Gill ionocytes of the Lake Magadi tilapia (Alcolapia grahami), an extremophilic teleost native to a highly alkaline environment

Gill ionocytes of the Lake Magadi tilapia (Alcolapia grahami), an extremophilic teleost native to a highly alkaline environment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gill ionocytes of the Lake Magadi tilapia (Alcolapia grahami), an extremophilic teleost native to a highly alkaline environment Jonathan Wilson, Chris Wood, Pierre Laurent, Claudine Chevalier, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8662004/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The Magadi tilapia thrives in arguably the most extreme aquatic environment on earth for fish, the hot springs of Lake Magadi in Kenya with its severe water chemistry: pH 10, alkalinity 300 mEq·L-1. This fish is 100% ureotelic yet has an osmoregulation pattern typical of marine teleosts, although the dominant anion is HCO 3- rather than Cl-. The gills must actively export basic equivalents (HCO 3-+CO 3 2-) and Na + against strong electrochemical gradients, yet simultaneously take up Cl-, for which a hypothetical “Laurent model” based on ionocyte structure alone was proposed. This model has been tested using immunohistochemistry to characterize ionocyte types based on ion transport protein expression patterns [CFTR anion channel, Na + :K + :2Cl-cotransporter (NKCC)/ Na + :Cl-cotransporter (NCC), Na + :HCO 3-co-transporter (NBC), Na + /K +-ATPase (NKA), and urea transporter (UT)]. A typical “seawater ionocyte” (Type IV) with apical CFTR and basolateral NKCC1 and NKA, is present validating key elements of the model. A “freshwater ionocyte” (Type II) is also present (apical NCC, weaker NKA and strong NBC1 basolateral staining). A third Type I ionocyte with only strong NKA staining was also identified. An acid excreting Type III ionocyte (apical NHE3 and basolateral NKA) was not present. The Magadi tilapia is unusual in having co-expression of both Type IV and Type II ionocytes, which are typically associated with Cl-excretion and uptake, respectively. Instead, we propose Type IV ionocytes are involved in basic equivalent and Na + excretion and Type II ionocytes in Cl-uptake. In these ureotelic fishes, the UT occurs only in lamellar pavement cells. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction The Magadi tilapia, with a current taxonomic name of Oreochromis (Alcolapia) grahami as recommended by Ford et al. ( 2019 ) (formerly Alcolapia grahami , formerly Oreochromis alcalicus grahami , formerly Sarotherodon alcalicus grahami , formerly Tilapia grahami ) lives in arguably the most extreme aquatic environment on earth for fish, Lake Magadi in the Rift Valley of Kenya. In addition to temperatures up to 45ºC in its environment, it survives extreme night-time hypoxia and anoxia, daytime hyperoxia, and some of the highest levels of reactive oxygen species (ROS) that have ever been recorded in natural waters (Coe, 1966 ; Narahara et al., 1996; Johannsson et al., 2014 ; Wood et al., 2016 ). However, its greatest challenge may be the highly unusual water chemistry in the hot springs where it lives around the edges of Lake Magadi. Tables listing the compositions of Lake Magadi water and the plasma of O. grahami can be found in Eddy et al. ( 1981 ), Eddy and Maloiy ( 1984 ), Maloiy et al. (1984); Wood et al. ( 1989 ), Laurent et al. ( 1995 ), Wood et al. ( 2002a ), Wood et al. ( 2002b ), Wilson et al. ( 2004 ), and Wood et al. ( 2012 ). Water values vary seasonally and among hot springs. However, the water pH is typically about 10 due to very high concentrations of HCO 3 − and CO 3 2− resulting in an alkalinity over 300 mmol·L − 1 at times and osmolality of about 500–600 mOsm·kg − 1 . By way of comparison, the typical pH of seawater is about 8, its alkalinity is 2–3 mmol·L − 1 and its osmolality is about 1050 mOsm kg − 1 . Moreover, unlike seawater where Na + and Cl − concentrations are both high (450–550 mmol·L − 1 ), Lake Magadi water has Na + concentrations (~ 356 mmol·L − 1 ) that are ~ 3-fold greater than Cl − concentrations (~ 110 mmol·L − 1 ), because the alkaline anions (HCO 3 − and CO 3 2− ) account for most of the negative charge. Surprisingly, the Magadi tilapia is able to maintain a plasma ionic composition and osmolality at levels that are very similar to those of typical teleosts in seawater. However, a particularly striking characteristic is that the fish excrete only urea and no ammonia, presumably an adaptation to the difficulty of excreting ammonia against a large pH gradient; O. grahami is the only 100% ureotelic teleost (Randall et al., 1989 ; Wood et al., 1989 ; Lindley et al., 1999 ). A specific facilitated diffusion urea transporter in Magadi tilapia (mtUT) has been cloned from the gills and tentatively localized to the pavement cells based on the presence of similar vesicles found in the gills of facultatively ureogenic Gulf toadfish ( Opsanus beta ) by transmission electron microscopy (TEM) (Laurent et al. 2000 ; Walsh et al., 2001 ). UT localization has been confirmed by IHC in the Gulf toadfish (Bucking et al. 2013 ). It has proven difficult to accurately measure the blood acid-base status in such small fish (typically < 5 g), but plasma pH and HCO 3 − concentrations appear to be elevated above values typical of other teleosts at comparable temperature (Johansen et al., 1975 ; Wood et al., 1994 ; Wood et al., 2002b ; Wood et al., 2012 ). This undoubtedly reflects the extreme alkalinity of the environment, which makes the water a “CO 2 vacuum” that renders the retention of dissolved CO 2 in the blood virtually impossible, while simultaneously presenting very high concentrations of basic equivalents to the external gill surface. Furthermore, in order to maintain body hydration in this hyperosmotic environment, Magadi tilapia drink the external water at a high rate (Maloiy et al., 1978 ; Wood et al., 2002a ; Bergman et al., 2003). However, rather than using the intestinal transport of Na + and Cl − to drive the absorption of the water in the manner of seawater teleosts, instead they transport Na + and mainly HCO 3 − or CO 3 2− , rather than Cl − in order to absorb water across the gut epithelium (Bergman et al., 2003). Thus, the influx of basic equivalents occurs at both intestine and gills. Detailed analyses of the electrochemical gradients across the gills have demonstrated that there is a very strong net gradient driving basic equivalents − inwards, a modest net gradient driving Na + inwards, and a smaller net gradient driving Cl − outwards (Eddy et al., 1981 ; Wood et al., 2004; Wood et al., 2012 ). Passive permeability of the gills to HCO 3 − is unusually low, which is presumably adaptive to reduce the loading of basic equivalents (Wood et al., 2012 ). Nevertheless, the cells are tasked with the unusual situation of excreting basic equivalents to the environment against a steep electrochemical gradient, while simultaneously excreting Na + yet taking up Cl − . The transepithelial potential (TEP) is positive inside, and this results from an inwardly directed Na + diffusion potential upon which is superimposed an electrogenic component thought to result from the secondary active export of basic equivalents (Wood et al., 2012 ). In standard marine teleosts, the TEP is also positive inside, but the electrogenic component results from the secondary active export of Cl − rather than basic equivalents (Potts, 1984 ; Potts and Hedges, 1991 ). There have been numerous morphological studies on the gill ionocytes using classical techniques (light, scanning, and TEM microscopy; Maina, 1990 ; Maina, 1991 ; Laurent et al., 1995 ; Wood et al., 2002a ; Wood et al., 2013 ; DeBoeck et al., 2019 ). Some of these studies suggest that more than one type of ionocyte is present, but the general picture that has emerged is that most of the ionocytes have a typical seawater morphology and location. These cells are rich in mitochondria with a complex tubular network penetrating the cytoplasm from the basolateral surface and are recessed at the bases of pits bordered by partially overlying pavement cells and flanked by interdigitating accessory cells. Based only on the morphology of these ionocytes, Laurent et al. ( 1995 ) proposed a scheme of passive Na + export through the paracellular “shunt” pathway (between ionocytes and accessory cells) energized by the secondary active excretion of basic equivalents through the transcellular pathway into the apical crypts. This would be powered by basolateral Na + /K + -ATPase, with basic equivalents entering the cell on a basolateral co-transporter analogous to NKCC (Na + :K + :2Cl − cotransporter) and leaving apically through anion channels. The scheme was a modification of the classic “chloride cell” model of Silva et al. ( 1977 ), but with basic equivalents replacing Cl − at every step. Later electrophysiological data (Wood et al., 2012 ) supported this model. Additionally, Laurent et al. ( 1995 ) proposed that Cl − uptake occurred via an apical Cl − /HCO 3 − exchanger on these cells. Immunohistochemistry (IHC) is a powerful tool to characterize ionocytes beyond the information provided by structure (Dymowska et al., 2012 ; Hiroi and McCormick, 2012 ; Wilson 2013). However, with the exception of its use in localizing ammonia transporters (Wood et al., 2013 ), this technique has not been applied to the gills of O.A. grahami. In other tilapine fishes, IHC has identified three (Dymowska et al., 2012 ) or four types (Hiroi et al., 2005 ; Hiroi et al., 2008 ; Choi et al., 2011 ; Hiroi and McCormick, 2012 ) of ionocyte. The model of Laurent et al. ( 1995 ) seems to match, at least in part, the Type IV “seawater ionocyte”, which is thought to be a transformation of the Type III ionocyte seen in freshwater and dilute salinities (Hiroi et al., 2005 ; Hiroi and McCormick, 2012 ; Dymowska et al., 2012 ; Inokuchi et al., 2022 ). In the present IHC study on the gills of the Magadi tilapia, we have applied a battery of antibodies that has proven useful in characterizing the various ionocytes in other teleosts. Our first goal was to determine whether the ionocytes fit into the three or four categories identified in other tilapine fishes (Dymowska et al., 2012 ; Hiroi et al., 2012; Inokuchi et al., 2022 ). A second objective was to evaluate the transport model of Laurent et al. ( 1995 ) for the most abundant type of ionocyte, and a third was to confirm the tentative localization of the mtUT urea transporter to the pavement cells (Walsh et al., 2001 ). 2 Materials and Methods 2.1 Animals Adult O.A. grahami (2–5 g) were collected by seine net from Fish Springs Lagoon (1°53′30.2″S, 36°18′09.9″E) at the edge of Lake Magadi, Kenya. The water chemistry of Fish Spring Lagoon measured at the time of capture is presented in Supplementary Table S1. Collections were carried out under permission from the Department of Fisheries, Ministry of Livestock and Fisheries (Kenya), and the Magadi Soda Foundation. The research complied with the laws of Kenya, and was performed under a research ethics clearance permit (NCST/RR1/12/1/MAS/99) from the National Commission for Science, Technology and Innovation (NACOSTI Kenya), under a research permit issued by the National Council for Science and Technology of the Ministry of Higher Education, Science, and Technology of the Republic of Kenya. Protocols were approved by the Animal Use and Ethics Committee of the Faculty of Veterinary Medicine, University of Nairobi. Fish were euthanized with an overdose of MS-222 (Syndel, Nanaimo, BC, Canada), wrapped in aluminum foil, and flash-frozen whole in liquid nitrogen, then transported at -80ºC in a dry shipper (CX100, Taylor Wharton, Columbus, OH, USA), and stored in an ultracold freezer at -80 ºC for later processing. 