decreasing the surface area of the gills dramatically. However, in the killifish, ILCM is decreased in SW-acclimated animals compared to FW-acclimated fish (LeBlanc et al., 2010). Ammonia exposure (Sinha et al., 2014), air exposure (Ong et al., 2007; Turko et al., 2011), or aluminum exposure in slightly acidic water (Mueller et al., 1991) also induce ILCM. On the contrary, it can occur when fish are exposed to hypoxia (Mitrovic et al., 2009; Sollid et al., 2005; Tzaneva et al., 2011), forced aerobic exercise (Fu et al., 2011; Perry et al., 2012), warmer (>15°C; Mitrovic & Perry, 2009), or colder water temperatures with ionocytes relocated on the edges of the ILCM (Bradshaw et al., 2012; Mitrovic & Dymowska, Nilsson, et al., 2009).

Therefore, the goal of this study was to evaluate the effects of different salinities (freshwater, seawater, and hypersaline condition) on the morphology and physiology of captive black-chinned tilapia *S. melanotheron* to evaluate the fish osmoregulatory and gill respiratory capacities, catalase activity, and apoptosis. Gills have a large surface area but, as multifunctional organs, a basic conflict exists between gas exchange and ion regulation and the balance between the “need of oxygen” and the “need of osmotic regulation” has defined as osmorespiratory compromise (Nilsson, 1986; Randall et al., 1972; Sardella & Brauner, 2007). This study aims at integrating various key physiological and functional parameters to determine what could be the preferred salinity when considering osmotic regulation, gill remodeling, and breathing capacities in normoxic and hypoxic conditions.

## 2 | MATERIALS AND METHODS

### 2.1 | Animal experimentation

Experiments on captive Black-chinned tilapia (*S. melanotheron*) were conducted at the aquatic experimental facilities of ISEM (PLATAX) (Montpellier). Juvenile fish (12–15 cm total length) maintained in freshwater (FW: 0.4 ppt; 12 mOsm kg⁻¹) were transferred to three different tanks. A control tank was kept in FW. For the other two tanks, salinity was gradually increased to seawater (SW: 32 ppt; 958 mOsm kg⁻¹) and concentrated SW (cSW: 65 ppt; 1920 mOsm kg⁻¹). Salinity increase was performed within a week with a different slope for the two experimental salinities (32 and 65 ppt). After 15 days at these different salinities, 12–13 fish per condition were killed with an

overdose of anesthetic (2-phenoxy-ethanol, 500 mg L⁻¹, Aquaveto), and then dissected on ice.

For each fish, blood was drawn using 1-mL syringe coated with heparin (Li-heparin; Sigma-Aldrich) inserted in the caudal vein. Blood samples were immediately used to estimate blood osmolality or flash frozen for subsequent ionic concentration determination. The first left gill arch was dissected for ex-vivo respirometric analyses. The second gill arch was flash frozen in liquid nitrogen to carry out biochemical measurements of antioxidant defenses and oxidative stress. The third gill arch was fixed in Bouin's solution for 24 h for histological analyses. All the experiments on captive animals and excised samples were conducted under the laboratory agreement for animal experimentation no. A-34-172-24 and the author's personal authorization for animal experimentation, both provided by the French government.

### 2.2 | Blood osmolality and chloride ion determinations

Immediately after blood collection, 20 µL samples were directly used to determine blood osmotic pressure by freezing point depression osmometry (Model 3320; Advanced Instruments, Inc.). This osmometer was calibrated using internal standards of 100, 300, and 1000 mOsm kg⁻¹. For each fish, blood osmolality was measured in duplicates. The same was conducted on water samples from the different rearing tanks. Blood osmotic values were plotted against the osmolality of the external medium.

To determine chloride concentration of the blood, 15 µl of plasma was used. Blood plasma was obtained following 8 min of centrifugation at 10,000 g at 4°C. The calibration factor for the blanks, the standards and the samples were obtained using a chloride titrator (AMINCO). This colometric titration method (Schales & Schales, 1941) enables to calculate the concentration of Cl⁻ in the samples (in mmol/L).