2.2 Histology and Immunohistochemistry Whole frozen fish were immersion-fixed in Dent’s fixative (20%DMSO/methanol) chilled to -80°C. After one week the solution was substituted with 100% methanol at -80°C and then slowly brought to 4°C over 3 days. Whole fish were then decapitated and the heads divided medially. After bringing the samples to room temperature, they were cleared with xylene, infiltrated with Type 6 paraffin (Richard-Allen Scientific, San Diego, CA, USA) and embedded for sectioning. Five-µm sections were collected onto either plain or APS (3-aminopropyl triethoxy silane) coated slides for respective histological staining with Periodic Acid Schiffs (PAS) and Alcian blue (pH 2.5) staining, or immunohistochemical staining for ion transporter proteins according to Wilson et al. ( 2007 ). Immunohistochemistry was performed with combinations of antibodies that included mouse monoclonal cystic fibrosis transmembrane regulator (CFTR), Na + /K + -ATPase (NKA) α subunit, and Na + : K + : 2 Cl − co-transporter-1 / Na + : Cl − co-transporter (NKCC1/NCC) antibodies plus rabbit polyclonal NKA, Na + : HCO 3 − co-transporter (NBC1), urea transporter [toadfish (tf) UT], the vacuolar type proton pump (VHA) B subunit, and NHE3b antibodies. See Table 1 for specific details on the antibodies. NBC, tfUT and NHE3b antibodies were kindly provided by Drs. S. Hirose (Tokyo Institute of Technology, Yokohama, Japan), P. Walsh (U. Ottawa, Ottawa Canada), and J. Hiroi (St. Marianna University School of Medicine, Kawasaki, Japan), respectively. Secondary antibodies used were goat anti-mouse Alexa 594 and goat anti-rabbit Alexa 488 conjugated antibodies (Thermo Fisher Scientific, Toronto, ON, Canada) diluted 1:500. Negative and null controls were performed, which consisted of primary antibody substitution with either normal rabbit serum or antibody dilution buffer, respectively. When possible, antigen pre-absorption controls were also performed. For monoclonal antibody negative controls, equivalently diluted iso-typed culture supernatant was used. In these experiments, the clone J3, which is specific for shark NKCC (Slc12a1), was used. Sections were counter-stained with DAPI (4 ', 6-diamidine-2'-phenylindole dihydrochloride) and a differential interference contrast (DIC) image collected along with fluorescence using a Leica DM6000B wide-field photomicroscope with a DFX340 camera and LAS IF software (Leica Microsystems GmbH, Wetzlar Germany). Images were assembled and enhanced (Photoshop CS3, Adobe San Jose CA USA) while maintaining the integrity of the data. Table 1 List of antibodies used to probe Oreochromis (Alcalicus) grahami gill sections. The antibody name, target, dilution in 1%Bovine Serum Albumin /Phosphate Buffered Saline with 0.05% Tween-20 and 1%Normal Goat Serum for Immunohistochemistry, host species, and reference information. Antibody Target IHC WB Host spp. Ref α5 NKA α subunit 1:100 1:500 Mo Takeyasu et al. 1988 αR1 NKA α subunit VTGVEEGRLIFDNLKKC 1:500 1:500 Rb Ura et al. 1996; Wilson et al. 2007 T4 NKCC/NCC human NKCC1 C-terminus (Met902-Ser1212) 1:100 1:1000 Mo Lytle et al., 1995 Hiroi et al. 2008 673 Mefugu T. obscurus mfNBCe1 [amino acid (aa) residues 985–1075] 1:100 1:1000 Rb Kurita et al. 2008 VHA B RKDHADVSNQLYACYA 1:500 1:1000 Rb Wilson et al. 2007 NHE3b Trout nhe3b position 755–769: GDEDFEFSEGDSASG; 818–839: PSQRAQLRLPWTPSNLRRLAPL 1:200 1:1000 Rb Wright et al. 2016 24 − 1 CFTR c-term 1:200 n/a Mo R&D systems Marshall et al. 2002 tfUT Slc14a2 SEIDLPKLFMSIPV 1:200 1:1000 Rb Bucking et al. 2013 3 Results Tissue morphology Flash freezing whole fish in liquid nitrogen, storage at -80°C and fixation in Dent’s fixative at low temperature (-80°C) resulted in tissue sections of reasonable quality for histological analysis (Fig. 1 ). Gill lamellae were largely devoid of large ionocytes which were limited to the gill filament along with large Alcian-blue staining goblet cells. There were some areas where freezing artefact could be identified and thus avoided for analysis. IHC On the basis of NKA α-subunit immunoreactivity using either the α5 mouse monoclonal or αR1 rabbit polyclonal antibodies, ionocytes could be easily recognized in the filament epithelium of the gills of Magadi tilapia with a dramatic increase in frequency towards the downstream or afferent edge (Figs. 2 , 3 ). These cells were readily identifiable by their apparent cytoplasmic staining which is indicative of the tubular system associated with the basolateral membrane in teleost ionocytes. Two types of ionocytes could be further characterized based on the strength of their NKA immunoreactivity and morphology. Ionocytes with strong NKA immunoreactivity were typically columnar-cuboidal in shape with a less pronounced apical crypt (indentation). In contrast, ionocytes with weaker NKA- immunoreactivity were characteristically ovoid-squamous in shape with a prominent apical crypt. These cells could be distinguished from pavement cells that were much more flattened and devoid of detectable NKA immunoreactivity. The NKA immunoreactive ionocytes were further characterized based on expression of other transporters including NKCC, NCC, CFTR and NBC. Localization of both the NKCC1 and NCC were done using the T4 mouse monoclonal antibody which recognizes both transporters although they can be differentiated based on subcellular localization (Hiroi et al. 2008 ). NKCC1 is identified by its basolateral localization similar in appearance to NKA. Weak NKCC1 immunoreactivity was found to co-localize with intense NKA- immunoreactivity in the columnar-cuboidal ionocytes corresponding to the type IV sub population (Fig. 3 ). In contrast, NCC immunoreactivity was identified on the basis of an apical localization to type II ionocytes. Strong NCC- immunoreactivity was present in either a shallow or deeper C-shaped concave crypt or pit in ovoid-squamous ionocytes which exhibited weak NKA- immunoreactivity. CFTR immunoreactivity was found apically in the columnar-cuboidal type IV ionocytes with strong NKA immunoreactivity (Fig. 2 ). The apical CFTR staining was restricted to a relatively small apical area in contrast to NCC and did not form a deep crypt or pit. CFTR- immunoreactivity was not seen in the ovoid-squamous ionocytes with weak NKA- immunoreactivity. The NBC1-like immunoreactivity was found associated with the ovoid-squamous ionocytes with weaker NKA- immunoreactivity (Fig. 3 ) and apical NCC (Fig. 4 ). We could not find colocalization of NBC1 and CFTR to the same cell type (Fig. 5 ). The tfUT- immunoreactivity was found in the lamellar epithelium in a punctiform pattern (Fig. 6 ). Staining was not associated with NKA- immunoreactive cells which were largely localized to the trailing edge of the filament epithelium. No immunoreactivity was found with the antibodies for V-type H + -ATPase and NHE3b (data not shown). Negative control staining of sections done in parallel showed negligible levels of background fluorescence (data not shown). Discussion With respect to our original objectives, we have identified three different types of ionocyte in the gills of the Magadi tilapia which fit clearly into three (Types I, II, and IV) of the four categories of ionocytes that have previously been characterized in other tilapine fishes (Hiroi et al., 2005 ; Hiroi et al., 2008 ; Choi et al., 2011 ; Dymowska et al., 2012 ; Hiroi and McCormick, 2012 ; Inokuchi et al., 2022 ). Furthermore, in the most abundant ionocyte (Type IV), our IHC results have confirmed many elements of the hypothetical model for Na + excretion plus basic equivalent excretion that was originally proposed by Laurent et al. ( 1995 ) on the basis of structure alone. Finally, we have also shown that the facilitated diffusion transporter UT is confined to the pavement cells, and does not occur in ionocytes, confirming the original conclusion of Walsh et al. ( 2001 ) that again was based on cell fine structure alone. Ionocyte types in the gills of O.A. grahami Based on IHC labelling, ionocytes corresponding to Types I, II, and IV of Hiroi et al. ( 2005 ), Hiroi et al. ( 2008 ), Choi et al. ( 2011 ), and Inokuchi et al. ( 2022 ) were found in the gills of the Magadi tilapia. This agrees with previous studies using classical techniques that have reported several types of ionocytes in the gills of this species, but without categorization (Maina, 1990 ; Maina, 1991 ; Wood et al., 2002a ). The localization of transporters within the ionocyte types is illustrated in Fig. 7 . Type I ionocytes stained only for NKA on the basolateral membranes and the vesicular network that extends from the base into most of the cytoplasmic component. The antibodies used in the present study did not identify any transporters on the apical membranes. However, there is evidence that in Mozambique tilapia, Type I ionocytes express ROMK (renal outer medullary K + ) channels on their apical membranes and are involved in K + excretion (Furukawa et al., 2014). This could be useful in Magadi tilapia which eat mainly blue-green algae such as Arthrospira (Coe, 1966 ) that is rich in K + (Cogne et al., 2002). Plasma K + concentrations (~ 5 mmol·L − 1 ; Wood et al., 2013 ) are about 2-fold environmental concentrations (Supplementary Table S1), and branchial K + excretion through ROMK channels would be further aided by the positive TEP inside the fish (Wood et al., 2012 ). However, since ROMK channels have also been found in Type III and IV cells in Mozambique tilapia (Furukawa et al., 2014; Inokuchi et al., 2022 ) the more general view is that Type I cells are immature ionocytes with limited or no contact with the outside water, waiting to differentiate into the other types (Inokuchi et al. ( 2022 ). There is evidence that O.A. grahami can be adapted to dilutions of Lake Magadi down to 1% (Wood et al., 2002a ; Wood et al., 2002b ) and even to true fresh water (Kavembe et al., 2015 ), and that other populations in the Magadi basin live naturally in much more concentrated water (see Table 5 in Wilson et al., 2004 ) than found in Fish Spring Lagoons (Supplementary Table 1), so this capability probably becomes very important under these conditions. Type II ionocytes were identified by apical NCC and basolateral NBC1 immunoreactivity, and the absence of apical CFTR immunoreactivity (Figs. 3 , 4 ). These combined characteristics were present only in the ovoid-squamous ionocytes which exhibited weak NKA- immunoreactivity and pronounced apical pits. Note that according to Choi et al. ( 2011 ) and Hiroi and McCormick ( 2012 ), the immunoreaction of the T4 antibody used in the present study is attributable to NCC when it appears in the apical membrane, and to NKCC1 when it appears in the basolateral membrane. In other tilapines, the Type II ionocyte is now considered to be a “freshwater ionocyte” responsible for Na + and Cl − uptake from dilute environments, (Hiroi et al., 2005 ; Hiroi et al., 2008 ; Dymowska, et al., 2012 ; Hiroi and McCormick, 2012 ; Inokuchi et al., 2022 ). Interestingly, Wood et al. ( 2002a ) reported that ionocytes with this sort of structure increased in abundance when Magadi tilapia were challenged with environmental dilution. Indeed, they became completely dominant when the fish were acclimated to 1% Lake Magadi water. In other tilapines, Type II cells decrease at higher salinities, yet they are abundant in O.A. grahami in Fish Springs Lagoon where the normal external osmolality is hypertonic and corresponds to about 57% of that of seawater (Supplementary Table S1). The explanation may lie in the unusual ionic composition of Lake Magadi water. In contrast to 57% seawater, Magadi water exhibits a relatively low Cl − concentration (Supplementary Table S1), much below that of the blood plasma. Indeed, taking the positive TEP inside into account, there is still an electrochemical gradient favouring the outward diffusion of Cl − at the gills (Wood et al., 2012 ). To satisfy the need for acquisition of this essential ion, the Magadi tilapia must use the secondarily active uptake of Cl − via the NCC co-transporters on the Type II ionocytes. The additional (counter-productive) uptake of Na + is an unavoidable consequence, which is exported to the internal environment by the basolateral NKA. In this species, we found no IHC evidence for either apical Na + / H + exchange or V-type H + -ATPase, the transporters usually associated with the active uptake of Na + and acidic equivalent excretion in the ionocytes of other non-tilapine teleosts (Evans et al., 2005 ; Dymowska et al., 2012 ). This is not surprising, as their function would presumably be maladaptive for Na + and acid-base homeostasis in the Magadi tilapia. Only NHE3 (and not V-type H + -ATPase) has been found in other tilapines, and only in Type III and IV ionocytes (Inokuchi et al., 2008 ; Choi et al., 2011 ). Type III ionocytes do not appear to be present in O.A. grahami . Type IV cells were by far the most abundant ionocytes in the gills of O.A. grahami and were recognized by strong NKA immunoreactivity throughout the vesicular network of the cytoplasm and along the basolateral membrane, coupled with strong apical expression of CFTR, and relatively weak expression of NKCC1 which followed the same pattern as that of NKA (Figs. 3 – 5 ). These characteristics were confined to the columnar-cuboidal ionocytes with less prominent apical pits. These cells clearly resemble the ionocytes that are dominant in other tilapines when acclimated to seawater (Hiroi and McCormick, 2012 ; Inokuchi et al., 2022 ). Note that when other tilapines are transferred from less saline to more saline waters, Type IV ionocytes may be generated by the transformation of Type III ionocytes, as well as by the differentiation of Type I ionocytes (Choi et al., 2011 ; Inokuchi et al., 2014). However, Type III ionocytes, which would have been identified by the presence of apical NHE and absence of apical CFTR, were not seen in the present study. The Type IV ionocytes identified in the present study appear to correspond to the most abundant ionocytes that were described in Magadi tilapia on the basis of morphology alone by Maina ( 1990 ), Maina ( 1991 ), Laurent et al. ( 1995 ) and Wood et al.(2002a). Laurent et al. ( 1995 ) also documented that they were bordered by accessory cells that play an important role in the model for NaCl excretion (Silva et al., 1977 ; Hiroi and McCormick, 2012 ) in seawater teleosts. Evaluation of the Laurent model for ion transport in Type IV ionocytes of O.A. grahami Very clearly, the model proposed Laurent and co-workers ( 1995 ) for Na + , Cl − , and basic equivalent transport across the gills of the Magadi tilapia was prescient. The first support for this model was provided by electrophysiological data showing that there is an inside positive TEP of partially electrogenic origin that is responsive in the expected fashion to experimental manipulations of HCO 3 − gradients (Wood et al., 2012 ). The present IHC data provide additional support for the presence of key elements in the Type IV ionocytes of Magadi tilapia, namely apical CFTR and basolateral NKCC1, powered by basolateral NKA. As noted in the Introduction, Laurent’s scheme was a modification of the classic “chloride cell” model of Silva et al. ( 1977 ), with basic equivalents replacing Cl − at every step. In 1995, the anion channel for apical exit of Cl − in fish ionocytes had not been characterized, and it was not until three years later that it was tentatively identified as CFTR (Singer et al., 1998 ), a concept that is now generally accepted (Marshall and Singer, 2002 ; Evans et al., 2005 ; Hiroi and McCormick, 2012 ). Laurent proposed that this unknown apical anion channel could serve as the exit pathway for basic equivalents, but it was only around that same time that the first evidence was emerging from mammalian studies that CFTR could also conduct HCO 3 − (Poulson et al., 1994; Linsdell et al., 1997 ). Laurent also proposed that a basolateral NKCC-like transporter, energized by basolateral NKA, could serve as the entry pathway for basic equivalents, together with Na + , into the ionocyte. Na + would be exported by NKA into the paracellular channels for exit into the external water through leaky junctions between the ionocytes and accessory cells. This export of Na + would create the electrochemical gradient for the operation of the NKCC-like transporter. NKA is highly abundant and NKCC1 is present in the basolateral membranes of Type IV ionocytes (Figs. 2 , 5 ), and accessory cells are also present, in accord with the Laurent model. However, we are aware of no evidence whether or not NKCC can transport basic equivalents such as HCO 3 − and CO 3 2− on its anion binding site, so further research is necessary to evaluate this idea. Certainly, basolateral NBC is not present in Type IV ionocytes (Fig. 5 ), which tends to eliminate this alternate pathway for HCO 3 − entry. Finally, Laurent proposed than an apical Cl − /HCO 3 − exchanger could account for Cl − uptake from the water. There is no IHC evidence for or against this idea in the present study or in previous work on other tilapine fishes. However, as argued earlier, the presence of apical NCC on Type II ionocytes (Fig. 3 , 4 ) may provide the pathway needed for Cl − uptake from the external water. Localization of UT exclusively to the pavement cells in the gills of O.A. grahami The fully ureotelic O.A. graham was only the second teleost species in which a UT-type facilitated diffusion transporter was cloned from the gills and characterized (Walsh et al., 2001 ). The initial discovery was in the facultatively ureogenic gulf toadfish ( Opsanus beta ) (Walsh et al., 2000 ). However, we now know that UTs are expressed in the gills of many teleosts, even though most are ammoniotelic (LeMoine and Walsh, 2015 ). In the Magadi tilapia, the presence of intense vesicular trafficking in the pavement cells, and its absence in the pavement cells of the closely related Nile tilapia, led to the tentative conclusion that the UTs were expressed in the pavement cells (Walsh et al., 2001 ). The present IHC observations confirm this conclusion, showing no UT in NKA-immunoreactive cells on the filament, but clear staining in pavement cells in the lamellar epithelium (Fig. 6 ). In this regard, the pattern is similar to the UT expression seen only in the pavement cells of the ammoniotelic plainfin midshipman ( Porichthys notatus ) (Bucking et al., 2013 ) but differs from that in its close relative, the ureogenic gulf toadfish, where UT expression is localized to NKA immunoreactive cells on the filament (Bucking et al., 2013 ). Similarly, in the ammoniotelic eel, UT is seen only within NKA-immunoreactive cells on the filament, and not in pavement cells (Mistry et al. 2001 ). Clearly, patterns vary amongst species, regardless of ureotelism versus ammoniotelism. However, as illustrated by Walsh et al. (2021), urea excretion rates in the 100% ureotelic Magadi tilapia are so much higher than in other fish that the much greater surface area of the pavement cells relative to the ionocytes may be required for efficient clearance. Future Directions The current IHC data show that the Magadi tilapia has three of the four basic ionocyte types that have been characterized in the gills other tilapines and suggest that they have been subtly re-engineered so as to function in such an unusual water chemistry. However, antibodies can reliably identify only proteins against which they are made. The recent full sequencing of the genome in this species (Bernardi et al., 2024 ) opens up the possibility to look for many more transport proteins in the gills, both by IHC and in situ hybridization. Another powerful avenue for future research is to employ the opercular epithelium of the Magadi tilapia, which is rich in ionocytes (Laurent et al., 1995 ), as a flat gill surrogate in vitro . Both classical (e.g. Ussing chamber; McCormick et al., 1992 ) and modern techniques (e.g. scanning ion-selective electrode technique or SIET; Horng et al., 2009 ) that have been successfully used on comparable preparations from other tilapines can then be employed to mechanistically characterize the ion transport mechanisms that allow this unique fish to live in such a hostile environment. Declarations Acknowledgements We thank the Magadi Soda Company, particularly John Ndonga and John Kabera, for their co-operation, the University of Nairobi for logistic support, and Dishon Muthee and the late George Muthee furnished invaluable assistance in the field. Drs. S. Hirose (Tokyo Institute of Technology, Yokohama, Japan), P.J. Walsh (U. Ottawa, Ottawa Canada) and J. Hiroi (St Marianna University School of Medicine, Kawasaki, Japan) for the gift of antibodies. The α5 and J3 monoclonal antibody developed by Drs. D. M. Fambrough (Johns Hopkins University, MD, USA) and C. Lytle (Division of Biomedical Sciences, University of California, Riverside, CA, USA) were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. CMW was supported by the Canada Research Chair Program. AB is a Research Fellow from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and was supported by the International Canada Research Chair Program from the International Development Research Centre (IDRC, Ottawa, Canada). Funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grants to CMW (RGPIN-2017-03843, RGPIN-2023-03714) and JW (RGPIN-2019-06838), a grant from the Brazilian CNPq to AB, and a grant from the National Research Foundation of South Africa to JM. References Bergman, A.N., Bergman, H.L., Laurent, P., Maina, J.N., Walsh, P.J., and Wood, C.M. (1996). 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Ojoo","email":"","orcid":"","institution":"University of Nairobi","correspondingAuthor":false,"prefix":"","firstName":"Rodi","middleName":"O.","lastName":"Ojoo","suffix":""}],"badges":[],"createdAt":"2026-01-21 16:23:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8662004/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8662004/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101303836,"identity":"5e144e6a-87f7-4c59-9025-55f784dbf2df","added_by":"auto","created_at":"2026-01-28 10:00:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2997324,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Sagital section through the head of \u003cem\u003eO\u003c/em\u003e.\u003cem\u003eA. grahami\u003c/em\u003e stained with PAS and Alcian Blue (pH 2.5). Higher magnification regions of sagital sections through the gill filaments revealing (b) lamellae and (c) the trailing edge. Scale bar (a) 0.5 cm or (b,c) 100 µm.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/a96b7c983a2bb0ae99304a84.png"},{"id":101302280,"identity":"0357a7c0-195c-48a6-b3c0-91502a7617eb","added_by":"auto","created_at":"2026-01-28 09:53:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2974701,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal section through the trailing edge of \u003cem\u003eO\u003c/em\u003e.\u003cem\u003eA. grahami\u003c/em\u003e gill filament double immunolabelled for \u003cstrong\u003eCFTR \u003c/strong\u003e(magenta; mouse monoclonal 24-1 )\u003cstrong\u003e \u003c/strong\u003eand \u003cstrong\u003eNKA α subunit \u003c/strong\u003e(green; rabbit polyclonal). Sections are counter stained with \u003cstrong\u003eDAPI\u003c/strong\u003e (blue) and overlaid with the DIC image. Insets are at 2x magnification. Scale bar 50 µm\u003c/p\u003e","description":"","filename":"Fig2Agram1gill41cch0003e.png","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/21fe92d0e7d100776eb1329c.png"},{"id":101302019,"identity":"df234b63-d6d5-4390-8bf9-2c98af37966d","added_by":"auto","created_at":"2026-01-28 09:53:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4385726,"visible":true,"origin":"","legend":"\u003cp\u003eDouble immunofluorescent labeling of \u003cstrong\u003eNKCC/NCC\u003c/strong\u003e (magenta; T4 monoclonal antibody) and \u003cstrong\u003eNKA α\u003c/strong\u003e \u003cstrong\u003esubunit\u003c/strong\u003e (green; rabbit polyclonal) in the trailing edge of the gills of \u003cem\u003eO\u003c/em\u003e.\u003cem\u003eA. grahami\u003c/em\u003e. See Fig.2 caption for additional information. Inset at 2x magnification. Scale bar 50 µm.\u003c/p\u003e","description":"","filename":"Fig3Agram1gill42cch0003d.png","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/a145f340464b38d66e064d91.png"},{"id":101303505,"identity":"afa372b8-9c0e-439f-8267-b0bf825ea48e","added_by":"auto","created_at":"2026-01-28 09:59:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2515259,"visible":true,"origin":"","legend":"\u003cp\u003eDouble immunofluorescent labeling of \u003cstrong\u003eNKCC/NCC\u003c/strong\u003e (magenta; T4 monoclonal antibody) and \u003cstrong\u003eNBC1 \u003c/strong\u003e(green; rabbit polyclonal) in the trailing edge of the gills of \u003cem\u003eO\u003c/em\u003e.\u003cem\u003eA. grahami\u003c/em\u003e. See Fig.1 caption for additional information. Inset at 2x magnification. Scale bar 50 µm.\u003c/p\u003e","description":"","filename":"Fig4Agram1gill52ach0003f.png","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/42b8631bf5d36557aef8b89a.png"},{"id":101302234,"identity":"caf1c61a-284b-4e20-8074-6982f1090d81","added_by":"auto","created_at":"2026-01-28 09:53:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2425074,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal section through the trailing side of the Magadi tilapia gill filament double labelled for both \u003cstrong\u003eCFTR \u003c/strong\u003e(magenta)\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e NBC1 \u003c/strong\u003e(green). See Fig.2 caption for additional information. Inset at 2x magnification. Scale bar 50 µm or 25 µm for the insets.\u003c/p\u003e","description":"","filename":"Fig5Agram1gill51cch0003d.png","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/1adbf58d1103513ed26bda55.png"},{"id":101397693,"identity":"6df03f9a-c694-4603-b1a5-ef965d2db404","added_by":"auto","created_at":"2026-01-29 09:35:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7655491,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical localization of the urea transporter (\u003cstrong\u003eUT; green\u003c/strong\u003e) double labelled with the \u003cstrong\u003eNKA\u003c/strong\u003e (magenta) in cryosections of the gills of the Magadi tilapia. Scale bar 100 µm.