### 2.3 | Gill morphology: The osmo-respiratory compromise

The third left gill arch of three fish from each experimental condition was fixed for 48 h in Bouin's fixative, embedded in Paraplast X-TRA® (Sigma-Aldrich; P3808) and cut longitudinally on a Leitz Wetzlar microtome. These histological sections (4 µm) were immunolabelled to detect Na⁺/K⁺-ATPase (NKA) proteins localized in the ionocytes using a standard protocol (Alix et al., 2017; Rind et al., 2017). To detect NKA, a primary polyclonal antibody against the α-subunit of the NKA was used (H-300; Santa Cruz Biotechnology Inc., 12 µg mL⁻¹) coupled to the Alexa Fluor® 488 conjugated goat anti-rabbit IgG (A-21202; Invitrogen) as secondary antibody. Immunolabeled sections were 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 software FW 4000 I (Leica Microsystems).
Ionocyte morphometry (number of ionocytes and cell area) were measured from selected fluorescent cells (19 per fish). Only cells sectioned through their nucleus were considered. Ionocyte density was obtained by counting the immunolabeled cells along a distance of 200 µm along the primary filament. The ratio between gas exchange and osmoregulation was identified on cross sections of the gill lamellae as the length of the thin lamellar epithelium and the length of the thicker lamellar epithelium where ionocytes were immunolabeled. These distances were collected from the tip of the gill lamellae to the junction between the lamellar arteriole blood vessel and the filament artery. All these morphometric parameters were collected using ImageJ (image processing program; National Institute of Health).

## 2.4 | Respirometry

Freshly extracted gills were used for ex-vivo respirometry. All measurements were conducted in a medium composed of diluted SW (dSW), filtered using a 2 µm mesh and supplemented with 25 mM NaHEPES to avoid pH shifts caused by the disappearance of O₂ within the respirometric chambers. The osmolality of the measuring medium was fixed to 350 mOsm kg⁻¹, ensuring that it was isosmotic with the blood. An average of 30 mg of gill filaments were introduced in a respirometric chamber, consisting in a well of 330 µL (maximum volume) and with an oxygen sensor spot glued to its inner side. Each chamber was completely filled with measurement media and closed to ensure that no bubbles of air remained within the chamber. Oxygen concentration was measured in each of the chambers every 3 s using a four-channel fiber optic oxygen meter (FireSting, PyroScience GmbH). Data were recorded through the Pyro Oxygen Logger software. All measurements started in fully saturated medium and lasted until oxygen was completely consumed in each of the chambers, which most often never took more than 1 h. After this time, samples were blotted dry and weighted to express respiration rates as nmol O₂ h⁻¹ mg (fresh weight).

## 2.5 | Antioxidant defenses

Gill samples were diluted in a 50 mM KPi buffer with 120 mM KCl and supplemented with 0.1% Triton X at a 1:15 (w:v) ratio. Samples were then homogenized using four stainless steel milling balls (Restch, no. FR0120) in a Mixer Mill MM 400 (Retsch GmbH) for 1 min at a rate of 30 beats s⁻¹. Antioxidant defenses were quantified as catalase (CAT) activity following the protocol originally described by Aebi (1984). Briefly, CAT activity in the sample is measured as the disappearance of externally added H₂O₂. CAT activities were expressed as U mg prot⁻¹, where 1 U of enzyme activity corresponds to the enzyme's catalytic activity that is, the conversion of 1 µm of substrate per minute under essay conditions. All measurements were carried out in triplicate in a microplate reader (Tecan Infinite M200; Tecan, Männedorf, Switzerland). Protein content measured after Bradford (1976).

## 2.6 | Cellular damage

Cellular damage was quantified as the activity of caspases 3/7 activities, using the Caspase-Glo® 3/7 kit (Promega Corporation), according to the manufacturer's instructions. For this purpose, gills samples were homogenized at 4°C at a ratio 1:100 (w:v) in a lysis buffer composed of 25 mmol l⁻¹ Hepes, 5 mmol l⁻¹, MgCl₂, 1 mmol l⁻¹, EGTA and 1 µl ml⁻¹ of each of the protease inhibitors leupeptin, pepstatin, and aprotinin (Rivera-Ingraham, Barri et al., 2016). Homogenization was conducted following the same procedure as for the CAT activity analyses. Caspase activity levels in supernatants was measured using Tecan microplate reader and values were recorded at 20°C and expressed as relative light units (RLU) referred to mg protein content (Bradford, 1976).

## 2.7 | Data processing and statistical analyses

Respiration rate data were analyzed using the RStudio software (R Core Team, 2014 Version 0.99891). All data sets were tested for normality and homoscedasticity using the Kolmogorov-Smirnov test and Levene tests, respectively. When these assumptions were met, one-way analysis of variance tests were applied followed by Student-Newman-Keuls a-posteriori multiple comparison test. Otherwise, Kruskal-Wallis tests were used followed by U-Mann Whitney pairwise comparisons. All statistical tests were conducted using the SPSS 15.0 (SPSS Inc.). Results are all expressed as average ± standard error of mean.