\u003c/p\u003e","description":"","filename":"Fig6Ag10861ach0003c.png","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/d6a65c841d7ddb4114e0b310.png"},{"id":101302440,"identity":"a54866bb-5f97-4595-bdd9-d824f1a12c0c","added_by":"auto","created_at":"2026-01-28 09:54:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":96952,"visible":true,"origin":"","legend":"\u003cp\u003eSummary figure of gill ion transporter expression patterns. Basolateral NKA (green) colocalizes with NKCC1 (red) in seawater (sw) type MRC. CFTR anion channels (red) are expressed apically in the cells, and we propose an apical AE (purple) is also present. Basolateral NBC (blue) was not found in these cells. A second freshwater (fw) type MRC with basolateral NKA and NBC and apical NKCC/NCC is also present. Based on evidence in \u003cem\u003eO. mossabicus\u003c/em\u003e, this is likely NCC rather than NKCC2. Urea transporter (UT, green) was observed apically in pavement cells (PVCs).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/0a18e24990b235fae5d03382.png"},{"id":101398847,"identity":"38033fe3-6460-4e81-abb4-e580f6070cfc","added_by":"auto","created_at":"2026-01-29 09:49:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20932904,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/314f223d-bd05-4c90-afe6-cec5d4ca015f.pdf"},{"id":101397803,"identity":"885bbcc0-ca94-4e67-80f6-cb1237eefdc8","added_by":"auto","created_at":"2026-01-29 09:37:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14139,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8662004/v1/60d82982f659923a490df354.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gill ionocytes of the Lake Magadi tilapia (Alcolapia grahami), an extremophilic teleost native to a highly alkaline environment","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe Magadi tilapia, with a current taxonomic name of \u003cem\u003eOreochromis (Alcolapia) grahami\u003c/em\u003e as recommended by Ford et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (formerly \u003cem\u003eAlcolapia grahami\u003c/em\u003e, formerly \u003cem\u003eOreochromis alcalicus grahami\u003c/em\u003e, formerly \u003cem\u003eSarotherodon alcalicus grahami\u003c/em\u003e, formerly \u003cem\u003eTilapia grahami\u003c/em\u003e) lives in arguably the most extreme aquatic environment on earth for fish, Lake Magadi in the Rift Valley of Kenya. In addition to temperatures up to 45\u0026ordm;C in its environment, it survives extreme night-time hypoxia and anoxia, daytime hyperoxia, and some of the highest levels of reactive oxygen species (ROS) that have ever been recorded in natural waters (Coe, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Narahara et al., 1996; Johannsson et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, its greatest challenge may be the highly unusual water chemistry in the hot springs where it lives around the edges of Lake Magadi.\u003c/p\u003e \u003cp\u003eTables listing the compositions of Lake Magadi water and the plasma of \u003cem\u003eO. grahami\u003c/em\u003e can be found in Eddy et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1981\u003c/span\u003e), Eddy and Maloiy (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), Maloiy et al. (1984); Wood et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), Laurent et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), Wood et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e), Wood et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002b\u003c/span\u003e), Wilson et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and Wood et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Water values vary seasonally and among hot springs. However, the water pH is typically about 10 due to very high concentrations of HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e resulting in an alkalinity over 300 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at times and osmolality of about 500\u0026ndash;600 mOsm\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. By way of comparison, the typical pH of seawater is about 8, its alkalinity is 2\u0026ndash;3 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and its osmolality is about 1050 mOsm kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Moreover, unlike seawater where Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e concentrations are both high (450\u0026ndash;550 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), Lake Magadi water has Na\u003csup\u003e+\u003c/sup\u003e concentrations (~\u0026thinsp;356 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) that are ~\u0026thinsp;3-fold greater than Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e concentrations (~\u0026thinsp;110 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), because the alkaline anions (HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) account for most of the negative charge. Surprisingly, the Magadi tilapia is able to maintain a plasma ionic composition and osmolality at levels that are very similar to those of typical teleosts in seawater.\u003c/p\u003e \u003cp\u003eHowever, a particularly striking characteristic is that the fish excrete only urea and no ammonia, presumably an adaptation to the difficulty of excreting ammonia against a large pH gradient; \u003cem\u003eO. grahami\u003c/em\u003e is the only 100% ureotelic teleost (Randall et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Lindley et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). A specific facilitated diffusion urea transporter in Magadi tilapia (mtUT) has been cloned from the gills and tentatively localized to the pavement cells based on the presence of similar vesicles found in the gills of facultatively ureogenic Gulf toadfish (\u003cem\u003eOpsanus beta\u003c/em\u003e) by transmission electron microscopy (TEM) (Laurent et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Walsh et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). UT localization has been confirmed by IHC in the Gulf toadfish (Bucking et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has proven difficult to accurately measure the blood acid-base status in such small fish (typically\u0026thinsp;\u0026lt;\u0026thinsp;5 g), but plasma pH and HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentrations appear to be elevated above values typical of other teleosts at comparable temperature (Johansen et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002b\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This undoubtedly reflects the extreme alkalinity of the environment, which makes the water a \u0026ldquo;CO\u003csub\u003e2\u003c/sub\u003e vacuum\u0026rdquo; that renders the retention of dissolved CO\u003csub\u003e2\u003c/sub\u003e in the blood virtually impossible, while simultaneously presenting very high concentrations of basic equivalents to the external gill surface. Furthermore, in order to maintain body hydration in this hyperosmotic environment, Magadi tilapia drink the external water at a high rate (Maloiy et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e; Bergman et al., 2003). However, rather than using the intestinal transport of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e to drive the absorption of the water in the manner of seawater teleosts, instead they transport Na\u003csup\u003e+\u003c/sup\u003e and mainly HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e or CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, rather than Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in order to absorb water across the gut epithelium (Bergman et al., 2003). Thus, the influx of basic equivalents occurs at both intestine and gills.\u003c/p\u003e \u003cp\u003eDetailed analyses of the electrochemical gradients across the gills have demonstrated that there is a very strong net gradient driving basic equivalents\u003csup\u003e\u0026minus;\u003c/sup\u003e inwards, a modest net gradient driving Na\u003csup\u003e+\u003c/sup\u003e inwards, and a smaller net gradient driving Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e outwards (Eddy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Wood et al., 2004; Wood et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Passive permeability of the gills to HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e is unusually low, which is presumably adaptive to reduce the loading of basic equivalents (Wood et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Nevertheless, the cells are tasked with the unusual situation of excreting basic equivalents to the environment against a steep electrochemical gradient, while simultaneously excreting Na\u003csup\u003e+\u003c/sup\u003e yet taking up Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e. The transepithelial potential (TEP) is positive inside, and this results from an inwardly directed Na\u003csup\u003e+\u003c/sup\u003e diffusion potential upon which is superimposed an electrogenic component thought to result from the secondary active export of basic equivalents (Wood et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In standard marine teleosts, the TEP is also positive inside, but the electrogenic component results from the secondary active export of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e rather than basic equivalents (Potts, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Potts and Hedges, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere have been numerous morphological studies on the gill ionocytes using classical techniques (light, scanning, and TEM microscopy; Maina, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Maina, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Laurent et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; DeBoeck et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Some of these studies suggest that more than one type of ionocyte is present, but the general picture that has emerged is that most of the ionocytes have a typical seawater morphology and location. These cells are rich in mitochondria with a complex tubular network penetrating the cytoplasm from the basolateral surface and are recessed at the bases of pits bordered by partially overlying pavement cells and flanked by interdigitating accessory cells. Based only on the morphology of these ionocytes, Laurent et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) proposed a scheme of passive Na\u003csup\u003e+\u003c/sup\u003e export through the paracellular \u0026ldquo;shunt\u0026rdquo; pathway (between ionocytes and accessory cells) energized by the secondary active excretion of basic equivalents through the transcellular pathway into the apical crypts. This would be powered by basolateral Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e-ATPase, with basic equivalents entering the cell on a basolateral co-transporter analogous to NKCC (Na\u003csup\u003e+\u003c/sup\u003e:K\u003csup\u003e+\u003c/sup\u003e:2Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e cotransporter) and leaving apically through anion channels. The scheme was a modification of the classic \u0026ldquo;chloride cell\u0026rdquo; model of Silva et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), but with basic equivalents replacing Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e at every step. Later electrophysiological data (Wood et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) supported this model. Additionally, Laurent et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) proposed that Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e uptake occurred via an apical Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e/HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e exchanger on these cells.\u003c/p\u003e \u003cp\u003eImmunohistochemistry (IHC) is a powerful tool to characterize ionocytes beyond the information provided by structure (Dymowska et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hiroi and McCormick, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wilson 2013). However, with the exception of its use in localizing ammonia transporters (Wood et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), this technique has not been applied to the gills of \u003cem\u003eO.A. grahami.\u003c/em\u003e In other tilapine fishes, IHC has identified three (Dymowska et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) or four types (Hiroi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hiroi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Choi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hiroi and McCormick, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) of ionocyte. The model of Laurent et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) seems to match, at least in part, the Type IV \u0026ldquo;seawater ionocyte\u0026rdquo;, which is thought to be a transformation of the Type III ionocyte seen in freshwater and dilute salinities (Hiroi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hiroi and McCormick, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dymowska et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Inokuchi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present IHC study on the gills of the Magadi tilapia, we have applied a battery of antibodies that has proven useful in characterizing the various ionocytes in other teleosts. Our first goal was to determine whether the ionocytes fit into the three or four categories identified in other tilapine fishes (Dymowska et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hiroi et al., 2012; Inokuchi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A second objective was to evaluate the transport model of Laurent et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) for the most abundant type of ionocyte, and a third was to confirm the tentative localization of the mtUT urea transporter to the pavement cells (Walsh et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eAdult \u003cem\u003eO.A. grahami\u003c/em\u003e (2\u0026ndash;5 g) were collected by seine net from Fish Springs Lagoon (1\u0026deg;53\u0026prime;30.2\u0026Prime;S, 36\u0026deg;18\u0026prime;09.9\u0026Prime;E) at the edge of Lake Magadi, Kenya. The water chemistry of Fish Spring Lagoon measured at the time of capture is presented in Supplementary Table S1.\u003c/p\u003e \u003cp\u003eCollections were carried out under permission from the Department of Fisheries, Ministry of Livestock and Fisheries (Kenya), and the Magadi Soda Foundation. The research complied with the laws of Kenya, and was performed under a research ethics clearance permit (NCST/RR1/12/1/MAS/99) from the National Commission for Science, Technology and Innovation (NACOSTI Kenya), under a research permit issued by the National Council for Science and Technology of the Ministry of Higher Education, Science, and Technology of the Republic of Kenya. Protocols were approved by the Animal Use and Ethics Committee of the Faculty of Veterinary Medicine, University of Nairobi.\u003c/p\u003e \u003cp\u003eFish were euthanized with an overdose of MS-222 (Syndel, Nanaimo, BC, Canada), wrapped in aluminum foil, and flash-frozen whole in liquid nitrogen, then transported at -80\u0026ordm;C in a dry shipper (CX100, Taylor Wharton, Columbus, OH, USA), and stored in an ultracold freezer at -80 \u0026ordm;C for later processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Histology and Immunohistochemistry\u003c/h2\u003e \u003cp\u003eWhole frozen fish were immersion-fixed in Dent\u0026rsquo;s fixative (20%DMSO/methanol) chilled to -80\u0026deg;C. After one week the solution was substituted with 100% methanol at -80\u0026deg;C and then slowly brought to 4\u0026deg;C over 3 days. Whole fish were then decapitated and the heads divided medially. After bringing the samples to room temperature, they were cleared with xylene, infiltrated with Type 6 paraffin (Richard-Allen Scientific, San Diego, CA, USA) and embedded for sectioning.\u003c/p\u003e \u003cp\u003eFive-\u0026micro;m sections were collected onto either plain or APS (3-aminopropyl triethoxy silane) coated slides for respective histological staining with Periodic Acid Schiffs (PAS) and Alcian blue (pH 2.5) staining, or immunohistochemical staining for ion transporter proteins according to Wilson et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Immunohistochemistry was performed with combinations of antibodies that included mouse monoclonal cystic fibrosis transmembrane regulator (CFTR), Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e-ATPase (NKA) α subunit, and Na\u003csup\u003e+\u003c/sup\u003e: K\u003csup\u003e+\u003c/sup\u003e: 2 Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e co-transporter-1 / Na\u003csup\u003e+\u003c/sup\u003e: Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e co-transporter (NKCC1/NCC) antibodies plus rabbit polyclonal NKA, Na\u003csup\u003e+\u003c/sup\u003e: HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e co-transporter (NBC1), urea transporter [toadfish (tf) UT], the vacuolar type proton pump (VHA) B subunit, and NHE3b antibodies. See Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for specific details on the antibodies. NBC, tfUT and NHE3b antibodies were kindly provided by Drs. S. Hirose (Tokyo Institute of Technology, Yokohama, Japan), P. Walsh (U. Ottawa, Ottawa Canada), and J. Hiroi (St. Marianna University School of Medicine, Kawasaki, Japan), respectively. Secondary antibodies used were goat anti-mouse Alexa 594 and goat anti-rabbit Alexa 488 conjugated antibodies (Thermo Fisher Scientific, Toronto, ON, Canada) diluted 1:500. Negative and null controls were performed, which consisted of primary antibody substitution with either normal rabbit serum or antibody dilution buffer, respectively. When possible, antigen pre-absorption controls were also performed. For monoclonal antibody negative controls, equivalently diluted iso-typed culture supernatant was used. In these experiments, the clone J3, which is specific for shark NKCC (Slc12a1), was used. Sections were counter-stained with DAPI (4 ', 6-diamidine-2'-phenylindole dihydrochloride) and a differential interference contrast (DIC) image collected along with fluorescence using a Leica DM6000B wide-field photomicroscope with a DFX340 camera and LAS IF software (Leica Microsystems GmbH, Wetzlar Germany). Images were assembled and enhanced (Photoshop CS3, Adobe San Jose CA USA) while maintaining the integrity of the data.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of antibodies used to probe \u003cem\u003eOreochromis (Alcalicus) grahami\u003c/em\u003e gill sections. The antibody name, target, dilution in 1%Bovine Serum Albumin /Phosphate Buffered Saline with 0.05% Tween-20 and 1%Normal Goat Serum for Immunohistochemistry, host species, and reference information.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHost spp.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eα5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNKA α subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTakeyasu et al. 1988\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eαR1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNKA α subunit\u003c/p\u003e \u003cp\u003eVTGVEEGRLIFDNLKKC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUra et al. 1996; Wilson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNKCC/NCC\u003c/p\u003e \u003cp\u003ehuman NKCC1 C-terminus (Met902-Ser1212)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLytle et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1995\u003c/span\u003e\u003c/p\u003e \u003cp\u003eHiroi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e673\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMefugu \u003cem\u003eT. obscurus\u003c/em\u003e mfNBCe1 [amino acid (aa) residues 985\u0026ndash;1075]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKurita et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVHA B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRKDHADVSNQLYACYA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWilson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNHE3b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrout nhe3b position 755\u0026ndash;769: GDEDFEFSEGDSASG; 818\u0026ndash;839: PSQRAQLRLPWTPSNLRRLAPL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWright et al. 2016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e24\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCFTR c-term\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eR\u0026amp;D systems\u003c/p\u003e \u003cp\u003eMarshall et al. 2002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003etfUT Slc14a2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSEIDLPKLFMSIPV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBucking et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e \u003cb\u003eTissue morphology\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFlash freezing whole fish in liquid nitrogen, storage at -80\u0026deg;C and fixation in Dent\u0026rsquo;s fixative at low temperature (-80\u0026deg;C) resulted in tissue sections of reasonable quality for histological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Gill lamellae were largely devoid of large ionocytes which were limited to the gill filament along with large Alcian-blue staining goblet cells. There were some areas where freezing artefact could be identified and thus avoided for analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIHC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOn the basis of NKA α-subunit immunoreactivity using either the α5 mouse monoclonal or αR1 rabbit polyclonal antibodies, ionocytes could be easily recognized in the filament epithelium of the gills of Magadi tilapia with a dramatic increase in frequency towards the downstream or afferent edge (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These cells were readily identifiable by their apparent cytoplasmic staining which is indicative of the tubular system associated with the basolateral membrane in teleost ionocytes. Two types of ionocytes could be further characterized based on the strength of their NKA immunoreactivity and morphology. Ionocytes with strong NKA immunoreactivity were typically columnar-cuboidal in shape with a less pronounced apical crypt (indentation). In contrast, ionocytes with weaker NKA- immunoreactivity were characteristically ovoid-squamous in shape with a prominent apical crypt. These cells could be distinguished from pavement cells that were much more flattened and devoid of detectable NKA immunoreactivity. The NKA immunoreactive ionocytes were further characterized based on expression of other transporters including NKCC, NCC, CFTR and NBC.\u003c/p\u003e \u003cp\u003eLocalization of both the NKCC1 and NCC were done using the T4 mouse monoclonal antibody which recognizes both transporters although they can be differentiated based on subcellular localization (Hiroi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). NKCC1 is identified by its basolateral localization similar in appearance to NKA. Weak NKCC1 immunoreactivity was found to co-localize with intense NKA- immunoreactivity in the columnar-cuboidal ionocytes corresponding to the type IV sub population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, NCC immunoreactivity was identified on the basis of an apical localization to type II ionocytes. Strong NCC- immunoreactivity was present in either a shallow or deeper C-shaped concave crypt or pit in ovoid-squamous ionocytes which exhibited weak NKA- immunoreactivity.\u003c/p\u003e \u003cp\u003eCFTR immunoreactivity was found apically in the columnar-cuboidal type IV ionocytes with strong NKA immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The apical CFTR staining was restricted to a relatively small apical area in contrast to NCC and did not form a deep crypt or pit. CFTR- immunoreactivity was not seen in the ovoid-squamous ionocytes with weak NKA- immunoreactivity. The NBC1-like immunoreactivity was found associated with the ovoid-squamous ionocytes with weaker NKA- immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and apical NCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We could not find colocalization of NBC1 and CFTR to the same cell type (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The tfUT- immunoreactivity was found in the lamellar epithelium in a punctiform pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Staining was not associated with NKA- immunoreactive cells which were largely localized to the trailing edge of the filament epithelium. No immunoreactivity was found with the antibodies for V-type H\u003csup\u003e+\u003c/sup\u003e-ATPase and NHE3b (data not shown). Negative control staining of sections done in parallel showed negligible levels of background fluorescence (data not shown).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith respect to our original objectives, we have identified three different types of ionocyte in the gills of the Magadi tilapia which fit clearly into three (Types I, II, and IV) of the four categories of ionocytes that have previously been characterized in other tilapine fishes (Hiroi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hiroi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Choi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Dymowska et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hiroi and McCormick, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Inokuchi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, in the most abundant ionocyte (Type IV), our IHC results have confirmed many elements of the hypothetical model for Na\u003csup\u003e+\u003c/sup\u003e excretion plus basic equivalent excretion that was originally proposed by Laurent et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) on the basis of structure alone. Finally, we have also shown that the facilitated diffusion transporter UT is confined to the pavement cells, and does not occur in ionocytes, confirming the original conclusion of Walsh et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) that again was based on cell fine structure alone.