# 3 | RESULTS

## 3.1 | Blood osmolality and plasma chloride ion concentrations

Significant differences were registered among animals under different salinities (F = 11.454; *p* < 0.001) (Figure 1a). The highest values were shown under cSW conditions with an average of 351 mOsm kg⁻¹, while animals under FW and SW conditions showed similar values (333 and 334 mOsm kg⁻¹, respectively). Chloride ion concentrations differed also among conditions (K = 10.130; *p* = 0.006) (Figure 1b): while no significant differences were recorded under FW and SW conditions, animals under cSW had an average of 15% more chloride ions.

## 3.2 | Gill structure, ionocyte morphology, and quantification of the osmo-respiratory compromise

Immunolabeled ionocytes possessing numerous membrane infoldings holding the sodium pump Na⁺/K⁺-ATPase (NKA), are differently located in fish kept at different salinities (Figures 2a-c). In FW condition (0.4 ppt; 12 mOsm kg⁻¹), ionocytes are only located along the gill filament (Figure 2a). In SW (32 ppt; 958 mOsm kg⁻¹), most of
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Blood osmolality (A) and chloride concentrations (b) registered for animals exposed to freshwater (FW), seawater (SW), and concentrated SW (cSW). For blood osmolality, a one-way analysis of variance test indicated significant differences among treatments. Values associated to the same letter belong to the same subgroup according to a Student-Newman-Keuls a-posteriori multiple comparison test. For chloride concentrations, a Kruskal-Wallis test was conducted and different letters indicate differences among conditions based on pair-wise U-Mann Whitney comparisons. Data are expressed as mean ± SD.</figcaption></figure>

the ionocytes are along the gill filaments but some cells appear more elongated with their extension filling the bottom part of the gill lamellae (Figure 2b). Some ionocytes are also packed at the bottom of these lamellae. In cSW (65 ppt; 1920 mOsm kg⁻¹), ionocytes are mostly filling the bottom part of the lamellae and are packed on top of each other in extended ILCM filling the space between the lamellae (Figure 2c). Ionocyte density appears to be salinity-dependent with a 42% increase from FW to CSW (Figure 2d). These cells are also bigger with increasing salinity (Figure 2e). Along the gill lamellae, the ratio between the thin respiratory epithelium and the thicker epithelium involved in osmoregulation was significantly different for fish kept at different salinities (F = 170; *p* < 0.001) (Figure 2f). Fish kept in cSW presented a significantly lower osmoregulatory ratio (with a 50% decrease) compared to fish kept in FW or SW. This is due to a drastic atrophy of the gill lamellae, the length of the respiratory epithelium being reduced by 70%.

## 3.3 | Respirometry

Excised gills are oxyconforming throughout the whole pO₂ range (Figure 3a). Under normoxic conditions (average values recorded for

the 80%-100% air saturation interval), respiration showed significant differences according to environmental salinity (SW > cSW > FW) (F = 82.468; *p* < 0.001). Under hypoxic conditions (here considered, for mere statistical purposes, as the 10%-30% air saturation interval), significant differences were also recorded (FW = SW > cSW) (F = 22.422; *p* < 0.001) (Figure 3b).

## 3.4 | Biochemical determinations

Protein concentrations did not differ among conditions (F = 0.351; *p* = 0.707). Equally, CAT activity did not show any significant differences among the treatments (F = 0.750; *p* = 0.480) (Figure 4a). Caspase 3/7 activity, used here as a proxy of cellular death, showed differences among environmental salinities (F = 3.547; *p* < 0.05) with SW values showing significantly higher values than animals exposed to FW or cSW (Figure 4b).