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIonocyte types in the gills of\u003c/b\u003e \u003cb\u003eO.A. grahami\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on IHC labelling, ionocytes corresponding to Types I, II, and IV of Hiroi et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), Hiroi et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), Choi et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and Inokuchi et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) were found in the gills of the Magadi tilapia. This agrees with previous studies using classical techniques that have reported several types of ionocytes in the gills of this species, but without categorization (Maina, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Maina, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e). The localization of transporters within the ionocyte types is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eType I ionocytes stained only for NKA on the basolateral membranes and the vesicular network that extends from the base into most of the cytoplasmic component. The antibodies used in the present study did not identify any transporters on the apical membranes. However, there is evidence that in Mozambique tilapia, Type I ionocytes express ROMK (renal outer medullary K\u003csup\u003e+\u003c/sup\u003e) channels on their apical membranes and are involved in K\u003csup\u003e+\u003c/sup\u003e excretion (Furukawa et al., 2014). This could be useful in Magadi tilapia which eat mainly blue-green algae such as \u003cem\u003eArthrospira\u003c/em\u003e (Coe, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1966\u003c/span\u003e) that is rich in K\u003csup\u003e+\u003c/sup\u003e (Cogne et al., 2002). Plasma K\u003csup\u003e+\u003c/sup\u003e concentrations (~\u0026thinsp;5 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Wood et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) are about 2-fold environmental concentrations (Supplementary Table S1), and branchial K\u003csup\u003e+\u003c/sup\u003e excretion through ROMK channels would be further aided by the positive TEP inside the fish (Wood et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, since ROMK channels have also been found in Type III and IV cells in Mozambique tilapia (Furukawa et al., 2014; Inokuchi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) the more general view is that Type I cells are immature ionocytes with limited or no contact with the outside water, waiting to differentiate into the other types (Inokuchi et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). There is evidence that \u003cem\u003eO.A. grahami\u003c/em\u003e can be adapted to dilutions of Lake Magadi down to 1% (Wood et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002b\u003c/span\u003e) and even to true fresh water (Kavembe et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and that other populations in the Magadi basin live naturally in much more concentrated water (see Table\u0026nbsp;5 in Wilson et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) than found in Fish Spring Lagoons (Supplementary Table\u0026nbsp;1), so this capability probably becomes very important under these conditions.\u003c/p\u003e \u003cp\u003eType II ionocytes were identified by apical NCC and basolateral NBC1 immunoreactivity, and the absence of apical CFTR immunoreactivity (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These combined characteristics were present only in the ovoid-squamous ionocytes which exhibited weak NKA- immunoreactivity and pronounced apical pits. Note that according to Choi et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Hiroi and McCormick (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), the immunoreaction of the T4 antibody used in the present study is attributable to NCC when it appears in the apical membrane, and to NKCC1 when it appears in the basolateral membrane. In other tilapines, the Type II ionocyte is now considered to be a \u0026ldquo;freshwater ionocyte\u0026rdquo; responsible for Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e uptake from dilute environments, (Hiroi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hiroi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Dymowska, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hiroi and McCormick, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Inokuchi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Interestingly, Wood et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e) reported that ionocytes with this sort of structure increased in abundance when Magadi tilapia were challenged with environmental dilution. Indeed, they became completely dominant when the fish were acclimated to 1% Lake Magadi water.\u003c/p\u003e \u003cp\u003eIn other tilapines, Type II cells decrease at higher salinities, yet they are abundant in \u003cem\u003eO.A. grahami\u003c/em\u003e in Fish Springs Lagoon where the normal external osmolality is hypertonic and corresponds to about 57% of that of seawater (Supplementary Table S1). The explanation may lie in the unusual ionic composition of Lake Magadi water. In contrast to 57% seawater, Magadi water exhibits a relatively low Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration (Supplementary Table S1), much below that of the blood plasma. Indeed, taking the positive TEP inside into account, there is still an electrochemical gradient favouring the outward diffusion of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e at the gills (Wood et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To satisfy the need for acquisition of this essential ion, the Magadi tilapia must use the secondarily active uptake of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e via the NCC co-transporters on the Type II ionocytes. The additional (counter-productive) uptake of Na\u003csup\u003e+\u003c/sup\u003e is an unavoidable consequence, which is exported to the internal environment by the basolateral NKA. In this species, we found no IHC evidence for either apical Na\u003csup\u003e+\u003c/sup\u003e/ H\u003csup\u003e+\u003c/sup\u003e exchange or V-type H\u003csup\u003e+\u003c/sup\u003e-ATPase, the transporters usually associated with the active uptake of Na\u003csup\u003e+\u003c/sup\u003e and acidic equivalent excretion in the ionocytes of other non-tilapine teleosts (Evans et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Dymowska et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This is not surprising, as their function would presumably be maladaptive for Na\u003csup\u003e+\u003c/sup\u003e and acid-base homeostasis in the Magadi tilapia. Only NHE3 (and not V-type H\u003csup\u003e+\u003c/sup\u003e-ATPase) has been found in other tilapines, and only in Type III and IV ionocytes (Inokuchi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Choi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Type III ionocytes do not appear to be present in \u003cem\u003eO.A. grahami\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eType IV cells were by far the most abundant ionocytes in the gills of \u003cem\u003eO.A. grahami\u003c/em\u003e and were recognized by strong NKA immunoreactivity throughout the vesicular network of the cytoplasm and along the basolateral membrane, coupled with strong apical expression of CFTR, and relatively weak expression of NKCC1 which followed the same pattern as that of NKA (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These characteristics were confined to the columnar-cuboidal ionocytes with less prominent apical pits. These cells clearly resemble the ionocytes that are dominant in other tilapines when acclimated to seawater (Hiroi and McCormick, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Inokuchi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Note that when other tilapines are transferred from less saline to more saline waters, Type IV ionocytes may be generated by the transformation of Type III ionocytes, as well as by the differentiation of Type I ionocytes (Choi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Inokuchi et al., 2014). However, Type III ionocytes, which would have been identified by the presence of apical NHE and absence of apical CFTR, were not seen in the present study. The Type IV ionocytes identified in the present study appear to correspond to the most abundant ionocytes that were described in Magadi tilapia on the basis of morphology alone by Maina (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), Maina (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), Laurent et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and Wood et al.(2002a). Laurent et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) also documented that they were bordered by accessory cells that play an important role in the model for NaCl excretion (Silva et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Hiroi and McCormick, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) in seawater teleosts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of the Laurent model for ion transport in Type IV ionocytes of\u003c/b\u003e \u003cb\u003eO.A. grahami\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVery clearly, the model proposed Laurent and co-workers (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) for Na\u003csup\u003e+\u003c/sup\u003e, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, and basic equivalent transport across the gills of the Magadi tilapia was prescient. The first support for this model was provided by electrophysiological data showing that there is an inside positive TEP of partially electrogenic origin that is responsive in the expected fashion to experimental manipulations of HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e gradients (Wood et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The present IHC data provide additional support for the presence of key elements in the Type IV ionocytes of Magadi tilapia, namely apical CFTR and basolateral NKCC1, powered by basolateral NKA.\u003c/p\u003e \u003cp\u003eAs noted in the Introduction, Laurent\u0026rsquo;s scheme was a modification of the classic \u0026ldquo;chloride cell\u0026rdquo; model of Silva et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), with basic equivalents replacing Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e at every step. In 1995, the anion channel for apical exit of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in fish ionocytes had not been characterized, and it was not until three years later that it was tentatively identified as CFTR (Singer et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), a concept that is now generally accepted (Marshall and Singer, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Evans et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hiroi and McCormick, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Laurent proposed that this unknown apical anion channel could serve as the exit pathway for basic equivalents, but it was only around that same time that the first evidence was emerging from mammalian studies that CFTR could also conduct HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (Poulson et al., 1994; Linsdell et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Laurent also proposed that a basolateral NKCC-like transporter, energized by basolateral NKA, could serve as the entry pathway for basic equivalents, together with Na\u003csup\u003e+\u003c/sup\u003e, into the ionocyte. Na\u003csup\u003e+\u003c/sup\u003e would be exported by NKA into the paracellular channels for exit into the external water through leaky junctions between the ionocytes and accessory cells. This export of Na\u003csup\u003e+\u003c/sup\u003e would create the electrochemical gradient for the operation of the NKCC-like transporter. NKA is highly abundant and NKCC1 is present in the basolateral membranes of Type IV ionocytes (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and accessory cells are also present, in accord with the Laurent model. However, we are aware of no evidence whether or not NKCC can transport basic equivalents such as HCO\u003csub\u003e3\u003c/sub\u003e \u003csup\u003e\u0026minus;\u003c/sup\u003e and CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e on its anion binding site, so further research is necessary to evaluate this idea. Certainly, basolateral NBC is not present in Type IV ionocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which tends to eliminate this alternate pathway for HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e entry.