# 4 | DISCUSSION

Given the key role of the gills in respiratory and osmoregulatory processes when fish are kept or transferred at different salinities (Li & Kültz, 2020), the integrated use in this study of gill morphology, biochemistry, and physiological measurements on isolated gills provided new insights on the physiological challenges and constraints that *S. melanotheron* face when environmental salinity changes. It reveals that animals under SW condition have the highest metabolic rates. The use of excised gills as a study model, is of special interest since we are able to analyze the energetic requirements of this tissue alone. This is not an uncommon practice in invertebrate studies, mainly bivalves, for which “excised intact gills are considered suitable models for studying the regulation of the energy metabolism in a working tissue” (Doeller et al., 1993). Some studies have carried out excise gill respiration rate (RR) in fish, but examples are scarcer: to the authors’ knowledge, there are no examples using tilapia species, but there are some studies done in other fish species notably regarding temperature (e.g., Evans et al., 1962) or, when fish are exposed to anti-parasite medication (Toovey et al., 1999). RRs were highest in gills collected from animals acclimated to SW, and values decreased as environmental salinity increased or decreased. Despite the fact that we, here, used excised gills, our results match those obtained in other euryhaline species such as the milkfish *Chanos chanos* or sheepshead minnow *Cyprinodon variegatus* who also reported highest RR under SW and comparably low values under reduced and increased salinities (Haney & Nordlie, 1997; Swanson, 1998). Swanson (1998) suggested that this could be due to fish activity level, which was the highest in SW-acclimated animals. In tilapias, most studies have focused on the acclimation from SW to FW and vice versa. Results are similar to those obtained in the present study with only the transfer from FW to SW and hypersaline SW. For example, in *O. mossambicus* (whole animal), RRs are lower in FW than in SW (Kammerer et al., 2010; Morgan et al., 1997). However, in this
<figure><img src="image_4.png" /><figcaption>FIGURE 2 (a-c), Immunolabelling of the NKA indicating the localization of the ionocytes along the gill filaments and gill lamellae of representative fish kept in FW (a), SW (b), and cSW (c). Note the location of the ionocytes along the gill filaments (a) at the bottom of the gill lamellae in SW (b) and in the interlamellae cell mass (ILCM) in cSW (c). Ionocyte hyperplasia and proliferation is observed with elevated salinity, strongly reducing the length of the respiratory part of the lamellae in cSW. (d), Ionocyte density (number of immunolabelled cells along 200 µm of primary filament) for fish kept in FW, SW, and cSW. (e), Ionocyte area (in µm²) obtained from cell sectioned through their nucleus. (f), Ratio between the lengths of the thin and thicker lamellar epithelium from the tip of the lamellae and the intersection between the lamellar and filament blood vessels. The areas corresponding to the thick lamellar epithelium where ionocytes are immunolabelled correspond to the ILCM. I, ionocyte; PF, primary filament; RBC, red blood cells; SL, secondary lamellae. Scale bar: 20 µm. For the graphs, one-way Analysis of variance tests indicated significant differences among treatments. Different letters indicate differences among conditions based on a Student-Newman-Keuls a-posteriori multiple comparison test. Data are expressed as mean ± SD.</figcaption></figure>

species, another study registered exactly the opposite pattern, RRs under SW being significantly lower than in FW and cSW (×1.6) (Iwama et al., 1997). However, as highlighted by these authors, these results are likely influenced by the fact that the experimental animals were initially raised in brackish water. Other studies support that the

salinity conditions in which the fish (different species or populations) have been raised, influence their acclimation capacity to salinity changes (Morgan & Iwama, 1991).

In our study, the increased respiration in SW is accompanied with increased apoptosis (measured as caspase 3/7 activities). It would be
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** Respiration rates results obtained from animals exposed to freshwater (FW), seawater (SW), and concentrated SW (cSW). Values are represented as average values in 1% pO₂ intervals (a), and as average values recorded under normoxic (80%-100% air saturation) and hypoxic (10%-30% air saturation) conditions (b). A one-way analysis of variance test indicated significant differences among treatments. Values associated to the same letter belong to the same subgroup according to a Student-Newman-Keuls a-posteriori multiple comparison test. Data are expressed as mean ± SD.</figcaption></figure>

interested to identify which cell type is apoptotic (the respiratory pavement cells and/or the branchial ionocytes). In green crabs exposed to different salinities, apoptosis only affects the pool of ionocytes (Rivera-Ingraham, Barri et al., 2016). With *S. melanotheron*, the same could occur inducing, therefore, a higher rate of cell renewal, the fish taking advantage of being in a typical, ancestral salinity condition that is not posing significant constraints for the organism; the fish being hypo-osmoregulating, that is, constantly drinking SW and excreting excess solutes through the gills and kidneys. Or, in contrast, since fish raised in FW were transferred to SW, this new environmental condition could impose a transient challenge, individuals requiring increased O₂ supply and, therefore, extra energy to fuel acclimation mechanisms. It could overall result in higher cell turnover with for example, the replacement of FW-type ionocytes along with an increased number of SW-type ionocytes. This has been detailed in Mozambique tilapias, freshwater fish possessing three populations of ionocytes (type I, II, and III), while type I and type IV ionocytes dominate in the gills of seawater-acclimated fish (Inokuchi et al., 2022).

This study also revealed that hypersalinity is the most challenging saline condition for *S. melanotheron*. Conditions of high osmolyte concentrations clearly impose an osmoregulatory constraint to the species, where individuals lose their hydro-mineral balance. This is evidenced by the increased blood osmotic pressure and chloride ion concentration, which are, respectively, 5.5 and 15% higher compared to the other environmental conditions. This could appear surprising given the high number of ionocytes located in the gills of cSW-acclimated animals. However, this hypersaline condition is probably so restrictive for the species that individuals must enter in metabolic depression, thus failing to deliver the necessary energy to fuel osmoregulation at the level of these cells. Such a decrease in (whole-animal) RR under increasing salinity have also been reported for other tilapia species (Sardella et al., 2004) as well as other euryhaline species such as *C. chanos* (Swanson, 1998) and *C. variegatus* (Haney & Nordlie, 1997). Nordlie et al. (1991) suggested that this metabolic depression may be caused by a decrease in the osmotic permeability of the gill membrane, which in turn reduces the potential for oxygen
<figure><img src="image_6.png" /><figcaption>**FIGURE 4** Results obtained from the biochemical analyses conducted: catalase (antioxidant) (a) and capase 3/7 (b) activities. For the latter, a one-way analysis of variance test indicated significant differences among treatments. Values associated to the same letter belong to the same subgroup according to a Student–Newman–Keuls *a-posteriori* multiple comparison test. Data are expressed as mean ± SD.</figcaption></figure>

uptake (see Nordlie et al., 1991 and references therein). This energetic disruption is coherent with other studies indicating that hypersalinity causes a reduction in activity (and hence, in feeding rates) in *C. chanos* (Swanson, 1998), and even, affects growth and reproduction rates, as observed in *S. melanotheron* (Panfili et al., 2004). The ILCM as observed in our study in cSW-acclimated animals could decrease osmotic permeability. Ionocytes that are at the surface of this ILCM are not in contact anymore with the blood vessels and those underneath are not connected to the outside medium. Furthermore, because metabolism under hypersalinity is partly shut down, the energy requirements under hypoxia must also be decreased. Under these conditions, we have no reason to believe that there is oxidative-stress mediated damage or constraints. A decreased respiration rate should also decrease the amount of ROS produced by mitochondria. There are not many reports of the amount of ROS produced by mitochondria under high salinity stress. In one of our studies, focusing on the marine platyhelminth

*Macrostomum lignano* exposed to 55 ppt (1617 mOsm kg⁻¹), it has been observed that DCF-reacting ROS and RNS species are significantly lower compared to animals maintained in diluted SW (Rivera-Ingraham, Nommick et al., 2016). Also, CAT activities remained undisturbed, further supporting that individuals are not suffering from redox unbalance. This is a relevant conclusion insofar that the highest number of ionocytes was observed in these hypersaline-acclimated platyhelminths. Altogether, this decrease in metabolism and redox stress could be a mechanism for individuals to withstand extreme hypersaline condition with minimum damage to promote survival until the return of more favorable conditions.

## 5 | CONCLUSION

Hypersalinity appeared to be the most challenging saline condition for *S. melanotheron*. It induces an osmotic imbalance and a profound morphological change with proliferating and hypertrophied ionocytes and a reduced apoptotic level. The thickening of the gill ILCM inducing the shortening of the lamellae, impaired respiratory capacity under both normoxic and hypoxic conditions. Animals under SW condition have the most elevated gill apoptotic level with also the highest metabolic rates. In freshwater condition, *S. melanotheron* can maintain their hydromineral balance with a low ionocyte density, a low apoptotic level and a drastically reduced O₂ consumption in normoxia that is maintained, however, in hypoxic condition. Therefore, acclimation to salinity fluctuations ranging from FW to SW is a phenotypic trait that can be sustained temporarily in hypersaline condition at the expanse of profound osmorespiratory and cell turnover imbalances.

## ACKNOWLEDGMENTS

The authors would like to acknowledge the support of the PLATAX research platform. Thanks also go to Kiam Barri and Caroline Ferrat for the support in the respiration analyses and to Eva Blondeau-Bidet for the caspase analyses. The study was partially funded by a Marie-Curie IF (FP7-PEOPLE-2013-IEF) awarded to G. A. Rivera-Ingraham (grant number 622087-"IAS-Life"). N'Golo Ouattara was funded by the Amruge French Government Scholarship in 2016 and 2022.

## DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## ORCID

Jehan-Hervé Lignot http://orcid.org/0000-0001-8164-3072

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**How to cite this article:** Ouattara, N., Rivera-Ingraham, G. A., & Lignot, J.-H. (2024). Salinity stress in the black-chinned tilapia *Sarotherodon melanotheron*. *Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, **341***, 553–562. https://doi.org/10.1002/jez.2798