\u003c/p\u003e \u003cp\u003eFinally, Laurent proposed than an apical Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e/HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e exchanger could account for Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e uptake from the water. There is no IHC evidence for or against this idea in the present study or in previous work on other tilapine fishes. However, as argued earlier, the presence of apical NCC on Type II ionocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e) may provide the pathway needed for Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e uptake from the external water.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLocalization of UT exclusively to the pavement cells in the gills of\u003c/b\u003e \u003cb\u003eO.A. grahami\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe fully ureotelic \u003cem\u003eO.A. graham\u003c/em\u003e was only the second teleost species in which a UT-type facilitated diffusion transporter was cloned from the gills and characterized (Walsh et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The initial discovery was in the facultatively ureogenic gulf toadfish (\u003cem\u003eOpsanus beta\u003c/em\u003e) (Walsh et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). However, we now know that UTs are expressed in the gills of many teleosts, even though most are ammoniotelic (LeMoine and Walsh, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the Magadi tilapia, the presence of intense vesicular trafficking in the pavement cells, and its absence in the pavement cells of the closely related Nile tilapia, led to the tentative conclusion that the UTs were expressed in the pavement cells (Walsh et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The present IHC observations confirm this conclusion, showing no UT in NKA-immunoreactive cells on the filament, but clear staining in pavement cells in the lamellar epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In this regard, the pattern is similar to the UT expression seen only in the pavement cells of the ammoniotelic plainfin midshipman (\u003cem\u003ePorichthys notatus\u003c/em\u003e) (Bucking et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) but differs from that in its close relative, the ureogenic gulf toadfish, where UT expression is localized to NKA immunoreactive cells on the filament (Bucking et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similarly, in the ammoniotelic eel, UT is seen only within NKA-immunoreactive cells on the filament, and not in pavement cells (Mistry et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Clearly, patterns vary amongst species, regardless of ureotelism versus ammoniotelism. However, as illustrated by Walsh et al. (2021), urea excretion rates in the 100% ureotelic Magadi tilapia are so much higher than in other fish that the much greater surface area of the pavement cells relative to the ionocytes may be required for efficient clearance.\u003c/p\u003e\n\u003ch3\u003eFuture Directions\u003c/h3\u003e\n\u003cp\u003eThe current IHC data show that the Magadi tilapia has three of the four basic ionocyte types that have been characterized in the gills other tilapines and suggest that they have been subtly re-engineered so as to function in such an unusual water chemistry. However, antibodies can reliably identify only proteins against which they are made. The recent full sequencing of the genome in this species (Bernardi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) opens up the possibility to look for many more transport proteins in the gills, both by IHC and \u003cem\u003ein situ\u003c/em\u003e hybridization. Another powerful avenue for future research is to employ the opercular epithelium of the Magadi tilapia, which is rich in ionocytes (Laurent et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), as a flat gill surrogate \u003cem\u003ein vitro\u003c/em\u003e. Both classical (e.g. Ussing chamber; McCormick et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and modern techniques (e.g. scanning ion-selective electrode technique or SIET; Horng et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) that have been successfully used on comparable preparations from other tilapines can then be employed to mechanistically characterize the ion transport mechanisms that allow this unique fish to live in such a hostile environment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Magadi Soda Company, particularly John Ndonga and John Kabera, for their co-operation, the University of Nairobi for logistic support, and Dishon Muthee and the late George Muthee furnished invaluable assistance in the field. Drs. S. Hirose (Tokyo Institute of Technology, Yokohama, Japan), P.J. Walsh (U. Ottawa, Ottawa Canada) and J. Hiroi (St Marianna University School of Medicine, Kawasaki, Japan) for the gift of antibodies. The α5 and J3 monoclonal antibody developed by Drs. D. M. Fambrough (Johns Hopkins University, MD, USA) and C. Lytle (Division of Biomedical Sciences, University of California, Riverside, CA, USA) were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. CMW was supported by the Canada Research Chair Program. AB is a Research Fellow from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and was supported by the International Canada Research Chair Program from the International Development Research Centre (IDRC, Ottawa, Canada). Funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grants to CMW (RGPIN-2017-03843, RGPIN-2023-03714) and JW (RGPIN-2019-06838), a grant from the Brazilian CNPq to AB, and a grant from the National Research Foundation of South Africa to JM.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBergman, A.N., Bergman, H.L., Laurent, P., Maina, J.N., Walsh, P.J., and Wood, C.M. \u0026nbsp;(1996). Respiratory physiology of the Lake Magadi tilapia (\u003cem\u003eOreochromis alcalilcus grahami\u003c/em\u003e), a fish adapted to a hot, alkaline, and frequently hypoxic environment. Physiol. Zool. 69: 1114-1136.\u003c/li\u003e\n \u003cli\u003eBernardi, G., Kavembe, G.D., Bergman, H.L., Bucciarelli, G. and Wood, C.M. (2024). The genome organization of the Lake Magadi tilapia, \u003cem\u003eOreochromis Alcolapia grahami\u003c/em\u003e, a cichlid extremophile. J. Great Lakes Res., 50(3), 102326.\u003c/li\u003e\n \u003cli\u003eBucking, C., Edwards, S. L., Tickle, P., Smith, C. P., McDonald, M. D., \u0026amp; Walsh, P. J. (2013). Immunohistochemical localization of urea and ammonia transporters in two confamilial fish species, the ureotelic gulf toadfish (\u003cem\u003eOpsanus beta\u003c/em\u003e) and the ammoniotelic plainfin midshipman (\u003cem\u003ePorichthys notatus\u003c/em\u003e).\u0026nbsp;Cell Tissue Res. 352, 623-637.\u003c/li\u003e\n \u003cli\u003eBurgess, D.W., Marshall, W.S., and Wood, C.M. (1998) Ionic transport by the opercular epithelia of freshwater acclimated tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) and killifish (\u003cem\u003eFundulus heteroclitus\u003c/em\u003e). Comp. Biochem. Physiol. A. 121 pp: 155-164\u003c/li\u003e\n \u003cli\u003eChoi, J. H., Lee, K. M., Inokuchi, M., and Kaneko, T. (2011). Morphofunctional modifications in gill mitochondria-rich cells of Mozambique tilapia transferred from freshwater to 70% seawater, detected by dual observations of whole-mount immunocytochemistry and scanning electron microscopy. Comp. Biochem. Physiol. A 158, 132\u0026ndash;142. doi:10.1016/j.cbpa.2010.09.019\u003c/li\u003e\n \u003cli\u003eCoe, M.J. (1966). The biology of \u003cem\u003eTilapia grahami\u003c/em\u003e Boulenger in Lake Magadi, Kenya. Acta Tropica 23, 146-177.\u003c/li\u003e\n \u003cli\u003eCogne, G., Lehmann, B., Dussap, C. G., and Gros, J. B. (2003). Uptake of macrominerals and trace elements by the cyanobacterium \u003cem\u003eSpirulina platensis\u003c/em\u003e (\u003cem\u003eArthrospira platensis\u003c/em\u003e PCC 8005) under photoautotrophic conditions: culture medium optimization. Biotech. Bioeng. 81(5), 588-593.\u003c/li\u003e\n \u003cli\u003eDe Boeck, G., Wood, C.M., Brix, K.V., Sinha, A., Matey, V., Johannsson, O.E., Bianchini, A., Bianchini, L.F., Maina, J.N., Kavembe, G.D., Papah, M.B., Kisipan, M.L., and Ojoo, R.O. (2019). Fasting in the ureotelic Lake Magadi tilapia, \u003cem\u003eAlcolapia\u003c/em\u003e\u003cem\u003egrahami\u003c/em\u003e, does not reduce its high metabolic demand. Conservation.Physiol. 10.1093/conphys/coz060.\u003c/li\u003e\n \u003cli\u003eDymowska, A. K., Hwang, P. P., \u0026amp; Goss, G. G. (2012). Structure and function of ionocytes in the freshwater fish gill.\u0026nbsp;Respir. Physiol. Neurobiol.\u0026nbsp;184(3), 282-292.\u003c/li\u003e\n \u003cli\u003eEddy,\u0026nbsp;F.B., Bamford,\u0026nbsp;O.S., and Maloiy,\u0026nbsp;G.M.O. 1981. Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003e effluxes and ionic regulation in \u003cem\u003eTilapia grahami\u003c/em\u003e, a fish living in conditions of extreme alkalinity. J. Exp. Biol.91, 349-353.\u003c/li\u003e\n \u003cli\u003eEddy, F.B. and Maloiy, G.M.O. 1984. Ionic content of body fluids and sodium efflux in O\u003cem\u003ereochromis alcalicus grahami\u003c/em\u003e, a fish living at temperatures above 30\u003csup\u003eO\u003c/sup\u003eC and in conditions of extreme alkalinity. Comp. Biochem. Physiol. 78A, 359-361.\u003c/li\u003e\n \u003cli\u003eEvans, D. H., Piermarini, P. M., and Choe, K. P. (2005). The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste.\u0026nbsp;Physiol. Rev.\u0026nbsp;85(1), 97-177.\u003c/li\u003e\n \u003cli\u003eFord, A. G., Bullen, T. R., Pang, L., Genner, M. J., Bills, R., Flouri, T., Ngatunga, B.P., R\u0026uuml;bere, L., Schlieweng, U.K., Seehausen, O., Shechonge, A, Stiassny, M.L.J., Turner, G.F., and Day, J. J. (2019). Molecular phylogeny of \u003cem\u003eOreochromis\u003c/em\u003e (Cichlidae: Oreochromini) reveals mito-nuclear discordance and multiple colonisations of adverse aquatic environments. Mol. Phylogenet. Evo. 136, 215-226.\u003c/li\u003e\n \u003cli\u003eFurukawa, F., Watanabe, S., Kimura, S., and Kaneko, T. (2012). Potassium excretion through ROMK potassium channel expressed in gill mitochondrion-rich cells of Mozambique tilapia.\u0026nbsp;Am J. Physiol.\u0026nbsp;302(5), R568-R576.\u003c/li\u003e\n \u003cli\u003eFurukawa F, Watanabe S, Inokuchi M, Kaneko T. (2011) Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities. Comp Biochem Physiol A 158: 468-476. 10.1016/j.cbpa.2010.12.003.\u003c/li\u003e\n \u003cli\u003eHiroi, J., and McCormick, S. D. (2012). New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish.\u0026nbsp;Resp. Physiol. Neurobiol.\u0026nbsp;184(3), 257-268.\u003c/li\u003e\n \u003cli\u003eHiroi, J., McCormick, S. D., Ohtani-Kaneko, R., and Kaneko, T. (2005). Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (\u003cem\u003eOreochromis mossambicus\u003c/em\u003e) embryos, by means of triple immunofluorescence staining for Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e-ATPase, Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e/2Cl\u003csup\u003e-\u003c/sup\u003e cotransporter and CFTR anion channel. J. Exp. Biol. 208, 2023\u0026ndash;2036. doi:10.1242/jeb.01611\u003c/li\u003e\n \u003cli\u003eHiroi, J., Yasumasu, S., McCormick, S.D., Hwang, P.P. and Kaneko, T., 2008. Evidence for an apical Na\u0026ndash;Cl cotransporter involved in ion uptake in a teleost fish. J. Exp. Biol. 211, 2584-2599.\u003c/li\u003e\n \u003cli\u003eHorng, J. L., Hwang, P. P., Shih, T. H., Wen, Z. H., Lin, C. S., \u0026amp; Lin, L. Y. (2009). Chloride transport in mitochondrion-rich cells of euryhaline tilapia (\u003cem\u003eOreochromis mossambicus\u003c/em\u003e) larvae.\u0026nbsp;Am. J. Physiol.\u0026nbsp;297(4), C845-C854.\u003c/li\u003e\n \u003cli\u003eInokuchi, M., Hiroi, J., \u0026amp; Kaneko, T. (2022). Why can Mozambique tilapia acclimate to both freshwater and seawater? Insights from the plasticity of ionocyte functions in the euryhaline teleost.\u0026nbsp;Front. Physiol.\u0026nbsp;13, 914277.\u003c/li\u003e\n \u003cli\u003eInokuchi M, Hiroi J, Watanabe S, Lee KM, Kaneko T. (2008). Gene expression and morphological localization of NHE3, NCC and NKCC1a in branchial mitochondria-rich cells of Mozambique tilapia (\u003cem\u003eOreochromis mossambicus\u003c/em\u003e) acclimated to a wide range of salinities. Comp Biochem Physiol A 151: 151\u0026ndash;158.\u003c/li\u003e\n \u003cli\u003eJohansen,\u0026nbsp;K., Maloiy,\u0026nbsp;G.M.O., and Lykkeboe,\u0026nbsp;G. 1975. A fish in extreme alkalinity. Respir. Physiol. 24,\u0026nbsp;156-162.\u003c/li\u003e\n \u003cli\u003eKavembe, G. D., Franchini, P., Irisarri, I., Machado-Schiaffino, G., \u0026amp; Meyer, A. (2015). Genomics of adaptation to multiple concurrent stresses: insights from comparative transcriptomics of a Cichlid fish from one of earth\u0026rsquo;s most extreme environments, the hypersaline soda Lake Magadi in Kenya, east Africa.\u0026nbsp;J. Mol. Evol. 81, 90-109.\u003c/li\u003e\n \u003cli\u003eKurita, Y., Nakada, T., Kato, A., Doi, H., Mistry, A.C., Chang, M.H., Romero, M.F. and Hirose, S., 2008. Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish.\u0026nbsp;Am. J. Physiol.\u0026nbsp;294(4), R1402-R1412.\u003c/li\u003e\n \u003cli\u003eJohannsson, O.E., Bergman, H.L., Wood, C.M., Laurent, P., Kavembe, D.G., Bianchini, A., Maina, J.N., Chevalier, C., Bianchini, L.F., Papah, M.B. and Ojoo, R.O. (2014) Air breathing in the Lake Magadi tilapia \u003cem\u003eAlcolapia grahami\u003c/em\u003e, under normoxic and hyperoxic conditions, and the association with sunlight and ROS. J. Fish Biol. 84:844-863.\u003c/li\u003e\n \u003cli\u003eLam, S.H., Lui, E.Y., Li, Z. et al. (2014) Differential transcriptomic analyses revealed genes and signaling pathways involved in iono-osmoregulation and cellular remodeling in the gills of euryhaline Mozambique tilapia, \u003cem\u003eOreochromis mossambicus\u003c/em\u003e. BMC Genomics 15, 921. https://doi.org/10.1186/1471-2164-15-921\u003c/li\u003e\n \u003cli\u003eLaurent, P., Maina, J. N., Bergman, H. L., Narahara, N., Walsh, P. J. and Wood, C. M. (1995). Gill structure of a fish from an alkaline lake: effect of short-term exposure to neutral conditions. Can. J. Zool 73, 1170\u0026ndash;1181.\u003c/li\u003e\n \u003cli\u003eLaurent, P., Wood, C. M., Wang, Y., Perry, S. F., Gilmour, K. M., P\u0026auml;rt, P. and Walsh, P. J.(2000). A new type of intracellular vesicular trafficking in the gill of urea-excreting fish (\u003cem\u003eOpsanus\u0026nbsp;\u003c/em\u003esp.) in the family Batrachoididae. Cell Tissue Res. 303(2), 197-210\u003c/li\u003e\n \u003cli\u003eLeMoine, C. M., \u0026amp; Walsh, P. J. (2015). Evolution of urea transporters in vertebrates: adaptation to urea\u0026apos;s multiple roles and metabolic sources.\u0026nbsp;J. Exp. Biol.\u0026nbsp;218(12), 1936-1945.\u003c/li\u003e\n \u003cli\u003eLindley, T.E., Scheiderer, C.L., Walsh, P.J., Wood, C.M., Bergman, H.G., Bergman, A.N., Laurent, P., Wilson, P., and Anderson, P.M. (1999). Muscle as a primary site of urea cycle enzyme activity in an alkaline lake-adapted tilapia, \u003cem\u003eOreochromis alcalicus grahami.\u003c/em\u003e J. Biol. Chem. \u003cstrong\u003e274\u003c/strong\u003e: 29858-29861.\u003c/li\u003e\n \u003cli\u003eLinsdell, P., Tabcharani, J. A., Rommens, J. M., Hou, Y. X., Chang, X. B., Tsui, L. C., Riordan, J.R., and Hanrahan, J. W. (1997). Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions.\u0026nbsp;J. Gen. Physiol.\u0026nbsp;110(4), 355-364.\u003c/li\u003e\n \u003cli\u003eLytle, C., Xu, J. C., Biemesderfer, D. and Forbush III, B.(1995). Distribution and diversity of Na\u0026ndash;K\u0026ndash;Cl cotransporter proteins: a study with monoclonal antibodies. Am. J. Physiol.269, C1496\u0026ndash;C1505.\u003c/li\u003e\n \u003cli\u003eMaina, J.N. 1990. A study of the morphology of the gills of an extreme alkalinity and hyperosmotic adapted teleost \u003cem\u003eOreochromis alcalicus grahami\u003c/em\u003e (Boulenger) with particular emphasis on the ultrastructure of the chloride cells and their modifications with water dilution. A SEM and TEM study. Anat. Embryol. 181, 83-98.\u003c/li\u003e\n \u003cli\u003eMaina JN (1991) A morphometric analysis of chloride cells in the gills of the teleosts \u003cem\u003eOreochromis alcalicus\u003c/em\u003e and \u003cem\u003eOreochromis niloticus\u003c/em\u003e and a description of presumptive urea-excreting cells in \u003cem\u003eO. alcalicus\u003c/em\u003e.J Anat 175: 131\u0026ndash;145\u003c/li\u003e\n \u003cli\u003eMaloiy, G.M.O., Lykkeboe, G., Johansen, K., and Bamford, O.S. 1978. Osmoregulation in \u003cem\u003eTilapia grahami\u003c/em\u003e: a fish in extreme alkalinity. \u003cem\u003eIn\u003c/em\u003e Comparative Physiology: Water, Ions and Fluid Mechanics. \u003cem\u003eEdited by\u003c/em\u003e K. Schmidt-Nielsen, L. Bolis, and S.H.P. Maddrell. Cambridge University Press, Cambridge, U.K. pp. 229-238.\u003c/li\u003e\n \u003cli\u003eMarshall, W. S., and Singer, T. D. (2002). Cystic fibrosis transmembrane conductance regulator in teleost fish.\u0026nbsp;Biochim. Biophys. Acta (BBA)-Biomembranes,\u0026nbsp;1566(1-2), 16-27.\u003c/li\u003e\n \u003cli\u003eMcCormick, S. D., Hasegawa, S., and Hirano, T. (1992). Calcium uptake in the skin of a freshwater teleost.\u0026nbsp;Proc. Nat. Acad. Sci.\u0026nbsp;89(8), 3635-3638.\u003c/li\u003e\n \u003cli\u003eMistry, A. C., Honda, S., Hirata, T., Kato, A., and Hirose, S. (2001). Eel urea transporter is localized to chloride cells and is salinity dependent. Am. J. Physiol. 281(5), R1594-R1604.\u003c/li\u003e\n \u003cli\u003ePoulsen, J. H., Fischer, H., Illek, B., and Machen, T. E. (1994). Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc. Nat. Acad. Sci. \u0026nbsp;91(12), 5340-5344.\u003c/li\u003e\n \u003cli\u003ePotts WTW (1984) Transepithelial potentials in fish gills. In: Hoar WS, Randall DJ (eds) Fish Physiology. Vol 10B Academic Press, Orlando, pp 105-128\u003c/li\u003e\n \u003cli\u003ePotts WTW, Hedges AJ(1991) Gill potentials in marine teleosts. J Comp Physiol B161: 401-405\u003c/li\u003e\n \u003cli\u003eRandall, D.J., Wood, C.M., Perry, S.F., Bergman, H.L., Maloiy, G.M.O., Mommsen, T.P., and Wright, P.A. (1989) Urea excretion as a strategy for survival in a fish living in a very alkaline environment. Nature: 337: 165-166.\u003c/li\u003e\n \u003cli\u003eSilva, P., Soloman, R., Spokes, K., and Epstein, F. H. (1977). Ouabain inhibition of gill Na\u0026apos;-K\u0026apos;-ATPase: Relationship to active chloride transport. J. Exp Zool. 199: 419-426.\u003c/li\u003e\n \u003cli\u003eSinger, T. D., Tucker, S. J., Marshall, W. S., and Higgins, C. F. (1998). A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost \u003cem\u003eFundulus heteroclitus\u003c/em\u003e. Am. J. Physiol. 274(3), C715-C723\u003c/li\u003e\n \u003cli\u003eWalsh, P.J., Grosell, M., Goss, G.G., Bergman, H.L., Bergman, A.N., Wilson, P., Laurent, P., Alper, S.L., Smith, C.P., Kamunde, C. and Wood, C.M., 2001. Physiological and molecular characterization of urea transport by the gills of the Lake Magadi tilapia (\u003cem\u003eAlcolapia grahami\u003c/em\u003e). J. Exp Biol. 204(3), 509-520.\u003c/li\u003e\n \u003cli\u003eWalsh, P.J., Heitz, M., Campbell, C.E., Cooper, G.J., Medina, M., Wang, Y.S., Goss,\u0026nbsp;G.G., Vincek, V., Wood, C.M., and Smith, C.P. (2000) Molecular identification of a urea transporter in gill of the ureotelic gulf toadfish (\u003cem\u003eOpsanus beta\u003c/em\u003e). J. Exp Biol. 203: 2357-2364\u003c/li\u003e\n \u003cli\u003eWatanabe, S., Niida, M., Maruyama, T., Kaneko, T., 2008. Na\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e exchanger isoform 3 expressed in apical membrane of gill mitochondrion-rich cells in Mozambique tilapia \u003cem\u003eOreochromis mossambicus\u003c/em\u003e. Fish. Sci. 74, 813\u0026ndash;821.\u003c/li\u003e\n \u003cli\u003eWilson, J.M., Leit\u0026atilde;o, A., Gon\u0026ccedil;alves, A.F., Ferreira, C., Reis-Santos, P., Fonseca, A.V., da Silva, J.M., Antunes, J.C., Pereira-Wilson, C. and Coimbra, J., 2007. Modulation of branchial ion transport protein expression by salinity in glass eels (\u003cem\u003eAnguilla anguilla\u003c/em\u003e L.). Mar. Biol. 151: 1633-1645.\u003c/li\u003e\n \u003cli\u003eWilson, P.J., Wood, C.M., Walsh, P.J., Bergman, A.L., Bergman, H.L., Laurent, P., and White, B.N. (2004) Discordance between genetic structure and morphological, ecological, and physiological adaptation in Lake Magadi tilapia. Physiol. Biochem. Zool. 77: 537-555.\u003c/li\u003e\n \u003cli\u003eWood C.M., Brix K.V., De Boeck, G., Bergman, H.L., Bianchini, A., Bianchini, L.F., Maina, J.N., Johannsson, O.E., Kavembe, G.D., Papah, M.B., Letura, K.M. and Ojoo, R.O. (2016). Mammalian metabolic rates in the hottest fish on earth. Sci. Reports 6: 26990.\u003c/li\u003e\n \u003cli\u003eWood, C.M., Nawata, C.M., Wilson, J.M., Laurent, P., Chevalier, C., Bergman, H.L., Bianchini, A., Maina, J.N., Johannson, O.E., Bianchini, L.F., Kavembe, G.D., Papah, M.B. and Ojoo, R.O. (2013). Rh proteins and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-activated Na\u003csup\u003e+\u003c/sup\u003eATPase in the Magadi Tilapia (\u003cem\u003eAlcolapia grahami\u003c/em\u003e), a 100% ureotelic teleost fish. J. Exp. Biol. 216: 2998-3007.\u003c/li\u003e\n \u003cli\u003eWood C.M., Bergman H.L., Bianchini A., Laurent P., Maina J., JohannssonO.E., Bianchini L.F., Chevalier C., Kavembe G.D., Papah M.B.,Ojoo R.O. (2012). Transepithelial potential in Magadi tilapia, a fish in extreme alkalinity. J. Comp. Physiol. B. 182: 247-258.\u003c/li\u003e\n \u003cli\u003eWood, C.M., Wilson, P.W., Bergman, H.L., Bergman, A.N., Laurent, P., Otiang\u0026rsquo;a-Owiti, G., and Walsh, P.J. (2002a). Ionoregulatory strategies and the role of urea in the Magadi tilapia (\u003cem\u003eAlcolapia grahami\u003c/em\u003e). Can. J. Zool. 80: 503-515.\u003c/li\u003e\n \u003cli\u003eWood, C.M., Wilson, P.W., Bergman, H.L., Bergman, A.N., Laurent, P., Otiang\u0026rsquo;a-Owiti, G., and Walsh, P.J. (2002b). Obligatory urea production and the cost of living in the Magadi tilapia revealed by acclimation to reduced salinity and alkalinity. Physiol. Biochem. Zool. 75: 111-122.\u003c/li\u003e\n \u003cli\u003eWood, C.M., Bergman, H.L., Laurent, P., Maina, J.N., Narahara, A., and Walsh, P. (1994). Urea production, acid-base regulation and their interactions in the Lake Magadi tilapia, a unique teleost adapted to a highly alkaline environment. J. Exp. Biol. 189: 13-36.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWood, C.M., Perry, S.F., Wright, P.A., Bergman, H.L., and Randall, D.J. (1989). Ammonia and urea dynamics in the Lake Magadi tilapia, a ureotelic teleost fish adapted to an extremely alkaline environment. Resp. Physiol. 77: 1-20.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8662004/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8662004/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Magadi tilapia thrives in arguably the most extreme aquatic environment on earth for fish, the hot springs of Lake Magadi in Kenya with its severe water chemistry: pH 10, alkalinity 300 mEq·L-1. This fish is 100% ureotelic yet has an osmoregulation pattern typical of marine teleosts, although the dominant anion is HCO 3- rather than Cl-. The gills must actively export basic equivalents (HCO 3-+CO 3 2-) and Na + against strong electrochemical gradients, yet simultaneously take up Cl-, for which a hypothetical “Laurent model” based on ionocyte structure alone was proposed. This model has been tested using immunohistochemistry to characterize ionocyte types based on ion transport protein expression patterns [CFTR anion channel, Na + :K + :2Cl-cotransporter (NKCC)/ Na + :Cl-cotransporter (NCC), Na + :HCO 3-co-transporter (NBC), Na + /K +-ATPase (NKA), and urea transporter (UT)]. A typical “seawater ionocyte” (Type IV) with apical CFTR and basolateral NKCC1 and NKA, is present validating key elements of the model. A “freshwater ionocyte” (Type II) is also present (apical NCC, weaker NKA and strong NBC1 basolateral staining). A third Type I ionocyte with only strong NKA staining was also identified. An acid excreting Type III ionocyte (apical NHE3 and basolateral NKA) was not present. The Magadi tilapia is unusual in having co-expression of both Type IV and Type II ionocytes, which are typically associated with Cl-excretion and uptake, respectively. Instead, we propose Type IV ionocytes are involved in basic equivalent and Na + excretion and Type II ionocytes in Cl-uptake. In these ureotelic fishes, the UT occurs only in lamellar pavement cells.\u003c/p\u003e","manuscriptTitle":"Gill ionocytes of the Lake Magadi tilapia (Alcolapia grahami), an extremophilic teleost native to a highly alkaline environment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 09:35:04","doi":"10.21203/rs.3.rs-8662004/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-13T12:19:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-13T10:03:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T06:39:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201411990226527037454578023671471065958","date":"2026-02-06T08:13:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161912550705295516333108790351778389353","date":"2026-01-30T15:40:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-30T14:58:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-26T07:37:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-26T07:34:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Research","date":"2026-01-21T16:06:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"43153a9a-1ee4-4fa3-9b3c-ce14b6c38c76","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T03:08:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 09:35:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8662004","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8662004","identity":"rs-8662004","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}